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Changes in cell morphology determined following hematoporphyrin monomethyl ether (HMME)-promising photodynamic therapy (PDT) treatment. CHMm cells were treated as indicated, after which images were acquired using a light microscope. Arrows indicate cells contained cytoplasmic vacuoles. Arrowheads show cells became round and smaller and detached. Scale bars = 100 m.
Source publication
Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis in vitro on canine breast cancer cells. However, the exact apoptosis pathway responsible for HMME-PDT in canine breast cancer cells still remains unknown. Our results showed that HMME-PDT induced signif...
Contexts in source publication
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... toxicity of HMME-PDT toward CHMm cells was observed by microscopy. The results revealed that HMME-Journal of Veterinary Science PDT caused morphological changes in cells at different time points after individual treatments (Fig. 1). When compared with mock treated cells, the growth of cells treated with HMME-PDT was significantly inhibited. Consistent with this observation, HMME-PDT treated cells demonstrated significantly more morphological changes than mock treated cells. A large number of HMME-PDT treated cells contained cytoplasm vacuoles (arrows in Fig. 1), ...
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... treatments (Fig. 1). When compared with mock treated cells, the growth of cells treated with HMME-PDT was significantly inhibited. Consistent with this observation, HMME-PDT treated cells demonstrated significantly more morphological changes than mock treated cells. A large number of HMME-PDT treated cells contained cytoplasm vacuoles (arrows in Fig. 1), and cells gradually became round, smaller and detached (arrowheads in Fig. 1). Cell death occurred in a time-dependent manner, showing sharply increased cell death and reduced density after 12 h of HMME and irradiation treatment relative to mock treated cells (Fig. 1). TUNEL staining was used to detect apoptosis induced by HMME-PDT ...
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... treated with HMME-PDT was significantly inhibited. Consistent with this observation, HMME-PDT treated cells demonstrated significantly more morphological changes than mock treated cells. A large number of HMME-PDT treated cells contained cytoplasm vacuoles (arrows in Fig. 1), and cells gradually became round, smaller and detached (arrowheads in Fig. 1). Cell death occurred in a time-dependent manner, showing sharply increased cell death and reduced density after 12 h of HMME and irradiation treatment relative to mock treated cells (Fig. 1). TUNEL staining was used to detect apoptosis induced by HMME-PDT in canine breast cancer cells. The nuclei of apoptosis cells (TUNEL-positive ...
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... large number of HMME-PDT treated cells contained cytoplasm vacuoles (arrows in Fig. 1), and cells gradually became round, smaller and detached (arrowheads in Fig. 1). Cell death occurred in a time-dependent manner, showing sharply increased cell death and reduced density after 12 h of HMME and irradiation treatment relative to mock treated cells (Fig. 1). TUNEL staining was used to detect apoptosis induced by HMME-PDT in canine breast cancer cells. The nuclei of apoptosis cells (TUNEL-positive cells) were labeled with fluorescein isothiocyanate, and their morphologic alterations associated with apoptosis were verified by fluorescence microscopy (Fig. 2). Apoptosis cells appearances at ...
Citations
... Recent studies have focused on the effectiveness of PDT or photodynamic detection (PDD) through various photosensitizers for CMT treatment [15][16][17][24][25][26][27][28]. However, there is no study evaluating the efficacy of 5-ALA-based PDT in treating different histological types of CMST alone. ...
... Furthermore, the anti-cancer activity of 5-ALA/PDT could result from apoptotic cell death via increased intracellular ROS level and overexpression of Bax and Caspase-9 and Caspase-3 mRNA and protein levels in CMST cells, particularly S1 and S2 cells. In the literature, HMME-based PDT causes apoptotic cell death in CMT cells through the up-regulation of Caspase-9, Caspase-3, and Bax levels [26,27]. Additionally, the molecular mechanisms of 5-ALA-mediated apoptosis have been revealed in different types of human cancer cell lines [29][30][31][32]. ...
Canine mammary sarcoma tumors (CMST) are the most aggressive tumors with poor prognosis in dogs. Due to inadequate treatment options for CMST, recent studies have focused on alternative treatment strategies. We previously determined the optimized protocol of 5-ALA-based photodynamic therapy (PDT) in canine liposarcoma. However, its molecular mechanisms in the treatment of different histological types of CMST remain unclear.
In this context, we, for the first time, assessed 5-aminolevulinic acid (5-ALA)-PDT–mediated anti-cancer activity and its molecular mechanism after continuous wave (CW) and pulse radiation (PR) on three different histological types (liposarcoma, chondrosarcoma, and osteosarcoma) of CMST cells by WST-1, Annexin V, ROS, acridine orange/propidium iodide staining, RT-PCR, and western blot analysis.
Our findings showed that 5-ALA/PDT significantly suppressed the proliferation of CMST cells (p < 0.01) and induced apoptosis via increased ROS level and overexpression of Caspase-9 and Caspase-3 mRNA and cleaved protein levels in especially liposarcoma and chondrosarcoma cells following CW and PR irradiation at 9 J/cm². However, the response of CMST cells to 5-ALA was different upon CW and PR irradiation due to differences in their origin.
Collectively, our findings provided the first evidence that 5-ALA-based PDT could be used as an alternative treatment strategy, especially liposarcoma and chondrosarcoma. However, further in vitro and in vivo studies are required to elucidate the underlying molecular mechanism of the efficacy of 5-ALA in CMST cells at the molecular level.
... The hematoporphyrin monomethyl ether was administered to canine mammary carcinoma cell lines [161][162][163]. Apoptosis plays an important role in the reduction induced by PDT (20 mg/mL; He-Ne laser, 2.8 J/cm 2 , 632.8 nm) in the viability of canine mammary carcinoma cells [161]. ...
... Treatment lead to loss of mitochondrial membrane potential [162]. Additionally, significant changes in cell morphology were observed, such as the formation of cytoplasmic vacuoles and the gradual rounding of cells, together with decreased size and detachment [163]. ...
Photodynamic therapy (PDT) in small animals’ oncology has been under research focus, pointing to new treatment possibilities. Moreover, several animal studies constitute experimental human disease models due to the similarity of tumor biology between animals and man. PDT uses photosensitizing compounds without toxicity per se. When subjected to a specific wavelength, the photosensitizers are activated, triggering the production of reactive oxygen species (ROS) that lead to cell death. Additionally, antiangiogenic effects and immune stimulation may also be elicited. PDT is minimally invasive, non-toxic, and does not induce carcinogenic or mutagenic side effects. Thus, it is safe for non-neoplastic tissues compared with other neoplasms treatment modalities. This review describes the applications of PDT in the cancer treatment of small animals, particularly dogs and cats, focusing on the respective photosensitizers and treatment protocols used in trials in this therapeutic modality.
... This provides a window for the treatment of PDT. At present, a large number of experiments have reported that HMME-mediated photodynamics (HMME-PDT) can induce apoptosis of various cancer cells, but the mechanism mainly focuses on HMME-PDT regulating the expression of single tumor suppressor genes or apoptosis-related proteins, such as upregulation of P53 or activation of caspase-3 [7][8][9]. However, the efficacy of HMME-PDT in OSCC is unclear. ...
... Previous studies revealed that PDT could upregulate the expression of P53 protein and mRNA in breast cancer cells, and the enhanced expression of P53 could inhibit the expression of downstream factor BCL-2, activated caspase-3, and induced cell apoptosis through the mitochondrial apoptosis pathway [9]. The posttranscriptional regulation of gene expression was mainly accomplished by miRNAs [35]. ...
The objective of this study was to determine the mechanism and effect of hematoporphyrin monomethyl ether mediated photodynamic therapy (HMME-PDT) on oral squamous cell carcinoma (OSCC). Human OSCC CAL-27 cells were randomly divided into four groups: control group, HMME group, laser group, and HMME-PDT group. Cell viability was detected by the CCK-8 method. Cell cycle distribution was evaluated by flow cytometry. GEO database was used to screen differentially expressed microRNAs (DEMs), and TCGA database was performed to verify DEM expression in OSCC and normal tissues. The effects of HMME-PDT on DEM expression were assayed by real-time PCR, and the expressions of miRNAs target genes were measured by western blot. Fluorescence probes were used to determine the production of singlet oxygen (¹O2). Compared with the other three groups, HMME-PDT dramatically inhibited CAL-27 cell proliferation and induced G0/G1 cycle arrest. The expressions of miR-21 and miR-155 were significantly upregulated in OSCC. HMME-PDT downregulated the expression of miR-21 but had no obvious effect on miR-155. HMME-PDT remarkably upregulated the levels of P53 and miR-21 target proteins, such as PDCD4, RECK, and SPRY2. ¹O2 was generated during HMME-PDT, and inhibition of ¹O2 production could reverse the regulation of HMME-PDT on P53, miR-21, and its target proteins, thus restoring cell viability. HMME-PDT can significantly inhibit the growth of OSCC cells, and the mechanism of this effect is related to the regulation of the P53-miR-21-PDCD4 axis via ¹O2 induced by HMME-PDT.
... Therefore, new treatment options including PDT, photodynamic detection (PDD), and photodynamic hyperthermia (PHT) have drawn attention for the treatment of canine tumors in veterinary medicine. In preclinical studies, different PS (5-ALA, verteporfin, G-Ce6, hematoporphyrin monomethyl ether (HMME), ICG, and HPPH) have been used for the treatment of various canine tumors including canine mammary carcinoma, canine oral squamous cell carcinoma, fibrosarcoma, liposarcoma, malignant shwannoma, and canine osseous tumors [22][23][24][25][26][27][28]. In clinical studies, PDT alone as well as the combination of PDT with PDD or PHT using different PS (HPPH, 5-ALA, ICG) have promising results in the treatment of canine tumors [17,[22][23][24][25]. ...
... However, there is limited evidence about the optimum PDT protocol and the underlying molecular mechanism of PDT in CMGTs, in vitro. The studies of Liu et al. [26] and Li et al. [27] state that HMME-based PDT induces apoptotic cell death through the mitochondrial apoptosis pathway including increased the level of caspase-9, caspase-3, and cytochrome c and mRNA and protein levels of Bax in canine mammary tumor cells after irradiation with He-Ne laser equipped with a 632.8 nm light source at a fluence of 2.8 J/cm 2 . However, the knowledge of pathology and subtype of these cells is not presented in these studies. ...
Canine mammary gland tumors (CMGTs) are heterogeneous disease and subclassified [sarcomas (S), carcinomas (C), and carcinosarcomas (CS)] according to histopathological differentiation. Photodynamic therapy (PDT) is a promising treatment strategy based on the use of a photosensitizer (PS) activated by light. However, the therapeutic potential of PDT in the treatment of CMGTs has not been investigated, yet. Therefore, the aim of this study was to determine the in vitro protocol of 5-ALA-based-PDT for the treatment of three subtypes of CMGTs, for the first time. The intracellular PpIX florescence intensity was measured for 5-ALA (0.5 and 1 mM). After irradiation with different light doses (6, 9, 12, 18, and 24 J/cm2) for two different modes [continuous wave (CW) and pulse radiation (PR)], the cytotoxic effects of 5-ALA (0.5 and 1 mM) on the subtypes (C, S, and CS) of CMGTs were analyzed by WST-1. Finally, the optimal PDT treatment protocol was validated through Annexin V and AO/EtBr staining. Our results showed that 1 mM 5-ALA for 4-h incubation was the best treatment condition in all subtypes of CMGTs due to higher intracellular PpIX level. After irradiation with different light doses, PR mode was more effective in S primary cells at 9 J/cm2. However, a significant decrease in the viability of C and CS cells was detected at 12 /cm2 in CW mode (p < 0.05). Additionally, 1 mM 5-ALA induced apoptotic cell death in each subtype of CMGTs. Our preliminary findings suggest that (i) each subtype of CMGTs differentially responds to PDT and (ii) the light dose and mode could play an important role in the effective PDT treatment. However, further studies are needed to investigate the role of the different light sources and PDT-based apoptotic cell death in CMGTs cells.
... The Bcl-2 gene family can be divided into 2 parts based on their actions: (1) activating apoptosis processes such as Bax, Bad, Bak and Bok; and (2) stopping apoptosis processes such as Bcl-2, Bcl-XL and Bcl-W [21]. Especially, the expression of Bax and Bcl-2 mRNA are generally approved as apoptosis indicators [22]. Thus, the study of these properties and mechanism in BmKn-2 and their effect on CMGTs is very important for developing new antitumor substances in the future. ...
... This research also studied BmKn-2 apoptosis-induction mechanisms in CMGT cell lines by observing the expression of Bax and Bcl-2 mRNA, which are generally approved apoptosis indicators [22]. The Bcl-2 gene family mostly regulates the apoptosis pathway [19] and controls mitochondrial integrity in cells [20]. ...
... The Bcl-2 gene family can be divided into 2 parts based on their actions: (1) activating apoptosis processes such as Bax, Bad, Bak and Bok; and (2) stopping apoptosis processes such as Bcl-2, Bcl-XL and Bcl-W [21]. Other studies have reported that anti-apoptosis action occurs when the Bcl-2 gene is overexpressed the formation of Bcl-2/Bax heterodimers in apoptotic cell death [22,32]. The properties of Bcl-2 genetic in dog may help to find the drug designing in cancers [36]. ...
The most common neoplasms in intact female dogs are CMGTs. BmKn-2, an antimicrobial peptide, is derived from scorpion venom and has published anticancer effects in oral and colon human cancer cell lines. Thus, it is highly likely that BmKn-2 could inhibit CMGT cell lines which has not been previously reported. This study investigated the proliferation and apoptotic properties of BmKn-2 via Bax and Bcl-2 relative gene expression in two CMGT cell lines, metastatic (CHMp-5b) and non-metastatic (CHMp-13a). The results showed that BmKn-2 inhibited proliferation and induced apoptosis in the CMGT cell lines. The cell morphology clearly changed and increased apoptosis in a dose dependent of manner. The half maximum inhibitory concentration (IC50) was 30 µg/mL for CHMp-5b cell line and 54 µg/mL for CHMp-13a cell line. The induction of apoptosis was mediated through Bcl-2 and Bax expression after BmKn-2 treatment. In conclusion, BmKn-2 inhibited proliferation and induced apoptosis in both CHMp-5b and CHMp-13a cell lines via down-regulation of Bcl-2 and up-regulation of Bax relative mRNA expression. Therefore, BmKn-2 could be feasible as candidate treatment for CMGTs.
... Caspase-3 is responsible for cell morphology and certain biochemical events including execution and completion of apoptosis [35]. As reported, caspase-3, activated by caspase-8 and caspase-9, cleaves vital cellular proteins, leading to apoptosis via mitochondria-dependent pathway [36]. In hepatocellular carcinoma cells, TM strongly enhanced the activation of caspase-3, caspase-8, and caspase-9. ...
Tricholoma matsutake , one of widely accepted functional mushrooms, possesses various pharmacological activities, and its antitumor effect has become an important research point. Our study aims to evaluate the cytotoxicity activities of T. matsutake aqueous extract (TM) in HepG2 and SMMC-7721 cells. In in vitro experiments, TM strikingly reduced cell viability, promoted cell apoptosis, inhibited cell migration ability, induced excessive generation of ROS, and caused caspases cascade and mitochondrial membrane potential dissipation in hepatocellular carcinoma cells. In in vivo experiments, 14-day TM treatment strongly suppressed tumor growth in HepG2 and SMMC-7721-xenografted nude mice without influence on their body weights and liver function. Furthermore, TM increased the levels of cleaved poly-ADP-ribose polymerase (PARP), Bad, and Bax and reduced the expressions of B-cell lymphoma 2 (Bcl-2) in treated cells and tumor tissues. All aforementioned results suggest that caspase-dependent mitochondrial apoptotic pathways are involved in TM-mediated antihepatocellular carcinoma.
Backgrounds
Canine mammary gland tumors (CMGTs) are heterogeneous tumors and share many similar features with human breast cancer. Despite the improvement of current treatment options, new treatment modalities are required to effectively kill tumor cells without general toxicity in the treatment of CMGTs. Photodynamic therapy (PDT) is a promising method for cancer treatment. However, there is a limited study evaluating the therapeutic efficacy of PDT in the treatment of CMGTs.
Methods
In this context, we, for the first time, investigated the therapeutic potential of 5-aminolaevulinic acid (5-ALA) mediated PDT at 6 and 12 J/cm² in two different subtypes [Tubulopapillary carcinoma (TPC) and carcinosarcoma (CS)] cells via different molecular analysis. The cytotoxic effects of 5-ALA/PDT on these cells were analyzed by intracellular PpIX level, WST-1 and ROS analysis. Furthermore, the underlying moleculer mechanism of 5-ALA/PDT mediated apoptotic effects on TPC and CS cells were evaluated Annexin V, AO/PI, RT-PCR and western blot analysis.
Results
The 5-ALA/PDT treatment upon irradiation considerably inhibited the viability of both TPC and CS cells (p<0.01) and caused apoptotic death through elevated ROS levels, the activation of Caspase-9, and Caspase-3, and the overexpression of Bax. However, the response of TPC and CS cells to 5-ALA/PDT was different.
Conclusions
Our preliminary in vitro findings provide novel insights into the molecular mechanisms underlying 5-ALA/PDT mediated apoptosis in both TPC and CS cells. However, the therapeutic response of CMGT cells to 5-ALA/PDT is limited.
