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Nuclear factor ??B binding activity in mouse L1210 cells following Photofrin II-mediated photosensitization
Abstract
Clinical photodynamic therapy (PDT) uses the photosensitizer photofrin II to produce singlet molecular oxygen and other reactive oxygen intermediates for localized tumor tissue cytotoxicity. In this report, we show that PDT enhances the DNA binding activity of nuclear factor kappa B (NF kappa B), a transactivator of cytokine gene expression. Photosensitization following a 16 h incubation of photofrin II induced NF kappa B binding activity in mouse leukemia L1210 cells 10-fold above that observed in exponentially growing cultures. Serum starvation, as well as drug-alone and light-alone controls, elevated basal NF kappa B binding activity two- to three-fold. Upstream stimulatory factor binding activity was not modulated by any of the cell treatments and was used to standardize gel mobility shift data. This study identifies porphyrin-mediated PDT as an inducer of NF kappa B binding activity, extending recent findings that NF kappa B activation is a general response to oxidative stress.
... 14 AP-1 is a dimer composed of subunits belonging to the Jun and Fos families. Photodynamic treatment has been reported to activate NF-κB [19][20][21][22][23] and AP-1 24,25 and stimulate their translocation into the nucleus, where they initiate gene expression. STAT-3 is a component of the Janus kinase (Jak)/STAT signaling pathway. ...
... 41 NF-κB and Ca 2þ -dependent enzymes, calpain and calcineurin, were involved in reperfusion-induced apoptosis of astrocytes. 42 PDT has been reported to activate NF-κB in various cell lines [19][20][21][22] and facilitate its binding to DNA. 22 PDT-induced activation of NF-κB caused apoptosis of cultured cancer cells. ...
... 42 PDT has been reported to activate NF-κB in various cell lines [19][20][21][22] and facilitate its binding to DNA. 22 PDT-induced activation of NF-κB caused apoptosis of cultured cancer cells. 21 The present proapoptotic effect of NF-κB on photosensitized crayfish glial cells is in line with these findings. ...
Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.
... 2−5 PDT induces cytotoxic reactive oxygen species (ROS) generation in the presence of photosensitizer (PS), molecular oxygen, and laser light. 6−8 As a noninvasive procedure, PDT has recently been shown to initiate the antitumor immune response by several mechanisms, including: (1) up-regulating expression of the chaperone heat shock protein 70 (HSP-70) 9 and transcription factor nuclear factor kappa B (NF-κB); 10 (2) stimulating the host immune response by promoting cytokine secretion; 11 and (3) facilitating antigen presentation to cytotoxic T lymphocytes (CTL). 12 Although PDT has been shown to be an effective therapeutic modality for a number of cancers, it has shown marginal to moderate effects in therapy against advanced or metastatic tumors. ...
... NF-κB p65 (Rel A) was also activated in laser-treated cells, implying that p65 is another crucial mediator for PDT induction of immune response. 10 To evaluate the therapeutic effect of combined PD-L1-KD and PDT in vivo, an antitumor study was performed by our group using a B16-F10 tumor xenograft in C57BL/6 mouse model. PBS or POP−NC showed negligible influence on the tumor growth, whereas PDT or PD-L1 KD alone significantly inhibited ∼73% and ∼65% of the tumor growth, respectively. ...
Photodynamic therapy (PDT) has emerged as a promising clinical modality for cancer therapy due to its ability to initiate an antitumor immune response. However, PDT-mediated cancer immunotherapy is severely impaired by tumor cell immunosuppression of host T-cell antitumor activity through the programmed cell death 1 ligand (PD-L1) and programmed cell death receptor 1 (PD-1) (PD-L1/PD-1) immune checkpoint pathway. Here, we demonstrate that PDT-mediated cancer immunotherapy can be augmented by PD-L1 knockdown (KD) in tumor cells. We rationally designed a versatile micelleplex by integrating an acid-activatable cationic micelle, photosensitizer (PS), and small interfering RNA (siRNA). The micelleplex was inert at physiological pH conditions and activated only upon internalization in the acidic endocytic vesicles of tumor cells for fluorescence imaging and PDT. Compared to PDT alone, the combination of PDT and PD-L1 KD showed significantly enhanced efficacy to inhibit tumor growth and distant metastasis in a B16-F10 melanoma xenograft tumor model. These results suggest that acid-activatable micelleplexes utilizing PDT-induced cancer immunotherapy are more effective when combined with siRNA-mediated PD-L1 blockade. This study could provide a general strategy to enhance the therapy efficacy of photodynamic cancer therapy.
... PDT with Photofrin II [224], methylene blue [218]; proflavin [217], aminopyropheophorbide (APP) [225], PMME [121,226], or verteporfin [227] activated NF-B in diverse cell lines. In these cases I B disappeared in the cytoplasm and NF-B heterodimer p50/p65 appeared in the nucleus. ...
... In these cases I B disappeared in the cytoplasm and NF-B heterodimer p50/p65 appeared in the nucleus. PDT also increased NF-B binding to DNA [224]. Photosensitization of human colon carcinoma HCT-116 cells with PMME or APP caused biphasic activation of NF-B. ...
Photodynamic therapy (PDT), in which stained cells are damaged by light in the presence of oxygen, is now widely used for tumor destruction. Photogenerated singlet oxygen and reactive oxygen species cause oxidative stress and cell death. The potential ROS sensors and following intracellular processes leading to cell death are considered. The cell death mode (necrosis or apoptosis) is shown to be controlled not only by PDT parameters (irradiation intensity, intracellular photosensitizer localization and its concentration) but also by signal transduction processes. Calcium and adenylate cyclase signaling pathways, receptor tyrosine kinases, MAP kinases, phosphatidylinositol 3-kinase pathway, various protein kinases and phosphatases, transcription factors, ceramide, NO, the plasma membrane, mitochondria and endoplasmic reticulum are involved in the cell response to photodynamic injury and following death. Combination of PDT and pharmacological modulators of signaling pathways can either enhance injury of malignant cells, or protect surrounding normal cells.
... However, DHA significantly abrogated the inductive effects of PDT on NF-κB activation. The activation of NF-κB induced by PDT was first found by Ryter et al. in their study on the Photofrinmediated PDT treatment of leukemia in 1993 [21]. Researchers have subsequently found this phenomenon in PDT mediated by other photosensitizers, such as 5-ALA and zinc phthalocyanine [22][23][24]. ...
Background/aims:
Although photodynamic therapy (PDT) can relieve esophageal obstruction and prolong survival time of patients with esophageal cancer, it can induce nuclear factor-kappa B (NF-κB) activation in many cancers, which plays a negative role in PDT. Dihydroartemisinin (DHA), the most potent artemisinin derivative, can enhance the effect of PDT on esophageal cancer cells. However, the mechanism is still unclear.
Methods:
We generated stable cell lines expressing the super-repressor form of the NF-κB inhibitor IκBα and cell lines with lentivirus vector-mediated silencing of the HIF-1α gene. Esophageal xenograft tumors were created by subcutaneous injection of Eca109 cells into BALB/c nude mice. Four treatment groups were analyzed: a control group, photosensitizer alone group, light alone group, and PDT group. NF-κB expression was detected by an electrophoretic mobility shift assay, hypoxia-inducible factor α (HIF-1α) and vascular endothelial growth factor (VEGF) by real-time PCR, NF-κB, HIF-1α, and VEGF protein by western blot, and Ki-67, HIF-1α, VEGF, and NF-κB protein by immunohistochemistry.
Results:
PDT increased NF-κB activity and the gene expression of HIF-1α and VEGF in vitro and in vivo. In contrast, the DHA groups, particularly the combined DHA and PDT treatment group, abolished the effect. The combined treatment significantly inhibited tumor growth in vitro and in vivo. NF-κB activity and HIF-1α expression were also reduced in the stable IκBα expression group, whereas the former showed no change in HIF-1α-silenced cells.
Conclusion:
DHA might increase the sensitivity of esophageal cancer cells to PDT by inhibiting the NF-κB/HIF-1α/VEGF pathway.
... These results agree with an earlier study reporting that the transcription factor AP-1 is involved in the in vitro expression of IL-6 following PDT (71). Gel mobility shift assays have also demonstrated that PDT can activate the transcription factor, nuclear factor kappa B, which is also involved in regulating the expression of numerous immunologically important genes (72). ...
... PDT produces an oxidative stress that can result in activation and translocation of NF-B to the nucleus, as originally shown for treatment of L1210 murine leukemia cells with Photofrin and light. [85]. The claim that NF-B may be a target for PDT has been convincingly demonstrated by Granville et al. [86]. ...
... It is a "rapid-acting" primary transcription factor that is present in cells in an inactive state and does not require new protein synthesis for activation 11 . In a number of studies, NF-κB has been reported to be activated during PDT [17][18][19][22][23][24][25] . ...
Photodynamic therapy (PDT) is used for selective destruction of cells, in particular, for treatment of brain tumors. However, photodynamic treatment damages not only tumor cells, but also healthy neurons and glial cells. To study the possible role of NF-κB in photodynamic injury of neurons and glial cells, we investigated the combined effect of photodynamic treatment and NF-κB modulators: activator betulinic acid, or inhibitors parthenolide and CAPE on an isolated crayfish stretch receptor consisting of a single neuron surrounded by glial cells. A laser diode (670 nm, 0.4 W/cm2) was used as a light source. The inhibition of NF-κB during PDT increased the duration of neuron firing and glial necrosis and decreased neuron necrosis and glial apoptosis. The activation of NF-κB during PDT increased neuron necrosis and glial apoptosis and decreased glial necrosis. The difference between the effects of NF-κB modulators on photosensitized neurons and glial cells indicates the difference in NF-κB-mediated signaling pathways in these cell types. Thus, NF-κB is involved in PDT-induced shortening of neuron firing, neuronal and glial necrosis, and apoptosis of glial cells.
... These results agree with an earlier study reporting that the transcription factor AP-1 is involved in the in vitro expression of IL-6 following PDT (71). Gel mobility shift assays have also demonstrated that PDT can activate the transcription factor, nuclear factor kappa B, which is also involved in regulating the expression of numerous immunologically important genes (72). ...
... NF-κB is one of the major transcription factors induced by PDT [194,195,[235][236][237][238][239], although in some instances NF-κB was also found to be downregulated following PDT, such as in nasopharyngeal carcinoma (hypericin as photosensitizer) and breast cancer cell lines (C-phycocyanin as photosensitizer) [240,241]. Despite the elusive NF-κB activation mechanism(s) in case of PDT, it is clear that NF-κB activation does occur after PDT on the basis of findings concerning at least two downstream targets of the NF-κB transcription factor, namely COX-2 and survivin. ...
Photodynamic therapy (PDT) has emerged as a promising alternative to conventional cancer therapies such as surgery, chemotherapy, and radiotherapy. PDT comprises the administration of a photosensitizer, its accumulation in tumor tissue, and subsequent irradiation of the photosensitizer-loaded tumor, leading to the localized photoproduction of reactive oxygen species (ROS). The resulting oxidative damage ultimately culminates in tumor cell death, vascular shutdown, induction of an antitumor immune response, and the consequent destruction of the tumor. However, the ROS produced by PDT also triggers a stress response that, as part of a cell survival mechanism, helps cancer cells to cope with the PDT-induced oxidative stress and cell damage. These survival pathways are mediated by the transcription factors activator protein 1 (AP-1), nuclear factor E2-related factor 2 (NRF2), hypoxia-inducible factor 1 (HIF-1), nuclear factor κB (NF-κB), and those that mediate the proteotoxic stress response. The survival pathways are believed to render some types of cancer recalcitrant to PDT and alter the tumor microenvironment in favor of tumor survival. In this review, the molecular mechanisms are elucidated that occur post-PDT to mediate cancer cell survival, on the basis of which pharmacological interventions are proposed. Specifically, pharmaceutical inhibitors of the molecular regulators of each survival pathway are addressed. The ultimate aim is to facilitate the development of adjuvant intervention strategies to improve PDT efficacy in recalcitrant solid tumors.
... The first demonstration that NF-κB activation can be achieved by photosensitization was done in 1993 by Ryter et al. who showed that a 10-fold increase of NF-κB activity can be detected in the nucleus of mouse leukemia L1210 cells after photofrin mediated photosensitization. 29 This work was rapidly followed by the studies of Legrand-Poels et al. 30 and of Piret et al. 31 In these two studies, NF-κB was shown to be fully activated in a T lymphoblastic cell line (ACH-2) by a DNA binding photosensitizer (proflavine) a lysosomotropic photosensitizer (Methylene Blue) and by a cytoplasmic photosensitizer (Rose Bengal). In these papers, NF-κB activation was demonstrated by the presence of the heterodimer RelA/p50 in the cell nucleus after irradiation using a band shift assay method and by following IκBα degradation by Western blot. ...
The response of tumours to photodynamic therapy (PDT) largely varies upon the intensity of the stress created in the cancer cells but also in the local environment. Singlet oxygen has been demonstrated, in many instances, as being the primary reactive oxygen species generated by PDT and responsible for most of the cellular effects. Cancer cells have developed various sensors which activate signalling pathways in response to PDT and the nature of the activated pathway varies with the PDT stress intensity. At low dose PDT, signalling pathways allow cancer cells to both proliferate and to switch on pro-survival responses such as autophagy. Above a certain level of PDT stress intensity, cancer cells cannot cope with the numerous damage and signalling pathways leading to cell death are activated. Two types of regulated cell death have been shown to be induced by PDT: apoptosis and necrosis. Signalling pathways activating NF-κB transcription factors have the peculiarity to be activated both at low and high doses of PDT; controlling the cross-talk with the immune system via the release of cytokines and chemokines but also an anti-cell death response via the control of apoptosis and necrosis. Therefore, NF-κB induced by PDT appears to play a positive role in educating the immune system to fight tumours but also a negative role in helping cancer cells to survive to the stress generated by singlet oxygen; explaining why NF-κB cannot easily be considered as a pharmacological target whose inhibition will favour tumour cells eradication by PDT.
... These results agree with an earlier study reporting that the transcription factor AP-1 is involved in the in vitro expression of IL-6 following PDT (71). Gel mobility shift assays have also demonstrated that PDT can activate the transcription factor, nuclear factor kappa B, which is also involved in regulating the expression of numerous immunologically important genes (72). ...
Photodynamic therapy involves administration of a tumorlocalizing photosensitizing agent, which may require metabolic synthesis
(i.e., a prodrug), followed by activation of the agent by light of a specific wavelength. This therapy results in a sequence
of photochemical and photobiologic processes that cause irreversible photodamage to tumor tissues. Results from preclinical
and clinical studies conducted worldwide over a 25-year period have established photodynamic therapy as a useful treatment
approach for some cancers. Since 1993, regulatory approval for photodynamic therapy involving use of a partially purified,
commercially available hematoporphyrin derivative compound (Photofrin®) in patients with early and advanced stage cancer of
the lung, digestive tract, and genitourinary tract has been obtained in Canada, The Netherlands, France, Germany, Japan, and
the United States. We have attempted to conduct and present a comprehensive review of this rapidly expanding field. Mechanisms
of subcellular and tumor localization of photosensitizing agents, as well as of molecular, cellular, and tumor responses associated
with photodynamic therapy, are discussed. Technical issues regarding light dosimetry are also considered. [J Natl Cancer Inst
1998;90:889-905]
... These results agree with an earlier study reporting that the transcription factor AP-1 is involved in the in vitro expression of IL-6 following PDT (71). Gel mobility shift assays have also demonstrated that PDT can activate the transcription factor, nuclear factor kappa B, which is also involved in regulating the expression of numerous immunologically important genes (72). ...
... 29 Deliberate production of reactive oxygen species within the cell, for example in photodynamic therapy, is an effective activator of NF-6B. [30][31][32] Consistent with the role of oxidative stress as an activating agent, treatment with antioxidants usually reduces the activation of the transcription factor. 33-35 ...
The cells of the retinal pigment epithelium (RPE) are subject to photo-oxidative stress arising from the interaction of incident light with lipofuscin, melanin, and other pigment granules in the RPE cytoplasm. Specific genotypic responses to these stressors are controlled by transcription factors, such as NF-kappaB (RelA/p50 dimer). The effects of CW laser exposures on NF-kappaB nuclear translocation have been studied in a line of human-derived RPE cells (hTERT-RPE) that develop melanin pigmentation in culture. The cells were exposed to the CW emission of an Argon-ion laser for 10 m at 0.5 W/cm2, a range previously shown to produce oxidation of cellular proteins, DNA, and antioxidants. NF-kappaB dimer was measured in nuclear extracts by an electrophoretic mobility shift assay. NF-kappaB nuclear translocation exhibited a modest, early peak at 1 h, and a larger, late peak at 24 h. NF-kappaB activation could be reduced only by some antioxidants; for example, 20 mM N-acetyl-L-cysteine or 100 muM pyrrolidine dithiocarbamate were ineffective, while 500 muM ascorbic acid was highly effective. These results indicate that interaction of the laser with the RPE melanin granules is a likely source of oxidative reactions, and that the induction of photoxidative stress activates NF-kappaB, but it remains to be determined if NF-kappaB is pro- or anti-apoptotic in the RPE cell.
... On the other hand, more often than not, if the treatment conditions are right, PDT could induce strong immune responses to enhance cancer treatment [4][5][6][7]. One component of PDT induced immune responses is through inflammatory reaction, which can initiate activation of the host antitumor immunity [8][9][10][11]. ...
Combination therapy using laser photothermal interaction and immunological stimulation has demonstrated its ability to induce immunological responses. Glycated chitosan (GC), an immunological stimulant, and imiquimod, a new type of immune response modifier (IRM), when used in conjunction with laser phototherapy, have shown to have a great immunological stimulation function. Specifically, imiquimod can help release cytokines from immunocompetent cells, stimulate TH1 lymphocyte responses (CD8+ T-cells), and recruit additional dendritic cells. To study the effects of immunoadjuvnats in combination of laser photo-irradiation, we treated animal tumors with laser-ICG-GC combination and late-stage melanoma patients with laser-ICG-imiquimod combination. At designated times, tumors, blood, and spleens in both treated and untreated animals were colleted for analysis. The major immunological indicators, such as IL-6, IL-12, IFN-gamma, CD4, and CD8 were analyzed. The same immunological analysis was also performed for melanoma patients treated by the laser-imiquimod combination.
... Activation of NF-κB upon photosensitization was first shown in studies using mouse leukemia L1210 cells and PF as a sensitizer [59]. PDT of the lymphocytic ACH-2 cell line with methylene blue led to the degradation of IκBa and increased NF-κB DNA-binding activity [60] and similar results were found using proflavine (a PS that intercalates into DNA) that did not cause AP-1 activation [61]. ...
Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In the second of a series of three reviews, we will discuss the mechanisms that operate in PDT on a cellular level. In Part I [Castano AP, Demidova TN, Hamblin MR. Mechanism in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization. Photodiagn Photodyn Ther 2004;1:279–93] it was shown that one of the most important factors governing the outcome of PDT, is how the photosensitizer (PS) interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. PS can localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes. An explosion of investigation and explorations in the field of cell biology have elucidated many of the pathways that mammalian cells undergo when PS are delivered in tissue culture and subsequently illuminated. There is an acute stress response leading to changes in calcium and lipid metabolism and production of cytokines and stress proteins. Enzymes particularly, protein kinases, are activated and transcription factors are expressed. Many of the cellular responses are centered on mitochondria. These effects frequently lead to induction of apoptosis either by the mitochondrial pathway involving caspases and release of cytochrome c, or by pathways involving ceramide or death receptors. However, under certain circumstances cells subjected to PDT die by necrosis. Although there have been many reports of DNA damage caused by PDT, this is not thought to be an important cell-death pathway. This mechanistic research is expected to lead to optimization of PDT as a tumor treatment, and to rational selection of combination therapies that include PDT as a component.
... e increased activity of these factors has been reported in several cancer cell lines photodynamically treated with different PSs. For example, NF B activation has been observed in L1210 mouse leukemia cells aer Photofrin-PDT [174], in lymphocytes or monocytes infected with HI�-1 aer pro�avine-PDT [175], in human colon carcinoma cells phototreated with pyropheophorbidea methyl ester (PPME) [176], and in human HL-60 cells aer PDT with benzoporphyrin-derivative-(BPD-) verterpor�n [177]. Similarly, AP-1 activation occurs in cervical carcinoma HeLa cells [178] and in epithelial PAM 212 cells [179] photosensitized with Photofrin. ...
Immunogenic Cell Death (ICD) could represent the keystone in cancer management since tumor cell death induction is crucial as well as the control of cancer cells revival after neoplastic treatment. In this context, the immune system plays a fundamental role. The concept of Damage-Associated Molecular Patterns (DAMPs) has been proposed to explain the immunogenic potential of stressed or dying/dead cells. ICD relies on DAMPs released by or exposed on dying cells. Once released, DAMPs are sensed by immune cells, in particular Dendritic Cells (DCs), acting as activators of Antigen-Presenting Cells (APCs), that in turn stimulate both innate and adaptive immunity. On the other hand, by exposing DAMPs, dying cancer cells change their surface composition, recently indicated as vital for the stimulation of the host immune system and the control of residual ill cells. It is well established that PhotoDynamic Therapy (PDT) for cancer treatment ignites the immune system to elicit a specific antitumor immunity, probably linked to its ability in inducing exposure/release of certain DAMPs, as recently suggested. In the present paper, we discuss the DAMPs associated with PDT and their role in the crossroad between cancer cell death and immunogenicity in PDT.
... Cytokines and various cell stresses, including irradiation (Criswell et al., 2003), oxidation (Marshall et al., 2000), and UV (Kato et al., 2003), induce NF-kB activation. Serum starvation also activates NF-kB in various cell lines (Ryter and Gomer, 1993;Grimm et al., 1996), indicating that serum contains unknown inhibitor(s) of NF-kB. ...
Several cell stresses induce nuclear factor-kappaB (NF-κB) activation, which include irradiation, oxidation, and UV. Interestingly, serum-starving stress-induced NF-κB activation in COS cells, but not in COS-A717 cells. COS-A717 is a mutant cell line of COS cells that is defective of the NF-κB signaling pathway. We isolated genes with compensating activity for the NF-κB pathway and one gene encoded the G protein β2 (Gβ2). Gβ2 is one of the G protein-coupled receptor signaling effectors. In COS-A717 cells, Gβ2 expression is significantly reduced. In Gβ2 cDNA-transfected COS-A717 cells, the NF-κB activity was increased along with the recovery of Gβ2 expression. Furthermore, serum-starving stress induced the NF-κB activity in Gβ2-transfected COS-A717 cells. Consistently, the serum-starved COS cells with siRNA-reduced Gβ2 protein expression showed decreased NF-κB activity. These results indicate that Gβ2 is required for starvation-induced NF-κB activation and constitutive NF-κB activity. We propose that serum contains some molecule(s) that strongly inhibits NF-κB activation mediated through Gβ2 signaling.
... sis (cIAPs), FLICE and members of Bcl-2 family. NF-B can also attenuate the apoptotic response to genotoxic anticancer drugs and radiation therapy [84]. PDT produces an oxidative stress that can result in activation and translocation of NF-B to the nucleus, as originally shown for treatment of L1210 murine leukemia cells with Photofrin and light. [85]. The claim that NF-B may be a target for PDT has been convincingly demonstrated by Granville et al. [86]. The photodynamic treatment of HL-60 cells with Verteporfin has no detectable effect on cellular IB levels by 1 h. Furthermore, lysates prepared up to 3 h post PDT did not contain detectable IB degradation products. However, the n ...
Photodynamic therapy (PDT) is an emerging cancer therapy that uses the combination of non-toxic dyes or photosensitizers (PS) and harmless visible light to produce reactive oxygen species and destroy tumors. The PS can be localized in various organelles such as mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes and this sub-cellular location governs much of the signaling that occurs after PDT. There is an acute stress response that leads to changes in calcium and lipid metabolism and causes the production of cytokines and stress response mediators. Enzymes (particularly protein kinases) are activated and transcription factors are expressed. Many of the cellular responses center on mitochondria and frequently lead to induction of apoptosis by the mitochondrial pathway involving caspase activation and release of cytochrome c. Certain specific proteins (such as Bcl-2) are damaged by PDT-induced oxidation thereby increasing apoptosis, and a build-up of oxidized proteins leads to an ER-stress response that may be increased by proteasome inhibition. Autophagy plays a role in either inhibiting or enhancing cell death after PDT.
... Nuclear-factor-kappa B (NF-kB) is a transcriptional factor and closely related to the expression of cytokines and chemokines in initial inflammation. There have been some reports that described that PDT could directly activate NF-kB464748. In our examination, however, NF-kB inhibitor (SN50) did not significantly decrease the effect of Pre-PDT (seeFigure S3, indicating that signaling cascades other than NF-kB activation were probably responsible for the Pre-PDT effect. ...
Local microbial infections induced by multiple-drug-resistant bacteria in the orthopedic field can be intractable, therefore development of new therapeutic modalities is needed. Photodynamic therapy (PDT) is a promising alternative modality to antibiotics for intractable microbial infections, and we recently reported that PDT has the potential to accumulate neutrophils into the infected site which leads to resolution of the infection. PDT for cancer has long been known to be able to stimulate the innate and adaptive arms of the immune system.
In the present study, a murine methicillin-resistant Staphylococcus aureus (MRSA) arthritis model using bioluminescent MRSA and polystyrene microparticles was established, and both the therapeutic (Th-PDT) and preventive (Pre-PDT) effects of PDT using methylene blue as photosensitizer were examined. Although Th-PDT could not demonstrate direct bacterial killing, neutrophils were accumulated into the infectious joint space after PDT and MRSA arthritis was reduced. With the preconditioning Pre-PDT regimen, neutrophils were quickly accumulated into the joint immediately after bacterial inoculation and bacterial growth was suppressed and the establishment of infection was inhibited.
This is the first demonstration of a protective innate immune response against a bacterial pathogen produced by PDT.
... Nuclear factor kappa B (NF-kB) is other transcription factor that is activated by 1 O 2 . Ryter and Gomer showed that 1 O 2 generated in mouse leukemia L1210 cells after PH-II-mediated photosensitization was able to induce an increase of DNA binding activity of NF-kB [193]. Following, Vile et al. studied the role of 1 O 2 on NF-kB activation on human skin fibroblasts (FEK-4) after treatment with UVA irradiation [194]. ...
Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.
Sonodynamic therapy (SDT) has been recognized as a spatial-temporal and noninvasive modality for the treatment of deep-seated tumors. However, current sonosensitizers suffer from low sonodynamic efficacy. Herein, we reported the design of nuclear factor kappa B (NF-κB) targeting sonosensitizers (TR1, TR2, and TR3) by integrating a resveratrol motif into a conjugated electron donor-acceptor (triphenylamine benzothiazole) skeleton. Among these sonosensitizers, TR2 with two resveratrol units in one molecule was the most potent for inhibiting NF-κB signaling. Owing to the synergy of high sonodynamic efficacy and NF-κB activation inhibition, TR2 displayed a remarkable sonocytotoxicity to MCF-7 breast cancer cells. Xenograft mice studies demonstrated that TR2 had excellent anticancer potency and biosafety. This study thus opens up a new avenue for the development of efficient organic sonosensitizers for cancer ablation.
Photodynamic therapy as a local therapeutic method shows great biocompatibility in tumor therapy. However, the poor accumulation of photosensitizer in tumors restricted the therapeutic efficacy. Different from traditional chemotherapeutic drugs, some additional issues will further decrease the photodynamic therapeutic efficacy including the oxygen concentration and the short half life of reactive oxygen species. Nanoparticles as drug carriers are currently under rapid development for tumor therapy, especially in photodynamic therapy. This review mainly focuses on the design of nanoparticles for photodynamic therapy, with special emphasis on optimizing the therapeutic efficacy via the tumor tissue/organelles target, oxygen supply, and combination therapy.
The nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκB levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκB levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκB is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)
Photodynamic therapy (PDT) exploits the biological consequences of localized oxidative damage inicted by photodynamic processes. A schematic outline of the major steps that lead to tumor destruction by PDT is given in Figure 1.1. ree critical elements are required for the initial photodynamic processes to occur: a drug that can be activated by a photosensitizer, light, and oxygen. Interaction of light at the appropriate wavelength with a photosensitizer produces an excited triplet state photosensitizer that can interact with ground state oxygen via two dierent pathways, designated as type I and type II. e individual steps of these pathways are shown in Figure 1.2. e type II reaction that gives rise to singlet oxygen (1O2) is believed to be the dominant pathway since the elimination of oxygen or scavenging of 1O2 from the system essentially eliminates the cytocidal eects of PDT.1-3 Type I reactions, however, may become important under hypoxic conditions or where photosensitizers are highly concentrated.1 e highly reactive 1O2 has a short lifetime (<0.04 µs) in the biological milieu and therefore a short radius of action (<0.02 µm).4 Consequently, 1O2-mediated oxidative damage will occur in the immediate vicinity of the subcellular site of photosensitizer localization. Depending on photosensitizer pharmacokinetics, these sites can be varied and numerous, resulting in a large and complex array of cellular eects. Similarly, on a tissue level, tumor cells as well as various normal cells can take up photosensitizer, which, upon activation by light, can lead to eects upon such targets as the tumor cells, the tumor and normal microvasculature, and the inammatory and immune host system. PDT eects.
Photodynamic therapy induces a highly complex series of changes in cells. It is likely to affect multiple cell targets, of which cell membranes and mitochondria may be of particular importance, but which may also include DNA and microtubules. Following exposure, which probably results in the generation of free radicals, cells characteristically experience a rapid increase in calcium concentration, which may be accompanied or followed by other electrolyte changes as membrane damage progresses. Sublethal damage may, via various signal transduction pathways, result in apoptosis. However, indirect effects, such as necrosis resulting from vascular damage, may also be important in vivo. The effects of PDT may be modulated by dose, or dose rate changes, conjugation of photosensitizers to lipoproteins or liposomes, or by the addition of chemotherapeutic agents. Many reports in the current literature are confusing, and often apparently contradictory. There is clearly scope for much greater understanding, and future studies should perhaps more systematically address phenomena in a range of cell types, photosensitizers, and treatment conditions. Nonetheless, there is ample ground for optimism, and such knowledge as we already have should effectively underpin the clinical research that is ongoing.
Photodynamic therapy is a therapeutic modality with a long history. It has been historically known in ancient India and China for the treatment of skin disorders. In Western medicine, the first experimental evidence of photodynamic therapy was reported by Raab et al. who observed the lethality of acridine dyes to paramecium in the presence of light [1]. The photodynamic effect was further demonstrated by Tappeiner and colleagues who reported killing of basal cell carcinoma using eosin and light illumination [2]. More recently, Dougherty et al. developed a hematoporphyrin derivative, which was shown to kill cancer cells in vitro and mammary tumors in mouse models in vivo [3]. Photodynamic therapy has evolved as an effective therapeutic modality for cancer treatment. In 1995, the FDA approved photodynamic therapy using Photofrin for the treatment of advanced esophageal cancer. Recent developments in biomedical imaging provide new opportunities for photodynamic therapy. Modern imaging technologies can accurately detect and diagnose malignant tumors at an early stage and can effectively assess tumor response to cancer photodynamic therapy. Combining photodynamic therapy with imaging can provide image guidance for accurate laser irradiation of tumor and timely assessment of therapeutic efficacy of photodynamic therapy. Image-guided photodynamic therapy is a new, minimally invasive cancer treatment modality that can further improve the efficacy of photodynamic therapy.
The transcription factor NF-κB is a central regulator of defensive responses which are mounted by cells against many potentially threatening environmental challenges. Bacteria, viruses, parasites, injury, radiation, oxidative stress, numerous chemical agents and many cytokines released in response to such challenges are all well-studied and potent activators of this transcription factor in a number of different cell types. Activated NF-κB then induces the expression of many genes whose encoded functions play critical roles for the defense of the organism. The induced proteins have a wide range of activities, including antibacterial or anti-viral functions, antigen recognition, cellular migration and adhesion as well as hematopoietic cell differentiation and proliferation. Aside from regulating cellular genes, several viruses, including the human immunodeficiency virus, have diverted this factor for their own purposes. Since many cytokines are regulated by NF-κB, this transcription factor appears critical to a coordinated defense response. In an evolutionary sense, the association between defensive responses and NF-κB dates at least as far back as insects.
Photodynamic TherapyThe Development of Photodynamic TherapyAminolevulinic Acid Photodynamic TherapyHeme Biosynthesis and Regulation During ALA-PDTIron and the Enhancement of ALA-PDTRedox Signaling in Photodynamic Therapy after Light IrradiationPhotosensitizers as Reactive Oxygen Species Generators in Redox ResearchReactive Oxygen Species Generation and Signaling in Clinical Photodynamic TherapyApoptosis in Photodynamic TherapySubcellular Localization of Reactive Oxygen Species ProductionChanges in Transcription Factor and Protein Levels Following Photodynamic TherapyIron and Iron Chelation Post Light Activation of PhotosensitizerVascular Damage, Hypoxia and Hypoxia Inducible FactorConclusion
References
Photodynamic therapy (PDT) is a therapeutic modality whose efficacy depends on several factors including the type of the photosensitizer, the light fluence, and cellular response. Cell recurrence and proliferation are problems that are unsolved in PDT; hence, new approaches are needed to increase the efficacy of PDT. Nitric oxide (NO), present in the tumor and in the tumoral microenvironment, has an important role as a modulator of PDT. Reports in the literature demonstrate that NO in tumors can exhibit pro- and anti-apoptotic properties by modulating different signalling pathways such as the NF-κB pathway and on downstream genes of the NF-κB/Snail/RKIP survival/anti-apoptotic circuitry. In this study, we present findings on the pivotal role played by NO in B78-H1 murine amelanocytic melanoma cells treated with pheophorbide α /PDT (Pbα/PDT). The NO released by Pbα/PDT modulated the NF-κB/Snail/RKIP loop leading to recurrence or death of the cells in a concentration-dependent manner. To sensitize cancer cells to photodynamic therapy, we demonstrate that the combined treatment of the photosensitizer Pheophorbide α with the NO donor, DETANONOate, resulted in tumor cell death.
Melanoma is a malignant, the most aggressive and dreaded skin cancer. This form of cancer arises from melanocytes and may grow rapidly and metastasize. Melanoma predominantly occurs in skin, but could also be found in the mouth, iris and retina of the eye. Melanoma is the most dangerous form of skin cancer, with a steeply rising incidence and a poor prognosis in its advanced stages. It is highly resistant to traditional chemotherapy and radiotherapy, although modern biological therapies are showing some promise. Photodynamic therapy (PDT), as a novel effective modality of the treatment of skin cancers, opens up new possibilities in melanoma treatment also. Many experimental photodynamic therapy studies were performed. The results of many experiments indicate that that photodynamic therapy may be a promising tool for adjuvant treatment in advanced melanoma.
Photodynamic therapy (PDT) is an elegant minimally invasive oncologic therapy. The clinical simplicity of photosensitizer (PS) drug application followed by appropriate illumination of target leading to the oxygen dependent tumor ablative Photodynamic Reaction (PDR) has gained this treatment worldwide acceptance. Yet the true potential of clinical PDT has not yet been achieved. This paper will review current mechanisms of action and treatment paradigms with critical commentary on means to potentially improve outcome using readily available clinical tools.
Topical 5-aminolevulinic acid-photodynamic therapy (5-ALA-PDT) causes a clinical inflammatory response in human skin. While histamine mediates the immediate reaction, the mediators of the prolonged erythema are unknown OBJECTIVE: To examine for involvement of pro-inflammatory mediators prostaglandin E2 (PGE2 ) and nitric oxide (NO) in topical PDT-induced erythema in human skin.
A series of studies were performed in forearm skin of healthy volunteers (n=35). Following definition of the erythemal time-course and dose-response to 5-ALA-PDT, duplicate 5-ALA dose-series were iontophoresed into the skin of each ventral forearm and exposed to 100J/cm(2) broadband red light. Within subject, arms were randomised to control or treatment with cyclooxygenase and nitric oxide synthase inhibitors, indomethacin and N(ω)-nitro-L-arginine methyl ester (L-NAME), respectively, and impact on 5-ALA-PDT induced erythema quantified. Additionally, release of PGE2 and NO was directly assessed by sampling dermal microdialysate at intervals following 5-ALA-PDT.
A 5-ALA dose-related delayed erythema occurred by 3h (r= 0.97, P<0.01), with erythema persisting to 48h post-PDT. Topical indomethacin applied immediately post-PDT reduced the slope of erythemal response at 3 and 24h (P<0.05). Intradermal injection of L-NAME into 5-ALA-PDT treated sites reduced the slope of response at 24h post-PDT (p<0.001) whilst significantly inhibiting erythema from 3-48h post-PDT (P<0.01). Analysis of dermal microdialysate showed release of NO and PGE2 following topical 5-ALA-PDT.
Topical 5-ALA-PDT upregulates PGE2 and NO in human skin, where they play a significant role in the clinical inflammatory response. The potential relevance of these mediators to PDT of human cutaneous pathology warrants study. This article is protected by copyright. All rights reserved.
Light has been used to treat diseases for hundreds of years. Convenient and powerful light sources such as lasers make photomedicine a major branch in diseases treatment and detection. Originally, light was often used for local treatment, using photomechanical, photochemical, photothermal reactions and photomodulation as the major mechanisms. More and more investigators have become interested in the systemic effects of light, particularly in its effects on immune systems. Much work has been done to activate and/or enhance the host immune system to combat cancer, either using light as a direct tool or as an adjuvant method. Light has long been used for assisting disease detection and diagnosis. Advances in light technology have made photo-diagnostics ever more precise spatially and temporally. Many techniques facilitate observation of bio-molecule interactions and other biological processes at the cellular level, hence providing opportunities to detect and monitor immune activities. This manuscript will review recent photo-immunological research in treatment of cancer. The recent development of combination therapies involving lasers will be presented. Specifically, the results of cancer treatment using laser photothermal interaction, either with or without additional immunological stimulation will be discussed. The immunological effects of photodynamic therapy (PDT), and of its combination with immunotherapy in cancer treatment will also be discussed. Much interest has been recently concentrated in the immunological responses after laser treatment. Such responses at cellular and molecular levels will be discussed. The effect of these treatment modalities on the distant metastases also showed promise of light induced antitumor immunity. The combination therapy and induced immunological responses appear to be the key for long-term control of tumors.
Historically, cancer has been the major therapeutic focus for PDT. The growing understanding of how PDT affects the behavior and viability of a variety of cell types has fostered the evaluation and use of PDT for non-oncologic indications, including ocular, cardiovascular, and immune diseases. The formulation, pharmacokinetics, and type of photosensitizer, the duration between its administration and light application, and the region of PDT on immune reactions. Among immune-mediated diseases, psoriasis is a promising candidate indication for PDT, since the skin lesions and the immunocytes associated with active disease are readily accessible to activating light. The beneficial effect of PDT on psoriasis may arise from a reduction in the number of pathogenic T cells, a modification of APC function, and/or an alteration of keratinocyte cytokine production within afflicted skin. Continued efforts to optimize PDT regimens will reveal the ultimate potential of this technique to effectively treat immune-mediated diseases. Likewise, there is potential to exploit the capacity of PDT to stimulate anti-tumor immunity. Both of these therapeutic areas will continue to provide opportunities to understand the underlying biological processes and develop improved treatment regimens.
Cells exposed to photodynamic therapy (PDT) respond with activation of molecular pathways that are involved in the regulation of gene expression, cell cycle and cell death. This chapter gives a review of the molecular responses that have been documented after PDT, including effects on cell fate decision, stress responses, and intra- and intercellular signaling. The subcellular localization of the photosensitizer is an important determinant for specific molecular response patterns and for the ultimate PDT effect. Divergent and sometimes, contradictory results of consequences of PDT at the molecular level are likely due to the variability of PDT regimens with respect to photosensitizers, PDT dose, and other variables of specific experimental model. We are beginning to understand a few of the molecular effects of PDT, and this learning process will form the basis for research that aims at developing improved PDT regimens. PDT may also be usful as a tool to analyze certain cellular-molecular pathways.
In human endothelial cells ECV 304 and HMEC-1 photosensitized by pyropheophorbide-a methylester (PPME) in sublethal conditions transcription factor Nuclear Factor kappa B (NF-κB) activation takes place for several hours. Activated NF-κB was functional because it stimulated the transcriptional activation of either a transfected reporter gene or the endogenous gene encoding interleukin (IL)-8. Concomitant with NF-κB activation, inhibitor of NF-κBα (IκBα) was degraded during photosensitization and IκBβ, p100, p105 and IκBε were slightly modified. Reactive oxygen species (ROS) were shown to be crucial intermediates in the activation because antioxidants strongly decreased NF-κB activation. Using both a fluorescent probe and isotope substitution, it was shown that ROS, and especially singlet oxygen (1O2), were important in the activation process. Because NF-κB activation in the presence of ROS was suspected to proceed through a pathway independent of the IκB kinases (IKK), we demonstrated that the IKK were indeed not activated by photosensitization but required an intact tyrosine residue at position 42 on IκBα, suggesting the involvement of a tyrosine kinase in the activation process. This was further reinforced by the demonstration that herbimycin A, a tyrosine kinase inhibitor, prevented NF-κB activation by photosensitization but not by TNFα, a cytokine known to activate NF-κB through an IKK-dependent mechanism.
Biological processes involving light may have both beneficial (photosynthesis) and destructive (photosensitization) consequences. Singlet molecular oxygen, 1O2, and other reactive oxygen species such as hydrogen peroxide and hydroxyl radical, arise during the interaction of light with photosensitizing chemicals in the presence of molecular oxygen. 1O2 oxidizes macromolecules such as lipids, nucleic acids, and protein, depending on its intracellular site of formation; and promotes detrimental processes such as lipid peroxidation, membrane damage, and cell death. Photochemical reactive oxygen species (ROS) generating systems induce the expression of several eukaryotic genes, which include stress proteins, early response genes, matrix metalloproteinases, immunomodulatory cytokines, and adhesion molecules. These gene expression phenomena may belong to cellular defensive mechanisms, or may promote further injury. Whereas the signal transduction pathways that link site-specific oxidative damage and gene expression are poorly understood, ROS may affect signalling components in the membrane, cytosol, or nucleus, leading to changes in phospholipase, cyclooxygenase, protein kinase, protein phosphatase, and transcription factor activities. Limited evidence for 1O2 involvement in gene activation phenomena consists of deuterium oxide solvent effects, inhibition by 1O2-quenchers, sensitization by porphyrins, chemical trapping methods, and comparative effects of photosensitizing dyes and thermolabile endoperoxides. The studies outlined in this review support an hypothesis that 1O2 and other ROS generated during photochemical processes such as ultraviolet-A (320–380 nm) radiation exposure, or photosensitizer mediated oxidation may have dramatic effects on eukaryotic gene expression.
Photodynamic therapy (PDT) is a new modality in cancer treatment. It is based on the tumour-selective accumulation of a photosensitizer followed by irradiation with light of a specific wavelength. PDT is becoming widely accepted owing to its relative specificity and selectivity along with absence of the harmful side-effects of chemo and radiotherapy. There are three known distinct mechanisms of tumour destruction following PDT, generation of reactive oxygen species which can directly kill tumour cells, tumour vascular shutdown which can independently lead to tumour destruction via lack of oxygen and nutrients and thirdly enhanced antitumour immunity.
A review based on the literature acquired from the PubMed database from 1983 with a focus on the enhanced antitumour immunity effects of PTD.
Tumour cell death is accompanied by the release of a large number of inflammatory mediators. These induce a non-specific inflammatory response followed by gradual adaptive antitumour immunity. Further, a combination of PDT with the immunological approach has the potential to improve PDT efficiency and increase the cure rate. This short review covers specific methods for achieving these goals.
Abstract— The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular signal transduction pathways in murine lymphoma L5178Y-R cells has been assessed by observing increases in protein tyrosine phosphorylation at early times post-PDT. Western blot analysis with an anti-phosphotyrosine antibody revealed a dramatic increase in phosphorylation of two major protein bands of Mr -80000 and -55000 in response to PDT. The increase was PDT dose-dependent, occurred as early as 20 s after initiation of light exposure of Pc 4-pre-loaded cells and was amplified by the presence of the protein tyrosine phosphatase inhibitor, sodium ortho-vanadate (NaV04). By immunoprecipitation, one of the Mr –80000 phosphorylated proteins has been identified as HS1, a substrate of nonreceptor-type protein tyrosine kinases. Although vanadate greatly enhanced the level and extent of PDT-induced phosphorylation, it had no influence on overall photocytotoxicity or on the rate of apoptotic DNA fragmentation. Genistein, an inhibitor of protein tyrosine kinases, diminished tyrosine phosphorylation of the Mr –80000 and other proteins and dramatically potentiated cell killing induced by PDT but did not significantly affect PDT-induced apoptosis. The results suggest that PDT rapidly activates a membrane-associated src family kinase(s) in L5178Y-R cells, one substrate of which is HS1, and that protein tyrosine phosphorylation is part of a stress response, protecting a portion of the cells from the lethal effects of PDT but not altering the mechanism by which they die.
Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4′,6-diamidine-2′-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-KB is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-KB turned out to be involved in an antia-poptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-KBα was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.
Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li–Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 ± 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 ± 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.
Photodynamic therapy (PDT) uses exogenously administered photosensitizers activated by light to induce cell death or modulation of immunological cascades, presumably via formation of reactive oxygen species (ROS). 5-Aminolevulinic acid (ALA) mediated photosensitization is increasingly used for the treatment of nonmelanoma skin cancer and other indications including benign skin disorders. Long-term side effects of this investigational modality are presently unknown. Just as tumor treatments such as ionizing radiation and chemotherapy can cause secondary tumor induction, PDT may potentially have a carcinogenic risk. Evaluation of the biological effects of ALA in absence of activating light and analysis of the mechanism of ALA-PDT and porphyrin-type photosensitizers mediated photosensitization indicate that this therapy has a pro-oxidant and genotoxic potential. However, porphyrin type molecules also possess antioxidant and antimutagenic properties. ALA-PDT delays photocarcinogenesis in mice, and topical ALA alone does not increase skin cancer incidence in these animals. Patients with increased tissue levels of ALA have an increased incidence of internal carcinoma, however, it is not clear whether this relationship is casual or causal. There is no evidence indicating higher rates of skin cancer in patients with photosensitivity diseases due to presence of high protoporphyrin IX (PP) levels in skin. Overall, the presently available data indicate that the risk for secondary skin carcinoma after topical ALA-PDT seems to be low, but further studies must be carried out to evaluate the carcinogenic risk of ALA-PDT in conditions predisposed to skin cancer.
Photodynamic therapy (PDT) is a therapeutic modality whose efficacy depends on several factors including type of photosensitizer, light fluence and cellular response. Cell recurrence is one of the problems still unsolved in PDT. In this work we found that in B78-H1 murine amelanotic melanoma cells there is a correlation between cell recurrence and the NF-κB/Snail/RKIP loop.
Proliferation and migration of surviving cells were analyzed by MTT and wound-scratch assays. The levels of ROS/NO in B78-H1 melanoma cells treated with pheophorbide a (Pba) and light (Pba/PDT) were measured by FACS, while expression of NF-κB, Snail and RKIP were determined by Western blots. The mechanism of cell death was investigated by caspase and microscopy assays.
Our data show that after a low-dose Pba/PDT treatment, B78-H1 cells are able to recover. This correlates with a low level of NO production, which blocks apoptosis via NF-κB pathway. Western blot analyses showed that a low-dose Pba/PDT increases the expression of NF-κB and anti-apoptotic Snail, but reduces the expression of pro-apoptotic RKIP. The role played by NF-κB in the modulation of Snail and RKIP was investigated using DHMEQ: a NF-κB inhibitor which behaves as NO donor. DHMEQ caused a decrease of Snail and an increase of RKIP expression. When B78-H1 cells were treated with a low dose Pba/PDT and DHMEQ, the NO level strongly increased, with the result that Snail was down-regulated and RKIP was upregulated, as observed with a high-dose Pba/PDT.
One major problem in PDT is the cellular rescue occurring in tissue regions receiving a low-dose PDT. To minimize this problem and sensitize cancer cells to PDT we propose a combined treatment in which the photosensitizer is delivered with a donor of NO acting on the NF-κB/Snail/RKIP loop.
The nuclear factor-kappa B (NF-kappaB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-kappaB principally resides within the cytoplasm in association with inhibitory kappa (IkappaB) proteins. The status of IkappaB and NF-kappaB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IkappaB proteins may be caspase substrates during apoptosis. However, the level of IkappaBbeta was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IkappaBalpha levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IkappaBalpha levels were transiently depressed after PDT. At these times, p50 and RelA NF-kappaB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a kappaB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-alpha, a well-characterized NF-kappaB activator. Productive NF-kappaB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IkappaBalpha is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)
Metastatic malignant melanoma remains one of the most dreaded skin cancers worldwide. Numerous factors contribute to its resistance to hosts of treatment regimes and despite significant scientific advances over the last decade in the field of chemotherapeutics and melanocytic targets, there still remains the need for improved therapeutic modalities. Photodynamic therapy, a minimally invasive therapeutic modality has been shown to be effective in a number of oncologic and non-oncologic conditions. Using second-generation stable, lipophilic photosensitizers with optimised wavelengths, PDT may be a promising tool for adjuvant therapy in combating melanoma. Potential targets for PDT in melanoma eradication include cell proliferation inhibition, activation of cell death and reduction in pro-survival autophagy and a decrease in the cellular melanocytic antioxidant system. This review highlights the current knowledge with respect to these characteristics and suggests that PDT be considered as a good candidate for adjuvant treatment in post-resected malignant metastatic melanoma. Furthermore, it suggests that primary consideration must be given to organelle-specific destruction in melanoma specifically targeting the melanosomes - the one organelle that is specific to cells of the melanocytic lineage that houses the toxic compound, melanin. We believe that using this combined knowledge may eventually lead to an effective therapeutic tool to combat this highly intractable disease.
Singlet oxygen, a metastable state of normal triplet oxygen, has been identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet oxygen trap is not toxic to the cells nor does it absorb the light responsible for hematoporphyrin activation. We have found that the singlet oxygen-trapping product, o-dibenzoylbenzene, is formed nearly quantitatively intracellularly when both the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of the TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet oxygen-trapping product indicates that singlet oxygen is the probable agent responsible for toxicity in this system.
Cultured endothelial cells from the human umbilical vein were incubated with low concentrations (1 microgram/ml) of the photosensitizer Photofrin II. Following a sublethal light exposure, a light dose-dependent release of von Willebrand factor (vWf) into the culture medium was observed. Analysis of the multimeric composition of the released protein indicated that it originated from the intracellular pool of large vWf multimers stored in the Weibel-Palade bodies. This release was detected as early as 1 h postirradiation. Release was inhibited at low temperature and was dependent upon the presence of extracellular calcium. Photosensitization resulted in an influx of calcium whose time course paralleled vWf release from the cells. Since vWf mediates platelet adhesion to the vascular subendothelium, it is possible that its photochemically stimulated release in vivo could contribute to platelet thrombus formation observed in tissue following photodynamic therapy.
Hydrogen peroxide and oxygen radicals are agents commonly produced during inflammatory processes. In this study, we show that micromolar concentrations of H2O2 can induce the expression and replication of HIV-1 in a human T cell line. The effect is mediated by the NF-kappa B transcription factor which is potently and rapidly activated by an H2O2 treatment of cells from its inactive cytoplasmic form. N-acetyl-L-cysteine (NAC), a well characterized antioxidant which counteracts the effects of reactive oxygen intermediates (ROI) in living cells, prevented the activation of NF-kappa B by H2O2. NAC and other thiol compounds also blocked the activation of NF-kappa B by cycloheximide, double-stranded RNA, calcium ionophore, TNF-alpha, active phorbol ester, interleukin-1, lipopolysaccharide and lectin. This suggests that diverse agents thought to activate NF-kappa B by distinct intracellular pathways might all act through a common mechanism involving the synthesis of ROI. ROI appear to serve as messengers mediating directly or indirectly the release of the inhibitory subunit I kappa B from NF-kappa B.
We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the kappa B enhancer. Constructs containing two or more copies of one of the kappa B enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like "Y box" and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.
The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.
Induction of rat heme oxygenase is mediated by at least two factors: its substrate heme and heat shock (Shibahara, S., Müller, R.M., and Taguchi, H. (1987) J. Biol. Chem. 262, 12889-12892). We have identified the cis-acting element of the rat heme oxygenase gene (HO gene), that was specifically bound by a nuclear protein prepared from rat glioma cells. We have termed this protein as heme oxygenase transcription factor (HOTF), whose estimated molecular weight is about 40,000. The element identified is CCACCACGTGACTCGAG (-51/-35) located just upstream of TATA-like sequence. The functional studies indicate that this sequence is required for the accurate and efficient initiation of transcription from the HO gene promoter. Since the HOTF-binding element is similar to the upstream promoter sequence (UPS) of the adenovirus 2 major late promoter (Ad2MLP), we examined whether rat HOTF is homologous to the upstream stimulatory factor (USF) or major late transcription factor previously identified in HeLa cells, which interacts with the UPS and activates the transcription from the Ad2MLP. HOTF specifically binds to the UPS of Ad2MLP, whereas HOTF failed to form a stable complex with the mutated HOTF-binding element lacking a GTGA sequence (-44/-42 of the HO gene), the sequence of which is identical to a core sequence of the USF-binding sites. Moreover, we show that like USF, HOTF is heat stable. These results suggest that HOTF may be homologous to USF or the major late transcription factor. Since USF is present in uninfected cells, it is conceivable that the expression of the HO gene is regulated at least in part by USF.
The enhancer-binding factor NF-kappa B, which is found only in cells that transcribe immunoglobulin light chain genes, has been purified from nuclear extracts of Namalwa cells (human Burkitt lymphoma cells) by sequence-specific DNA affinity chromatography. The purified NF-kappa B has been identified as a 51-kDa polypeptide by UV-crosslinking analysis. "Footprint" and methylation-interference analyses have shown that purified NF-kappa B has a binding activity specific for the kappa light chain enhancer sequence. The purified factor activated in vitro transcription of the human immunodeficiency virus type I promoter by binding to an upstream NF-kappa B-binding site.
We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription
initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using
the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus
promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA
and Ad 2 VA).
Normal human foreskin fibroblasts were used to examine transcriptional induction by IL-1 and TNF-alpha of the novel cytokine gro (melanoma growth-stimulating activity). Gro mRNA was expressed at levels 100-fold above background within 45 min of exposure to either IL-1 or TNF-alpha, in growing or serum-starved cells and a similar response was shown by IL-6. In contrast, as shown previously, gro mRNA was elevated only 10-fold by serum in starved but not in growing cells, similar to fos. Thus gro expression appears to be regulated by at least two signal transduction systems: a cytokine pathway, and a growth-related pathway. Three closely related gro genes (alpha, beta, and gamma) have been described. Their proximal 5' regulatory sequences presented here show close similarity in the region to -136, which includes the NF-kappa B site at -66 to -76 in gro alpha and gro gamma, and -64 to -74 in gro beta, and sequence diversity further upstream. Transient transfection of HeLa cells with CAT constructs localized the cytokine response to a region between -84 and -65 in gro beta. Gel retardation studies with FS-2 cells identified a cytokine-induced protein binding at the NF-kappa B site in all three gro genes as shown by competition studies with a pair of oligonucleotides representing wild-type and mutant sequences of the NF-kappa B binding site. Neither serum nor PMA induced a detectable gel shift at NF-kappa B or upstream to position -723. These results demonstrate conservation of the cytokine response element, NF-kappa B, in the three genes, consistent with the conservation of sequence in this region; and suggest that differential expression of the three gro genes may depend upon interactions with other sites located in the divergent upstream region.
Singlet oxygen, a metastable state of normal triplet oxygen, has been identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet oxygen trap is not toxic to the cells nor does it absorb the light responsible for hematoporphyrin activation. We have found that the singlet oxygen-trapping product, o-dibenzoylbenzene, is formed nearly quantitatively intracellularly when both the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of the TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet oxygen-trapping product indicates that singlet oxygen is the probable agent responsible for toxicity in this system. © 1976, American Association for Cancer Research. All rights reserved.
Crosslinkage of the B cell antigen receptor by anti-mu beads or SAC results in the selective induction of hsp70. We have observed that activated cells, having enhanced expression of hsp70, survive lethal stimuli much better than their unactivated counterparts. These results are in accordance with the proposal that hsp70 is essential for cells to survive lethal environmental stresses. Moreover, the activation event itself primes B cells thereby enabling them to increase the expression of both hsp70 mRNA and protein. This is the first demonstration that triggering of B cells via crosslinkage of sIg is accompanied by the induction of thermotolerance without the need for a prior sublethal heat treatment.
We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the kappa B enhancer. Constructs containing two or more copies of one of the kappa B enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like "Y box" and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.
Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca2+]i, from about 0.2 μM to 1 μM within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca2+]i. These findings raise the possibility that changes in [Ca2+]i after photodynamic treatment may be relevant to cell killing and/or other biological responses of the cells, e.g. release of eicosanoids.
The cell-damaging photochemistry of hematoporphyrin derivative (HPD) has been investigated using isolated erythrocyte membranes as a test system. Irradiation of membranes in the presence of the tumor-localizing fraction of HPD resulted in formation of singlet molecular oxygen (1O2) as measured by the phosphorescence at 1268 nm. The authentic product of 1O2 attack on cholesterol, 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide, was identified in this system. Relatively insignificant amounts of free radicalderived hydroperoxides were detected. These results suggest that 1O2 plays a major role in the HPD-sensitized photokilling of tumor cells in vivo.
The effects of the antitumor drugs daunorubicin, doxorubicin and their complexes with Fe(III) on phosphoinositide hydrolysis, lipid peroxidation and protein kinase C (PKC) activation were measured in intact human platelets. Doxorubicin and the Fe(III) complexes of both doxorubicin and daunorubicin quickly induced lipid peroxidation [as measured by the thiobarbituric acid (TBA) assay], phosphorylation of the 40 K substance of PKC, and increased levels of phosphatidic acid and inositol phosphates. Fe(III) alone or complexed to acetohydroxamic acid induced high levels of TBA-reactive material but did not affect either PKC activation or phosphoinositide turnover. In contrast, daunorubicin, which was ineffective per se, inhibited all these doxorubicin- and anthracyclines/Fe(III)-induced biochemical events. We suggest that phosphoinositide hydrolysis determined by anthracyclines, and consequently PKC activation, could be due to lipid peroxidation, thus triggering the activity of phospholipase C.
Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca2+], from about 0.2 microM to 1 microM within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca2+]i. These findings raise the possibility that changes in [Ca2+]i after photodynamic treatment may be relevant to cell killing and/or other biological responses of the cells, e.g. release of eicosanoids.
Porphyrin mediated photosensitization can enhance the transcription and translation of several oxidative stress genes. In this study, we report on the enhanced expression of the gene encoding for heme oxygenase in Chinese hamster fibroblasts by; (1) incubation in Photofrin II; (2) Photofrin II mediated photosensitization; and (3) photosensitization induced by Rose Bengal. Increased expression of heme oxygenase mRNA was accompanied by a concomitant increase in the synthesis of the 34 kDa heme oxygenase protein. Western blot analysis using antibody to heme oxygenase confirmed the immunoreactivity of the 34 kDa protein induced by Photofrin II and PDT. These results demonstrate that heme oxygenase can be activated by non-metalloporphyrins as well as by photosensitization associated with singlet oxygen mediated subcellular injury.
Normal human foreskin fibroblasts were used to examine transcriptional induction by IL-1 and TNF-alpha of the novel cytokine gro (melanoma growth-stimulating activity). Gro mRNA was expressed at levels 100-fold above background within 45 min of exposure to either IL-1 or TNF-alpha, in growing or serum-starved cells and a similar response was shown by IL-6. In contrast, as shown previously, gro mRNA was elevated only 10-fold by serum in starved but not in growing cells, similar to fos. Thus gro expression appears to be regulated by at least two signal transduction systems: a cytokine pathway, and a growth-related pathway. Three closely related gro genes (alpha, beta, and gamma) have been described. Their proximal 5' regulatory sequences presented here show close similarity in the region to -136, which includes the NF-kappa B site at -66 to -76 in gro alpha and gro gamma, and -64 to -74 in gro beta, and sequence diversity further upstream. Transient transfection of HeLa cells with CAT constructs localized the cytokine response to a region between -84 and -65 in gro beta. Gel retardation studies with FS-2 cells identified a cytokine-induced protein binding at the NF-kappa B site in all three gro genes as shown by competition studies with a pair of oligonucleotides representing wild-type and mutant sequences of the NF-kappa B binding site. Neither serum nor PMA induced a detectable gel shift at NF-kappa B or upstream to position -723. These results demonstrate conservation of the cytokine response element, NF-kappa B, in the three genes, consistent with the conservation of sequence in this region; and suggest that differential expression of the three gro genes may depend upon interactions with other sites located in the divergent upstream region.
The purpose of this study was to determine if there was an early increase in intracellular Ca++ which preceded generalized lysis of thymocytes during photodynamic permeabilization. A method was developed that facilitated the simultaneous measurement in real time of permeabilization of the thymocyte cell membrane to Ca++, Mn++, and ethidium bromide during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to determine how soon and to what extent intracellular Ca++ changed following illumination. In the presence of extracellular manganese, the same system was used to determine how soon the cells became permeable to Mn++ or quin-2. It was determined that the fluorescence emission of the ethidium bromide-DNA complex was strong enough to be measured in the presence of the calcium-quin-2 complex. This enabled the concomitant determination of the elapsed time following illumination before ethidium bromide entered the cell. It was established that increased intracellular Ca++ was an early event in the photodynamic permeabilization of thymocytes that preceded permeabilization of the cell membrane to ethidium bromide, Mn++ or quin-2, or lysis.
Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.
Photodynamic therapy (PDT) involves the treatment of tumors in the presence of sensitizer, light, and oxygen, causing energy-dependent cytotoxicity. A vascular effect that causes hemorrhagic tumor necrosis has been described with PDT, but its basis remains undefined. To investigate the possible role of tumor necrosis factor (TNF) production in the generation of such a vascular effect and/or a direct tumor effect, we treated thioglycollate-elicited murine macrophages with PDT, and we measured the possible production of TNF using the L929 assay. An energy-dependent production of TNF by macrophage treated with PDT, stimulated or unstimulated with endotoxin, was demonstrated, and TNF production was inhibited at the highest treatment energy levels. These data represent the first description of cytokine production by PDT-treated macrophages, and may serve as another mechanism of PDT cytotoxicity in vivo, either directly by TNF-mediated tumor necrosis, or indirectly by vascular effects on tumor vessels.
Photodynamic treatment in vitro, using the photosensitizer Photofrin II and light at 630 nm, was found to liberate large amounts of prostaglandin E2 (PGE2) from mouse radiation-induced fibrosarcoma tumor cells and peritoneal macrophages, but not from L929 fibroblasts. PGE2 release was dose dependent and directly related to cell membrane disruption. It occurred rapidly and was complete within 30 min of treatment. PGE2 release could be inhibited by indomethacin, meclofenamate and extended prior exposure to dexamethasone, indicating that it was due to new production involving both the phospholipase and cyclooxygenase enzyme systems. Removal of calcium ions, necessary for phospholipase activation, from the medium did not inhibit the photodynamically induced elevated PGE2 production, possibly because of Ca2+ resupply from leaking intracellular pools.
Photodynamic therapy (PDT) is the treatment of malignant lesions with visible light following the systemic administration of a tumor-localizing photosensitizer. Pharmacological and photochemical properties of the photosensitizer are combined with precise delivery of laser-generated light to produce a treatment which can offer selective tumoricidal action. Hematoporphyrin derivative (HD) and a purified component called Photofrin II are currently being used in clinical PDT. Initial patient results have been encouraging, and considerable interest has developed in the synthesis and evaluation of new photosensitizers with improved photochemical and pharmacological characteristics. In addition, there has been a gradual increase in knowledge related to in vitro and in vivo mechanisms of action of PDT. This report provides an overview of the properties and applications of PDT. Information and data related to drug development, photochemistry, subcellular targets, in vivo responses, and clinical trials of PDT are presented.
Photodynamic therapy (PDT) is being utilized in the treatment of a wide variety of malignant tumors. Results using PDT have been encouraging, and controlled clinical trials are currently being performed. The procedure exploits both the tumor-localizing and -photosensitizing properties of hematoporphyrin derivative or its purified component, Photofrin II. When this porphyrin mixture is administered systemically, it is retained preferentially in tumor tissue as compared to surrounding normal tissue. Localized tumor destruction induced by PDT results from the photochemical generation of cytotoxic oxygen species within the tumor. This review will provide a summary of historical and current research pertaining to molecular, cellular, and tissue responses induced by PDT. Emphasis is placed on information related to the chemistry of current photosensitizers, subcellular targets, preclinical treatment parameters, and clinical responses following PDT.
Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III and art genes. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-kappa B, with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-kappa B acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).
The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.
In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.
Recent studies indicate that human immunodeficiency virus type 1 (HIV) gene expression can be dramatically enhanced by certain heterologous viral and chemical agents, implicating these as potential reactivating agents of latent virus infection. A common denominator shared by these agents is their ability to cause stress responses in cells. In an effort to determine whether stress responses affect HIV gene expression, we examined the effects of ultraviolet light (UV) and mitomycin C, on HIV gene expression as well as on viral growth and development. We demonstrate that these agents enhance HIV gene expression up to 150-fold. These levels are similar to those obtained by the tat gene product, the HIV trans-activating factor responsible for enhancing viral gene expression. The increase in gene expression after UV irradiation appears to require transcription but not de novo protein synthesis, and correlates with an accumulation of stable mRNA. Most importantly, UV irradiation of human T-cells prior to viral infection significantly shortens the viral growth cycle. Apparently, UV-induced cellular stress is highly conducive for viral replication and growth. We further demonstrate that even direct sunlight can activate HIV gene expression. These results demonstrate that DNA damaging agents, and perhaps other agents which elicit SOS-like stress responses in mammalian cells, can activate HIV expression thereby enhancing viral replication and development.
In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found
in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate.
The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein
(I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated
cells--in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-kappa B and I kappa
B was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase
C, cause dissociation of a cytoplasmic complex of NF-kappa B and I kappa B by modifying I kappa B. this releases active NF-kappa
B which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive
regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.
- Ryter S.