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Growth Inhibition and Induction of Apoptosis by Fenretinide in Small-Cell Lung Cancer Cell Lines
Abstract
Lung cancer is the major cause of cancer-related death in the United States, with small-cell lung cancer (SCLC) constituting approximately 20% of all cases of lung cancer. Numerous epidemiologic and molecular studies have suggested that alterations in retinoid-signaling pathways play a role in the pathogenesis of lung cancer. Fenretinide [N-(4-hydroxyphenyl)retinamide; HPR] is a synthetic retinoid with minimal toxicity and favorable pharmacokinetics during long-term administration to patients in clinical trials.
The aim of this investigation was to study the effect of HPR on the growth of SCLC cells in vitro.
Seven SCLC cell lines (NCI-H69, NCI-H82, NCI-H146, NCI-H209, NCI-H345, NCI-H446, and NCI-H510A) were exposed continuously to a broad range of concentrations of HPR or all-trans-retinoic acid (RA), and cell viability was determined on day 3 and day 7 by the trypan blue dye exclusion assay. The growth of these cells was compared with that of control vehicle-treated cells to determine survival fraction and the dose resulting in a 50% inhibition of growth when compared with growth of control cells (IC50). The induction of apoptosis was evaluated by fluorescent microscopy, DNA content analysis, and a terminal deoxyribonucleotidyl transferase-based assay that labels 3'-hydroxyl ends of DNA fragments (TUNEL assay) combined with flow cytometric analysis.
HPR inhibited growth of a panel of SCLC cell lines at IC50 values that ranged from 0.1 to 3.0 microM (concentrations that are clinically achievable). In all cell lines tested, HPR was a more potent growth inhibitor than RA. By use of fluorescent microscopy, HPR was found to induce morphologic changes consistent with apoptosis in NCI-H82 SCLC cells, including cellular shrinkage, chromatin condensation, and nuclear fragmentation. Flow cytometric analysis revealed decreased DNA content, and TUNEL assay showed increased digoxigenin-uridine triphosphate incorporation in HPR-treated NCI-H82 SCLC cells; these findings are consistent with the induction of apoptosis.
HPR inhibited the in vitro growth of SCLC cells. In NCI-H82 cells, HPR inhibited growth via the induction of apoptosis.
... The anticancer activity of fenretinide results from its ability to induce apoptosis in tumor cells by improving diverse signaling molecules including reactive oxygen species, ceramide and ganglioside GD3. In particular fenretinide has been shown to possess cytotoxic activity on a wide variety of experimental models belonging to different types of cancer, including lung cancer [50][51][52][53]. ...
... To better evaluate the presence of apoptosis in the dead areas TUNEL assay was carried out in sections of tumor mass obtained from treated and control samples. Results showed the presence of several apoptotic cells in the treated sample compared to the untreated sample suggesting that 4HPR-HSA induced cell death mainly by apoptosis in agreement with previous data demonstrating the apoptotic effect of 4-HPR [50][51][52][53]. ...
... The anticancer activity of fenretinide results from its ability to induce apoptosis in tumor cells by improving diverse signaling molecules including reactive oxygen species, ceramide and ganglioside GD3. In particular fenretinide has been shown to possess cytotoxic activity on a wide variety of experimental models belonging to different types of cancer, including lung cancer [50][51][52][53]. ...
... To better evaluate the presence of apoptosis in the dead areas TUNEL assay was carried out in sections of tumor mass obtained from treated and control samples. Results showed the presence of several apoptotic cells in the treated sample compared to the untreated sample suggesting that 4HPR-HSA induced cell death mainly by apoptosis in agreement with previous data demonstrating the apoptotic effect of 4-HPR [50][51][52][53]. ...
Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and Acsvl3 mainly connected with tumor growth.
Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and Acsvl3 suggested a reduction of tumor growth.
In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.
... The relatively new synthetic retinoid HPR has been reported to markedly inhibit the growth of malignant myeloid, lymphoid, small cell lung cancer, ovarian carcinoma and neuroblastoma cell lines through the induction of apoptosis, even in cell lines resistant to retinoic acid (Chan et al., 1997;Delia et al., 1993;Kalemkerian et al., 1995;Lotan, 1995;Ponzoni et al., 1995). In humans, the potential of HPR to prevent relapse in a variety of cancers is under evaluation in several clinical trials, though its role has not been unquestionably defined (Modiano et al., 1990;Moon et al., 1994). ...
... The above studies have determined that serum concentrations of HPR between 1 and 3 µM are attainable in humans with minimal toxicity (Formelli et al., , 1998, while concentrations of at least 10 µM HPR are necessary to completely inhibit the growth of ovarian carcinoma cells (Supino et al., 1996) or small cell lung cancer cells (Kalemkerian et al., 1995) in vitro. We have noted that different neuroblastoma cell lines are partially resistant to 1 to 3 µM HPR (Pagnan et al., 1998). ...
Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitro modulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitro to increasing concentrations of HPR. Dose-dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR-treated melanoma cells were shown to undergo apoptosis. However, IC50 values ranged from 5 to 28 μM, while IC90 values were between 10 and 45 μM. These last concentrations are approximately 10-fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour-specific antigen extensively expressed by neuroectoderma-derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR-containing immunoliposomes followed by culture in drug-free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines. Int. J. Cancer 81:262–267, 1999. © 1999 Wiley-Liss, Inc.
... Fenretinide has been widely used in clinical trials for breast cancer chemoprevention ( 5 ) and for treatment of neuroblastoma and Ewing sarcoma ( 6 ). It has been shown that fenretinide inhibits the proliferation of many tumor cell lines (7)(8)(9)(10)(11), whereas it manifests minimal cytotoxicity to nonmalignant cells ( 7,12 ). Early studies of fenretinide supported the hypothesis that fenretinide increased the de novo synthesis of ceramide by stimulating SPT (13)(14)(15). ...
... However, we observed a 21% decrease of total anti-infl ammatory role against A . a stimulation. Although fenretinide has inhibited the proliferation of many tumor cell lines (7)(8)(9)(10)(11), it has been shown to have minimal cytotoxicity to nonmalignant cells ( 7,12 ). In our study, fenretinide (2.5-5 M) did not induce toxicity in Raw 264.7 cells after up to 16 h treatment (n = 4; P > 0.05; data not shown), which confi rmed that fenretinide had minimal cytotoxicity. ...
Abstract Ceramides play an essential role in modulating immune signaling pathways and pro-inflammatory cytokine production in response to infectious pathogens, stress stimuli, or chemotherapeutic drugs. In this study, we demonstrated that Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the pathogen for aggressive periodontitis, induced de novo synthesis of ceramide in Raw 264.7 cells. In addition, we identified that fenretinide, a synthetic retinoid, suppressed the de novo synthesis of ceramide induced by A. actinomycetemcomitans. Moreover, fenretinide attenuated interleukin (IL)-1β, IL-6 and cyclooxygenase (COX)-2 mRNA expression induced by A. actinomycetemcomitans. Fenretinide also decreased IL-1β, IL-6, and prostaglandin E2 pro-inflammatory cytokine levels in Raw 264.7 cells induced by A. actinomycetemcomitans. However, fenretinide had no significant effects on TNF-α mRNA or protein levels. Furthermore, we showed that fenretinide inhibited the JAK-STAT, PI3K-Akt, PKC and NF-κB signaling pathways, whereas fenretinide up-regulated MAPK signaling pathways after bacterial stimulation. In conclusion, this study emphasizes the de novo ceramide synthesis pathway in response to bacterial stimulation and demonstrates the anti-inflammatory role of fenretinide in bacteria-induced immune response.
... A promising achievement in this field is represented by HPR, which has been reported to markedly inhibit the growth of different tumor cell lines, including NB and melanoma, through the induction of apoptosis (Kalemkerian et al., 1995;Lotan, 1995;Ponzoni et al., 1995;Montaldo et al., 1999). However, concentrations of at least 10 µM HPR are necessary to completely inhibit the growth of ovarian carcinoma cells (Supino et al., 1996) or small-cell lung cancer cells (Kalemkerian et al., 1995) in vitro. ...
... A promising achievement in this field is represented by HPR, which has been reported to markedly inhibit the growth of different tumor cell lines, including NB and melanoma, through the induction of apoptosis (Kalemkerian et al., 1995;Lotan, 1995;Ponzoni et al., 1995;Montaldo et al., 1999). However, concentrations of at least 10 µM HPR are necessary to completely inhibit the growth of ovarian carcinoma cells (Supino et al., 1996) or small-cell lung cancer cells (Kalemkerian et al., 1995) in vitro. We have noted that various NB and melanoma cell lines are partially resistant to 1-3 µM HPR (Pagnan et al., 1998;Montaldo et al., 1999). ...
Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non-specific isotype-matched Abs or Ab-free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. When melanoma cells were incubated with 30 μM HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR. Int. J. Cancer 81:268–274, 1999.
... This may be due in part to defects in the retinoid signaling pathways detected in many lung carcinoma cells (31). However, the retinoid 4-hydroxyphenyl retinamide (4-HPR) is an exception, and has been shown to inhibit the proliferation of small-cell carcinoma cell lines but not non-small-cell carcinoma cell lines (32). This growth-inhibitory effect is not mediated by an RAR-or RXR-dependent signaling pathway, but by an unknown mechanism. ...
... The inhibition of cell proliferation was not restricted to a specific type of lung tumor, since small-cell carcinoma, adenocarcinoma as well as squamous carcinoma cell lines were inhibited. The effects of retinoids on the growth of human lung cancer cell lines have been examined by several investigators (28)(29)(30)32). These studies have shown that most lung carcinoma cell lines are rather resistant to the growth-inhibitory effects of RA (28,29). ...
In this study, we investigated the effect of the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) on the growth of human lung carcinoma cell lines. AHPN inhibits the proliferation of all cell lines tested, irrespective of the lung tumor type, in a concentration- and time-dependent manner. A dramatic reduction in cell number was observed in adenocarcinoma H460 cells, and was shown to be related to an induction of apoptosis. Bromodeoxyuridine (BrdU) incorporation and flow-cytometric analyses indicated that treatment of H460 cells with AHPN induces cell-cycle arrest at the G1 phase. We therefore investigated the effect of AHPN on several regulatory proteins of the G1 phase of the cell-cycle. The cell-cycle arrest induced by AHPN was accompanied by an inhibition of the hyperphosphorylation of the retinoblastoma (Rb) protein, an indication of G1 arrest. Furthermore, two cyclin-dependent kinases, cdk2 and cdk4, which are normally involved in the phosphorylation of Rb, were shown to have decreased activity. In some cell lines, the decrease in cdk activity may be partly related to an increase in p21(WAF1/Cip1) (p21), an inhibitor of cyclin-dependent kinases. No changes were observed in the cyclin-dependent kinase inhibitor p27(Kip1). The observed increase in p53 in response to AHPN could at least to some extent be responsible for the increased levels of p21. The increase in p53 expression was found to be regulated at a post-transcriptional level. Our results suggest that the growth inhibition of certain lung carcinoma cell lines by AHPN is at least partly related to an increase in p21. However, in other cell lines, different mechanisms appear to be involved. The specificity with which AHPN and other retinoids induce growth arrest and p21 expression indicates that the action of AHPN is not mediated by RAR or RXR receptors, but involves a novel signaling pathway.
... 5,6 Subsequent preclinical studies have shown that 4-HPR has chemopreventative activity in several animal models, 7-10 as well as cytotoxic activity in a variety of human cancer cell lines in vitro, including head and neck, non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, neuroblastoma, Ewing's family of tumor, leukemia, multiple myeloma, and pancreatic cancer at concentrations of 1-10 mmol/L. [11][12][13][14][15] In neuroblastoma and leukemia cell lines that are resistant to ATRA or 13-cis retinoic acid, 4-HPR has shown activity as well. 11, 16 4-HPR has also been tested clinically both as a chemoprevention agent in breast, 17 bladder, 18,19 oral mucosal, [20][21][22] and prostate cancers; 23 and more recently as a chemotherapeutic agent in pediatric [24][25][26][27] and adult malignancies. ...
Fenretinide (4-HPR) is a synthetic retinoid that has cytotoxic activity against cancer cells. Despite substantial in vitro cytotoxicity, response rates in early clinical trials with 4-HPR have been less than anticipated, likely due to the low bioavailability of the initial oral capsule formulation. Several clinical studies have shown that the oral capsule formulation at maximum tolerated dose (MTD) achieved <10 µmol/L concentrations in patients. To improve bioavailability of 4-HPR, new oral powder (LYM-X-SORB®, LXS) and intravenous lipid emulsion (ILE) formulations are being tested in early-phase clinical trials. ILE 4-HPR administered as five-day continuous infusion achieved over 50 µmol/L at MTD with minimal systemic toxicities; multiple complete and partial responses were observed in peripheral T cell lymphomas. The LXS oral powder 4-HPR formulation increased plasma levels approximately two-fold at MTD in children without dose-limiting toxicities and demonstrated multiple complete responses in recurrent neuroblastoma. The clinical activity observed with new 4-HPR formulations is attributed to increased bioavailability. Phase I and II clinical trials of both LXS 4-HPR and ILE 4-HPR are in progress as a single agent or in combination with other drugs.
Impact statement
One of the critical components in drug development is understanding pharmacology (especially pharmacokinetics) of the drugs being developed. Often the pharmacokinetic properties, such as poor solubility leading to poor bioavailability, of the drug can limit further development of the drug. The development of numerous drugs has often halted at clinical testing stages, and several of them were due to the pharmacological properties of the agents, resulting in increased drug development cost. The current review provides an example of how improved clinical activity can be achieved by changing the formulations of a drug with poor bioavailability. Thus, it emphasizes the importance of understanding pharmacologic characteristics of the drug in drug development.
... We have previously reported that retinoic acid receptors mediate the growth inhibitory action and the induction of TGase II activity by RA in mouse fibroblasts (5,6). The synthetic retinoid 4-hydroxyphenylretinamide (4-HPR) inhibits cell proliferation and induces apoptosis even in RAresistant cell lines (7,8). 4-HPR has been suggested to function through retinoic acid receptor (RAR) α. ...
Since 4-HPR binds poorly to the retinoic acid receptors, the issue of whether 4-HPR exerts its biological actions via classical retinoid receptor pathways remains to be resolved. We have previously reported that stable expression of a truncated retinoic acid receptor α, RARα403, transduced in NIH 3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and induction of tissue transglutaminase (TGase II). Here, we report that stable expression of the dominant negative construct RARα403 fails to blunt growth inhibition and TGase II induction by 4-HPR, a potent chemopreventive retinoid, in the same cells. These data show that retinoic acid receptors do not mediate either growth inhibition or induction of TGase II activity by 4-HPR in mouse fibroblast cells.
... Fenretinide (4-HPR) is one of the most promising clinically tested retinoids. 4-HPR demonstrated a significant cytotoxic activity of tumour cells through the induction of apoptotic and nonapoptotic cell death [67] in breast [68,69], prostate, bladder, skin [52,[70][71][72][73][74], colon-rectal [75], head and neck [76], ovarian cancers [77,78], both small cell and non-small cell lung cancer [79][80][81], neuroblastoma [82][83][84], and leukaemia cell lines [85,86]. 4-HPR has activity against tumours by generating reactive oxygen species [76,87,88], increasing dihydroceramide production [87,[89][90][91] and natural killer cell activity [92,93] and inhibiting angiogenesis [89,94]. ...
Retinol and vitamin A derivatives influence cell differentiation, proliferation, and apoptosis and play an important physiologic role
in a wide range of biological processes. Retinol is obtained from foods of animal origin. Retinol derivatives are fundamental for
vision, while retinoic acid is essential for skin and bone growth. Intracellular retinoid bioavailability is regulated by the presence
of specific cytoplasmic retinol and retinoic acid binding proteins (CRBPs and CRABPs). CRBP-1, the most diffuse CRBP isoform,
is a small 15KDa cytosolic protein widely expressed and evolutionarily conserved in many tissues. CRBP-1 acts as chaperone and
regulates the uptake, subsequent esterification, and bioavailability of retinol. CRBP-1 plays a major role in wound healing and
arterial tissue remodelling processes. In the last years, the role of CRBP-1-related retinoid signalling during cancer progression
became object of several studies. CRBP-1 downregulation associates with a more malignant phenotype in breast, ovarian, and
nasopharyngeal cancers. Reexpression of CRBP-1 increased retinol sensitivity and reduced viability of ovarian cancer cells in vitro.
Further studies are needed to explore newtherapeutic strategies aimed at restoringCRBP-1-mediated intracellular retinol trafficking
and the meaning of CRBP-1 expression in cancer patients’ screening for a more personalized and efficacy retinoid therapy.
... Fenretinide (4-HPR) is one of the most promising clinically tested retinoids. 4-HPR demonstrated a significant cytotoxic activity of tumour cells through the induction of apoptotic and nonapoptotic cell death [67] in breast [68,69], prostate, bladder, skin [52,[70][71][72][73][74], colon-rectal [75], head and neck [76], ovarian cancers [77,78], both small cell and non-small cell lung cancer [79][80][81], neuroblastoma [82][83][84], and leukaemia cell lines [85,86]. 4-HPR has activity against tumours by generating reactive oxygen species [76,87,88], increasing dihydroceramide production [87,[89][90][91] and natural killer cell activity [92,93] and inhibiting angiogenesis [89,94]. ...
Retinol and vitamin A derivatives influence cell differentiation, proliferation, and apoptosis and play an important physiologic role in a wide range of biological processes. Retinol is obtained from foods of animal origin. Retinol derivatives are fundamental for vision, while retinoic acid is essential for skin and bone growth. Intracellular retinoid bioavailability is regulated by the presence of specific cytoplasmic retinol and retinoic acid binding proteins (CRBPs and CRABPs). CRBP-1, the most diffuse CRBP isoform, is a small 15 KDa cytosolic protein widely expressed and evolutionarily conserved in many tissues. CRBP-1 acts as chaperone and regulates the uptake, subsequent esterification, and bioavailability of retinol. CRBP-1 plays a major role in wound healing and arterial tissue remodelling processes. In the last years, the role of CRBP-1-related retinoid signalling during cancer progression became object of several studies. CRBP-1 downregulation associates with a more malignant phenotype in breast, ovarian, and nasopharyngeal cancers. Reexpression of CRBP-1 increased retinol sensitivity and reduced viability of ovarian cancer cells in vitro. Further studies are needed to explore new therapeutic strategies aimed at restoring CRBP-1-mediated intracellular retinol trafficking and the meaning of CRBP-1 expression in cancer patients' screening for a more personalized and efficacy retinoid therapy.
... EPI JSCC-2 SCC2095sc 4-HPR, at levels comparable with those used in this study, induced apoptosis in a variety of cultured human cancer cells, including head and neck, ovary, and small-cell lung carcinomas (27)(28)(29). Following 4-HPR treatment in these studies, execution phase caspase induction occurred in half of the cell lines. ...
The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys pro-survival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC's co-morbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities e.g. mTOR and retinol binding protein interactions. These studies employed a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated fenretinide has ~200-fold greater binding affinity relative to the natural ligand (ATP) at FAK's kinase domain. Fenretinide also shows intermediate binding at FAK's FERM domain and interacts at the ATP-binding site of the closest FAK analogue, Pyk2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2/M cell cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, while FAK's FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA treated cells showed an intermediate cell migration capacity; data which suggest co-contribution of the established migrating-enhancing Pyk2. Our data imply that fenretinide is uniquely capable of disrupting FAK's and Pyk2's pro-survival and mobility-enhancing effects and further extend fenretinide's chemopreventive contributions beyond induction of apoptosis and differentiation.
Copyright © 2015, American Association for Cancer Research.
... In particular, 4-HPR has proved to hold cytotoxic activity on a wide variety of experimental models belonging to different types of cancer, including lung cancer. [13][14][15][16] In spite of its excellent pharmacological profile, a major limitation to the clinical use of fenretinide is represented by its low bioavailability due to insolubility in water and thus in aqueous body fluids. Numerous attempts have been made to develop a fenretinide formulation capable of increasing its bioavailability to a level that can elicit a therapeutic response. ...
The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilisation effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favoured albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumour xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation.
Copyright © 2014. Published by Elsevier Inc.
... As such, it is emerging as one of the most promising antitumor agents [17]. Studies have demonstrated that fenretinide can induce cytotoxicity in multiple human cancer cell lines in vitro [18][19][20][21][22][23]. Clinically, fenretinide has been used as both a chemopreventive agent in breast [24], bladder [25] and oral mucosal cancers [26], and as a chemotherapeutic agent in pediatric [27,28] and adult cancers [29][30][31]. ...
Resistance to progestin treatment is a major hurdle in the treatment of advanced and reoccurring endometrial cancer. Fenretinide is a synthetic retinoid that has been evaluated in clinical trials as a cancer therapeutic and chemo-preventive agent. Fenretinide has been established to be cytotoxic to many kinds of cancer cells. In the present study, we demonstrate that fenretinide decreased cell viability and induced apoptosis in Ishikawa cells, which are an endometrial cancer cell line, in dose dependent manner in-vitro. This effect was found to be independent of retinoic acid nuclear receptor signaling pathway. Further, we have shown that this induction of apoptosis by fenretinide may be caused by increased retinol uptake via STRA6. Silencing of STRA6 was shown to decrease apoptosis which was inhibited by knockdown of STRA6 expression in Ishikawa cells. Results of an in-vivo study demonstrated that intraperitoneal injections of fenretinide in endometrial cancer tumors (created using Ishikawa cells) in mice inhibited tumor growth effectively. Immunohistochemistry of mice tumors showed a decrease in Ki67 expression and an increase in cleaved caspase-3 staining after fenretinide treatment when compared to vehicle treated mice. Collectively, our results are the first to establish the efficacy of fenretinide as an antitumor agent for endometrial cancer both in-vitro and in-vivo, providing a valuable rationale for initiating more preclinical studies and clinical trials using fenretinide for the treatment of endometrial cancer.
... One such agent is N-(4-hydroxyphenyl)retinamide (fenretinide) (HPR), which has exhibited minimal toxicity and favorable pharmacokinetics in clinical trials. 36 HPR induces apoptosis in a variety of malignant cell types, including SCLC, at clinically achievable concentrations 37 and acts synergistically with both cisplatin and etoposide against SCLC cells in vitro. 38 Although RA in combination with cisplatin and etoposide, as administered in this trial, does not appear to be more active than chemotherapy alone in patients with extensive stage SCLC, further studies utilizing improved schedules or more active and less toxic retinoids may be warranted in light of promising preclinical data. ...
BACKGROUND
The dysregulation of both myc gene expression and retinoid signaling pathways commonly occurs in small cell lung carcinoma (SCLC). Because preclinical data showed that all-trans-retinoic acid (RA) inhibited SCLC growth, altered myc expression, and blocked transition to a treatment-resistant phenotype, a Phase II trial was designed to determine the effects of the combination of RA, cisplatin, and etoposide in patients with SCLC.METHODS
Patients with untreated, extensive stage SCLC were treated with up to 8 cycles of cisplatin, 60 mg/m2, intravenously (i.v.) on Day 1 and etoposide, 120 mg/m2, i.v. on Days 1-3 in addition to up to 1 year of oral RA, 150 mg/m2/day.RESULTSOf 22 assessable patients 1 had a complete response and 9 had a partial response, for an overall response rate of 45% (95% confidence interval, 24-68%). The median survival was 10.9 months and the 1-year survival was 41%. The median duration of chemotherapy was 6 cycles and the median duration of RA treatment was 2.8 months. Thirteen patients discontinued RA prematurely due to toxicity and only 4 responders were receiving RA at the time of recurrence. Toxicity-limiting RA treatment mainly was comprised of mucocutaneous changes and headaches.CONCLUSIONSRA at a dose of 150 mg/m2/day was tolerated poorly in combination with cisplatin plus etoposide, leading to early discontinuation of RA in the majority of patients. The hematologic toxicity, response rate, and survival were similar to those associated with cisplatin and etoposide in prior trials. Further studies with more active and less toxic agents will be required to determine the role of retinoids in the treatment of SCLC. Cancer 1998;83:1102-1108. © 1998 American Cancer Society.
... To analyze the effects of RA on the induction of apoptosis or programmed cell death in RAR--expressing SCLC cells, we measured apoptotic cells by specific 3' end labeling of DNA ends in situ. As we previously reported (Kalemkerian et al., 1995), RA did not significantly increase the percentage of H82 and H209 cells undergoing apoptosis (Table I). The percentage of apoptotic H209-RAR- cells after RA treatment was only moderately increased compared with untreated cells (9.9% vs. 3.6%), indicating that RA-induced growth inhibition in H209-RAR- and H82 cells is caused primarily by mechanisms interfering with G 1 /Stransition, and not with the induction of programmed cell death. ...
Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor β (RAR-β) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-β in the growth regulation of SCLC by retinoids, we restored RAR-β expression in RAR-β-negative H209 SCLC cells by retroviral transduction (H209-RAR-β). We found that H209-RAR-β, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-β cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-β cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-β, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-β cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-β gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-β. Int. J. Cancer 80:935–943, 1999. © 1999 Wiley-Liss, Inc.
... This agent is also effective in reducing tumor load in animal model systems (11)(12)(13). Although it has been reported that the antiproliferative effects of 4HPR in cultured cancer cell lines are mediated through the induction of apoptosis (7,8,14,15), the mechanism of action of 4HPR remains poorly understood. ...
The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4HPR) can inhibit the growth of tumor cells. Preliminary results from a clinical trial suggest that 4HPR may reduce ovarian cancer incidence. We examined the growth-inhibitory effects of 4HPR on gynecologic cancer cell lines in vitro and the role of retinoid receptors in modulating this effect.
Twelve human gynecologic cancer cell lines (the ovarian cell lines--A224, AD10, UCI 101, UCI 107, SKOV3, 222, CP70, ML3B, and ML5; the cervical cell lines--HT3 and ME180; and the endometrial cell line--Hec 1A were tested for sensitivity to 4HPR (by assaying cell proliferation rates). Gel electrophoretic analysis of DNA fragmentation was used to measure programmed cell death (apoptosis). Specific retinoid receptor (retinoic acid receptor [RAR] and retinoid X receptor) messenger RNA (mRNA) levels were measured by northern blot hybridization. AD10 cells were stably transfected with human RARbeta complementary DNA, and the effect of 4HPR on cell proliferation was examined.
4HPR inhibited the growth of all 12 cell lines, but to varying degrees; IC50 values (i.e., concentrations that inhibit proliferation by 50%) ranged from 0.3 to 9 microM. Following 4HPR treatment, ovarian cancer cells that were sensitive to 4HPR (222, CP70, and UCI 101; IC50 <3 microM) contained higher levels of RARbeta transcripts than more resistant cells (AD10, ME180, Hec 1A, and A224; IC50 > or =3 microM) (2.8-fold; two-sided P = .006). Anchorage-independent growth of transfected AD10 cells expressing high levels of RARbeta was totally abolished, even in the absence of 4HPR; transfectants expressing low levels of RARbeta exhibited lower levels of anchorage-independent growth and grew more slowly in the presence of 4HPR than control untransfected AD10 cells.
4HPR inhibited the proliferation of ovarian cancer cells in vitro; RARbeta expression appeared to be associated with this effect.
... Fenretinide (N-(4-hydroxyphenyl)retinamide; 4-HPR) is a synthetic analog of all-trans retinoic acid (ATRA) that exhibits cytotoxic activity against a variety of human cancer cell lines in vitro at concentrations of 1 – 10 µM (Delia et al., 1993; Kalemkerian et al., 1995; Mariotti et al., 1994; O'Donnell et al., 2002; Oridate et al., 1996). 4-HPR has also been studied clinically both as a chemopreventative agent in breast (Veronesi et al., 1999), bladder (Sabichi et al., 2008), and oral mucosal cancers (Chiesa et al., 2005), and more recently as a chemotherapeutic agent in pediatric (Garaventa et al., 2003; Villablanca et al., 2006) and adult cancers (Puduvalli et al., 2004; Reynolds et al., 2007; Vaishampayan et al., 2005). ...
High plasma levels of fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4-HPR has been limiting its therapeutic applications. This study characterized metabolism of 4-HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4-HPR plasma levels in mice and to increase the low bioavailability of 4-HPR.
4-HPR metabolites were identified by mass spectrometric analysis and levels of 4-HPR and its metabolites [N-(4-methoxyphenyl)retinamide (4-MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR)] were quantified by high-performance liquid chromatography (HPLC). Kinetic analysis of enzyme activities and the effects of enzyme inhibitors were performed in pooled human and pooled mouse liver microsomes, and in human cytochrome P450 (CYP) 3A4 isoenzyme microsomes. In vivo metabolism of 4-HPR was inhibited in mice.
Six 4-HPR metabolites were identified in the plasma of patients and mice. 4-HPR was oxidized to 4-oxo-4-HPR, at least in part via human CYP3A4. The CYP3A4 inhibitor ketoconazole significantly reduced 4-oxo-4-HPR formation in both human and mouse liver microsomes. In two strains of mice, co-administration of ketoconazole with 4-HPR in vivo significantly increased 4-HPR plasma concentrations by > twofold over 4-HPR alone and also increased 4-oxo-4-HPR levels.
Mice may serve as an in vivo model of human 4-HPR pharmacokinetics. In vivo data suggest that the co-administration of ketoconazole at normal clinical doses with 4-HPR may increase systemic exposure to 4-HPR in humans.
Disease signature-based drug repositioning approaches typically first identify a disease signature from gene expression profiles of disease samples to represent a particular disease. Then such a disease signature is connected with the drug-induced gene expression profiles to find potential drugs for the particular disease. In order to obtain reliable disease signatures, the size of disease samples should be large enough, which is not always a single case in practice, especially for personalized medicine. On the other hand, the sample sizes of drug-induced gene expression profiles are generally large. In this study, we propose a new drug repositioning approach (HDgS), in which the drug signature is first identified from drug-induced gene expression profiles, and then connected to the gene expression profiles of disease samples to find the potential drugs for patients. In order to take the dependencies among genes into account, the human protein complexes (HPC) are used to define the drug signature. The proposed HDgS is applied to the drug-induced gene expression profiles in LINCS and several types of cancer samples. The results indicate that the HPC-based drug signature can effectively find drug candidates for patients and that the proposed HDgS can be applied for personalized medicine with even one patient sample.
In multicellular organisms, a balance between differentiation, proliferation and cell death maintains tissue homeostasis in adult tissues and directs normal development during embryonic morphogenesis. Vitamin A is one of the critical factors regulating the molecular events involved in these processes. The requirement of retinoids for normal development and tissue homeostasis has been known since Wolbach and Howe first reported on the defects that occurred in vitamin A-deficient animals in 1926. A large literature has since appeared describing the role of retinoids in promoting proliferation, differentiation or apoptosis in vivo (see also Chaps. 12–15, this volume), in a variety of cell culture systems (see also Chaps. 3, 9, 10, this volume) and more recently through studies using retinoid receptor knock-out strategies (Taneja et al. 1996; Sapin et al. 1997). In addition, these investigations have provided insight into the mechanisms by which retinoids induce teratogenic effects and inhibit neoplastic development, and hold promise for new therapeutic applications of retinoids, particularly in cancer.
PURPOSE: 13 cis Retinoic acid (isotretinoin) is a retinoid with preclinical evidence of anti–prostate cancer activity. This phase II, cross-over, randomized study of advanced, predominantly androgen-dependent prostate cancer patients was designed to assess primarily the effect on prostate-specific antigen (PSA) decline and toxicity of adding isotretinoin to hormonal therapy and, secondarily, the potential antitumor activity of the combination.
PATIENTS AND METHODS: Thirty-seven D0 to D2 patients were randomized soon after initiating luteinizing hormone–releasing hormone agonist with antiandrogen treatment to add (arm 1) or not (arm 2) isotretinoin from weeks 1 to 12. After cross-over on week 13, patients in arm 1 discontinued while patients in arm 2 added isotretinoin from weeks 14 to 25. Observation on hormonal therapy alone continued until week 49.
RESULTS: Baseline and randomization median PSA for 30 assessable patients were, respectively, 34 and 18.2 ng/mL for arm 1 and 31 and 13.4 ng/mL for arm 2. Median PSA at week 13 was 0.5 ng/mL (range, < 0.05 to 136 ng/mL) for arm 1 and 0.7 ng/mL (range, < 0.05 to 4.4 ng/mL) for arm 2; at week 25, 0.1 ng/mL (range, < 0.05 to 121 ng/mL) and 0.4 ng/mL (range, < 0.05 to 3.1 ng/mL), respectively. At week 49, arm 1 had median PSA of 0.1 ng/mL (range, < 0.05 to 345 ng/mL) and arm 2, 0.3 ng/mL (range, < 0.05 to 8.8 ng/mL); seven of 15 and three of 15 patients, respectively, had undetectable PSA levels (P = .12). Frequent isotretinoin-related toxicity included grade 1 cheilitis (76%), skin dryness (43%), and elevated triglycerides (50%).
CONCLUSION: Isotretinoin does not impair PSA decline or add significant toxicity to hormonal therapy. An adequately powered, randomized study would be required to determine whether the combination is superior to standard hormonal treatment.
A simple and accurate high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the determination of N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and its metabolites, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) and N-(4-methoxyphenyl)retinamide (4-MPR), in human plasma. Plasma samples were prepared using protein precipitation with ethanol. Chromatographic separation of the three analytes and N-(4-ethoxyphenyl)retinamide (4-EPR), an internal standard, was achieved on a Zorbax SB-C18 column (3.5 μm, 50 × 2.1 mm) using gradient elution with the mobile phase of 0.1% formic acid in water and acetonitrile (pH* 2.4) at a flow rate of 0.5 mL/min. Electrospray ionization (ESI) mass spectrometry was operated in the positive ion mode with multiple reaction monitoring (MRM). The calibration curves obtained were linear over the concentration range of 0.2–50 ng/mL with a lower limit of quantification of 0.2 ng/mL. The relative standard deviation of intra-day and inter-day precision was below 7.64%, and the accuracy ranged from 94.92 to 105.43%. The extraction recoveries were found to be higher than 90.39% and no matrix effect was observed. The analytes were stable for the durations of the stability studies. The validated method was successfully applied to the analyses of the pharmacokinetic study for patients treated with 4-HPR in a clinical trial.
Sphingolipids have been extensively studied as signal transducers regulating stress response, proliferation, and cell death, yet agents that target sphingolipid signaling have been slow to advance to clinical studies in oncology. The role of sphingolipids as critical components of membrane synthesis, combined with the cytotoxicity achieved by ceramides and dihydroceramides, indicate that targeting sphingolipids has significant potential for novel cancer therapeutics. The discovery that high concentrations of the semi-synthetic retinoid, N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR), can stimulate production of cytotoxic dihydroceramides led to preclinical studies focused on the use of high-dose 4-HPR as a dihydroceramide inducer for cancer therapeutics both as a single agent and in various ceramide-modulating drug combinations. Subsequent studies showed that 4-HPR increased dihydroceramides by simultaneously stimulating the de novo dihydroceramide synthesis pathway while inhibiting dihydroceramide desaturase 1 (DES1), preventing forward metabolism of dihydroceramides to ceramides. The high clinical therapeutic index of 4-HPR is due in part to the ability of 4-HPR to stimulate production of dihydroceramides in susceptible cancer cells but not in normal cells. The minimal cytotoxicity of 4-HPR for normal cells combined with improved formulations has enabled 4-HPR plasma concentrations of 10–60 μM to be achieved in clinical trials with minimal systemic toxicity. Durable complete clinical responses to 4-HPR have been observed in recurrent neuroblastoma, recurrent peripheral T cell lymphoma, and recurrent cutaneous T cell lymphoma, with clinical signals of activity also observed in ovarian cancer and gastrointestinal cancers. Future clinical trials will not only employ 4-HPR as a single agent, but will also test 4-HPR in combination with traditional cytotoxic agents, such as vincristine, and with other agents that target sphingolipid metabolism, such as safingol.
The retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR), mediates p53-independent cytotoxicity and can increase reactive oxygen species and ceramide In solid tumor cell lines. We determined changes In ceramide and cytotoxicity upon treatment with 4-HPR (3-12 muM) In six human acute lymphoblastic leukemia (ALL) cell lines: T cell (MOLT-3, MOLT-4, CEM), pre-B-cell (NALM-6, SMS-SB), and null cell (NALL-1). Exposure to 4-HPR (12 muM) for 96 h caused 4.7 (MOLT-3), 3.5 (MOLT-4), 3.9 (CEM), 2.9 (NALM-6), 4.7 (SMS-SB), AND 4.5 (NALL-1) logs of cell kill. The average 4-HPR concentration that killed 99% of cells (LCgg) for all six lines was 4.8 muM (range: 1.5-8.9 muM). Treatment with 4-HPR (9 muM) for 24 h resulted in an 8.9 +/- 1.0-fold (range: 4.9-15.7-fold) increase of ceramide. Ceramide increase was time- and dose-dependent and abrogated by inhibitors of do novo ceramide synthesis. Concurrent inhibition of ceramide glycosylation/acylation by d,1-threo-(1-phenyl-2-hexadecanoyl-amino-3-morpholino-1-propanol) (PPMP) further increased ceramide levels, and synergistically increased 4-HPR cytotoxicity in four of six ALL cell lines. 4-HPR was minimally cytotoxic to peripheral blood mononuclear cells and a lymphoblastoid cell line, and increased ceramide <2-fold. Thus, 4-HPR was cytotoxic and increased ceramide in ALL cell lines, but not in nonmalignant lymphoid cell types.
Purpose:
Retinoic acid is a substance that has previously been reported to increase radiosensitivity, but at concentrations likely to inhibit cell growth or to induce celluar differentiation. We choose head and neck cancer cell lines to investigate the role of retinoic acid as a radiosensitizer and to elucidate the mechanism through the changes in the expression of retinoid receptors and squamous cell differentiation marker.
Materials and methods:
Three cell lines (PCI-50, SqCC/ Y1 and UMSCC-11B) were used. 7-AAD staining for apoptosis and Western blot analysis for RAR-alpha, beta, gamma, RXR-alpha, beta, gamma and involucrin were performed after various treatments (control, beta-all-trans-retinoic acid (t-RA) only (10 6 M), radiation only (3 Gy), radiation with t-RA).
Results:
The synergistic radiosensitivity effect of t-RA was seen only radioresistant UMSCC-11B cell line. Expression of RAR-beta was induced by t-RA in maily UMSCC- 11B cell line. RAR-alpha,gamma, and RXR-alpha, beta, gamma expression were not changed in all cell lines tested. Expression of involucrin was inhibited by t-RA in PCI-50 cell line but other two cell lines were not changed by t-RA treatment.
Conclusion:
We found that only radioresistant cell line (UMSCC-11B) showed synergistic radiosensitivity effect by t-RA and this mechanism may be through RAR-beta expression induction.
Fat-soluble small molecule hormones and vitamins have long attracted the curiosity of molecular biologists, since they appeared to regulate gene expression in eukaryotic cells via similar mechanisms as certain signal molecules that had been studied extensively in prokaryotic systems. Vitamin A and its natural and synthetic analogues and derivatives (the retinoids) were of particular interest because of a variety of reasons. Starting a century ago it became more and more obvious that vitamin A played a very central role in the regulation and timing of many important biological processes, including development, differentiation, morphogenesis, growth, metabolism and homeostasis. Because of their central biological roles it appeared possible that retinoids serve as therapeutic agents, increasing the desire and need to understand their molecular mechanism of action. This became particularly important when synthetic vitamin A derivatives were sought with fewer undesirable side effects. Thus not surprisingly, as soon as the molecular biology technologies became available, a rapid progress was made in the deciphering of molecular signal trans- duction pathways of small fat soluble hormones and vitamins including the retinoids.
The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 μM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 μM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 μM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 μM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.
Human neuroblastoma (NB) tumours represent a major therapeutic challenge due to the lack of drugs effective in controlling cell proliferation. We previously reported that the synthetic retinoid Fenretinide (HPR) inhibits NB cell growth through the induction of programmed cell death. More recently, various NB cell lines have been shown to be partially resistant, in vitro, to HPR used at in vivo achievable concentrations (1-3 μmol/L). To significantly increase the dose, half-life, and stability of this promising anticancer agent we studied a system of conventional or long-circulating liposomes.
In this study, we showed that HPR can be efficiently and stable encapsulated in conventional (CL-HPR) and stabilized liposomes (SL-HPR). Since the leakage of the drug from the liposomes under the experimental conditions used is negligible, it seems that HPR is entering cells via uptake of intact liposomes. Liposome-entrapped HPR completely arrested the growth of NB cells. The effect was dose- and time-dependent. Indeed, SL-HPR at 30 (imol/ L induced, in the cell lines partially resistant to free HPR, a very rapid (24-48 h) fall in thymidine uptake (> 95 %), whereas at 3 μmol/L it exhibited cytostatic effects.
Time lapse photomicroscopy showed that NB cells treated with SL-HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with SL-HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses at the nuclear periphery, a typical feature of apoptotic cells. These findings were confirmed by electronic microscopy, DNA fragmentation assay, DNA content analysis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. HPLC analysis showed that HPR did not become metabolized after uptake into NB cells cultured in vitro, thus indicating that SL-HPR-induced apoptosis results from the action of HPR, itself, and not from its metabolite(s). In conclusion, our study demonstrates that Fenretinide entrapped in conventional or sterically stabilized liposomes dramatically suppresses NB cell growth by inducing programmed cell death.
Retinoid drugs find major applications in the topical treatment of cutaneous proliferative disorders such as acne, psoriasis, and photoaging and cutaneous cancers such as cutaneous T-cell lymphoma (CTCL) and Kaposi's sarcoma. Oral retinoids are successfully used to treat nodular/cystic acne, systemic CTCL, and acute promyelocytic leukemia. This overview of available retinoid drugs and those in the preclinical or clinical pipeline covers their development, indications, clinical trial results, adverse effects, and pharmacology/metabolism. Retinoids have been described historically and functionally in the context of interaction with their nuclear receptors—retinoic acid receptors (RARs) and retinoid X receptors (RXRs)—in inducing gene transcription. RAR-selective retinoids or their prodrugs include first-generation all-trans-retinoic acid (tretinoin) and its 13-cis isomer (isotretinoin), and second-generation acitretin and its ethyl ester (etretinate), both having an aromatic 2,3,6-trimethyl-4-methoxyphenyl terminus. Third-generation more aromatic analogs include ethyl ester tazarotene (Tazorac®) and adapalene (Differin®), both selective for RAR subtypes β and γ, and RARα-selective pipeline candidates Am80 and NRX 195183, which are in clinical trials. RAR and RXR transcriptional panagonist 9-cis-retinoic acid is next and is followed by RXR-selective bexarotene (Targretin™) and two RXR-selective clinical trial candidates, 9cUAB and NRX 194204. The fourth generation includes three compounds that may have applications in the treatment or prevention of cancer. While originally considered as retinoids, such as N-(4-hydroxyphenyl) retinamide, or as retinoid-related or derived, such as ST1926 (AHPC) and SHetA2, they subsequently were found to function independently of the RARs and RXRs in inhibiting cancer cell proliferation and inducing apoptosis.
Retinoids serve as physiologic and pharmacologic mediators of proliferation, differentiation and apoptosis in normal and malignant cell types. All-trans-retinoic acid (tRA), a natural metabolite of vitamin A, induces differentiation and subsequent apoptosis in several types of malignant cells with immature phenotypes. Clinically, tRA has been approved for the treatment of patients with acute promyelocytic leukemia. Several synthetic retinoids induce apoptosis without differentiation in a variety of malignant epithelial cells in vitro. The synthetic derivative, N-(4-hydroxyphenyl)retinamide (HPR), shows significant promise as a chemo-preventive and therapeutic anti-cancer agent in light of its minimal toxicity and broad activity in experimental cancer models representing common human malignancies. This paper reviews the role of retinoids as mediators of differentiation and apoptosis in malignant cells, and the impact this activity could have on clinical oncology.
N-(4-hydroxyphenyl)retinamide (4HPR-fenretinide) is a synthetic amide of all-trans retinoic acid (RA) which was synthesised in 1978 [1]. After the important observation that RA reduced mouse skin carcinogenesis, many analogs, now termed retinoids, were synthesised
and tested for their efficacy. The major problem that arose in testing retinoids as therapeutic agents was their toxicity.
The ratio of efficacy to toxicity, i.e. the “therapeutic index” became an important consideration in assessing the potential
clinical utility of different retinoids. Since the first in vivo studies on inhibition of experimentally induced carcinogenesis, 4HPR proved to be one of the most effective retinoids with
relatively low toxicity. It has then been proven to be active in the prevention and treatment of a variety of tumors in animals
and, by inducing apoptosis, it has shown in vitro growth inhibitory activity against a large number of human tumor cell lines from different tissue origins. For all these
reasons it was selected to enter into clinical trials. 4HPR was first tested for the treatment of skin disease and it is currently
under investigation as a chemopreventive and therapeutic agent.
Apoptosis, a genetically controlled process of cell death, plays an important role in embryogenesis, development, homeostasis, and in many diseases, such as cancer, certain neurodegenerative, and immune disorders. An increasing number of studies have demonstrated that retinoids, natural and synthetic analogs of vitamin A, have profound effects on the apoptotic process. The effect of retinoids on apoptosis is dependent on the chemical nature of the retinoid and the cell type. In most instances, retinoids induce apoptosis, while in a few cell types, retinoids exert an inhibitory effect. The mechanisms by which retinoids regulate apoptosis are just emerging during the last year. In most cases, activation of nuclear retinoid receptors, either retinoic acid (RA) receptors (RARs) or retinoid X receptors (RXRs) or both, is involved in retinoid-regulated apoptosis. The induction of apoptosis by N-(4-hy-droxyphenyl)retinamide (4-HPR) is not mediated by retinoid receptors and may involve the generation of reactive oxygen intermediates. Induction of apoptosis by the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) also occurs through a retinoid receptor-independent pathway. Induction of apoptosis may be one mechanism by which retinoids control morphogenesis during normal embryonic development. It may also be responsible for some of the teratogenic effects induced by retinoids at high, nonphysiological concentrations. In addition, the induction of apoptosis may be part of the mechanism by which retinoids inhibit cell proliferation in various carcinoma cells in vitro and suppress the development of tumors. Defects in retinoid signaling pathways have been shown to cause resistance of certain carcinoma cells to retinoid-induced growth-inhibition and apoptosis, and contribute to the malignant phenotype of cancer cells. Insight into the mechanisms by which retinoids regulate apoptosis may lead to more effective strategies in the prevention and therapy of disease as well as a better understanding of the regulatory role retinoids play in development.
To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.
Patients received 7 days of fenretinide: 2,475 mg/m(2)/d divided TID (<18 years) or 1,800 mg/m(2)/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only).
Sixty-two eligible patients, median age 5 years (range 0.6-19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17-506) for stratum 1 and 48 days (range 17-892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40-56%) at day 7 course 1. Toxicities were mild and reversible.
Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies.
Intravenous fenretinide (4-HPR), a cytotoxic retinoid, is being evaluated as part of a phase I clinical trial for patients with hematologic malignancies. In its orally administered form, it is also being evaluated for the treatment of various malignancies and geographic atrophy in subjects with the dry form of age-related macular degeneration. The authors report a case of acute large subretinal and intraretinal hemorrhage noted immediately after initiation of intravenous fenretinide therapy in a patient with hairy cell leukemia. This case highlights the importance of considering multilayered retinal hemorrhage as a possible side effect of fenretinide therapy, especially in patients with underlying hematologic abnormalities.
Alterations in retinoid signaling appear to be involved in the pathogenesis of small cell lung cancer (SCLC). Fenretinide [N-(4-hydroxyphenyl)retinamide], a synthetic retinoid, inhibits the growth of SCLC cells in vitro via the induction of apoptosis. Since these data suggested that SCLC is the adult solid tumor that is most susceptible to fenretinide, a trial to evaluate the clinical activity of fenretinide in patients with SCLC was considered the definitive test of its clinical potential in adult oncology.
Patients with progressive SCLC after one or two prior chemotherapy regimens and a performance status of 0-2 were eligible for the study. Patients with stable, treated brain metastases were eligible. Fenretinide 900 mg/m(2) twice daily was administered orally on days 1-7 of each 21-day cycle. Blood and saliva were collected pre-treatment and on day 7 of cycle 1 to measure fenretinide and retinol levels by high-pressure liquid chromatography (HPLC).
Nineteen patients were enrolled. Fifteen patients had one prior chemotherapy regimen and four patients had two prior regimens. The median time from diagnosis to enrollment was 10 months. A median of two cycles of fenretinide was administered. There were no objective responses, but four of 17 evaluable patients (24%) had stable disease after 2-17 cycles. The median time to treatment failure was 5.7 weeks overall, while the four patients with stable disease demonstrated treatment failure at 11, 13, 19, and 52 weeks. Median survival was 25 weeks, with one patient alive 22 months after the start of treatment. The 1-year survival rate was 29%. Toxicity included mild, reversible visual changes (haziness, altered night vision), grade 1-3 nausea/vomiting, and grade 1-2 diarrhea. The mean day 7 plasma fenretinide level was 2.90 +/- 1.66 μg/ml (7.40 +/- 4.25 muM; n = 14). The mean pre-treatment and day 7 plasma retinol levels were 0.47 +/- 0.16 μg/ml and 0.05 +/- 0.07 μg/ml (n = 8), respectively. The mean day 7 salivary fenretinide level was 0.08 +/- 0.18 μg/ml, with no correlation between salivary and plasma drug levels.
Fenretinide is well tolerated in patients with SCLC and stabilization of disease was noted in 24% of patients with this aggressive disease. However, after the first stage of enrollment, the response rate did not meet criteria to proceed with full trial accrual. Plasma concentrations of fenretinide that induce cytotoxicity in vitro in SCLC cell lines are clinically achievable, but there were no objective responses. Non-invasive drug monitoring using saliva underestimates systemic exposure.
Fenretinide is a synthetic retinoid that is cytotoxic to a variety of cancers. We conducted a phase II trial of oral fenretinide in patients with biochemically recurrent prostate cancer.
Eligible patients had histologically confirmed prostate cancer and a confirmed rising prostate-specific antigen (PSA) >or= 2 ng/mL following either radical prostatectomy and/or pelvic radiation therapy, without clinical or radiographic evidence of metastasis. The primary endpoint was PSA response, which was defined as a confirmed decrease by >or=50%, and >or=5 ng/mL, from the pretreatment value. Treatment comprised oral fenretinide 900 mg/m2 twice daily for 1 week, every 3 weeks, for 1 year.
After a median follow-up of 17.7 months, out of 23 patients, 7 (30%) patients had PSA stable disease (SD), 11 (48%) patients had PSA progression within 3 months, 4 patients had minimal increases over 3 months that did not qualify as SD or progression (17%), and one patient (4%) was not evaluable. Median time to PSA progression was 4.6 months (95% CI, 3.2-8.2 months). Observed grade 3 toxicities included fatigue, pain, hypermagnesemia, a rise in lipase, and nyctalopia.
Although well-tolerated, oral fenretinide did not meet prespecified PSA criteria for response in biochemically recurrent prostate cancer; however, 30% of patients had SD, which suggests modest single-agent clinical activity. The role of different formulations of fenretinide, which might allow for higher serum concentrations of the drug, is currently under investigation.
Lung cancer in the United States leads other cancers in both incidence and mortality (1). The severe morbidity of lung cancer and the discouraging 15% overall 5-year survival rate have not improved despite advancements in treatment modalities. Fur- thermore, the incidence of lung cancer among women is increas- ing. Therefore, novel approaches to control this dreadful disease are urgently needed. Biological agents such as differentiati on- inducing agents are being evaluated in clinical trials for their potential use for therapy and prevention of a variety of malig- nancies, including lung cancer (2,5). Among these novel agents are vitamin A analogues (retinoids) that modulate the growth and differentiation of a variety of normal and malignant celjs in vitro and in vivo (4). All-fra/w-retinoic acid (ATRA) was found to be effective in the treatment of patients with acute promyelocytic leukemia by inducing granulocytic differentia- tion of the leukemia cells in vivo (5). 13-c/s-Retinoic acid (13CRA) has demonstrated efficacy in suppressing premalig- nant oral lesions and in preventing the development of second primary cancers in the upper aerodigestive tract in patients with head and neck cancer (6). Likewise, retinyl palmitate was found
N-(4-Hydroxyphenyl)retinamide (4-HPR, Fenretinide) is a retinoid derivative with antineoplastic activity in various tumor types including prostate carcinoma. The mechanism of action of 4-HPR toxicity is unknown. 4-HPR induces apoptosis in leukemia- and lymphoma-derived cells, neuroblastoma, and small cell lung cancers. The present study was designed to investigate: (a) the mechanism of 4-HPR cytotoxicity in prostate cancer cells; and (b) correlate increased expression of transforming growth factor beta 1 (TGF beta 1) with induction of apoptosis. 4-HPR exposure to PC-3 cells in vitro was associated with apoptosis as evidenced by increased incidence of hypodiploid nuclei in propidium iodide fluorescence histograms and DNA fragmentation. An increase in the percentage of nuclei in the G1 phase of the cell cycle preceded induction of apoptosis. TGF beta 1-increased expression was noted in mRNA levels and in secretion of active TGF beta 1 into culture media. TGF beta 1 and TGF-beta receptor type II detected immunohistochemically were increased in 4-HPR-treated PC-3 cells. Furthermore, 4-HPR-induced cytotoxicity in PC-3 cells was abrogated by the addition of anti-TGF beta 1 antibody. In BT-20 cells, a 4-HPR-resistant breast carcinoma cell line, apoptosis was not observed after exposure to 4-HPR nor was TGF beta 1 expression enhanced in stained cells or in conditioned media. It is concluded that 4-HPR induces the expression of TGF beta 1 in association with the induction of apoptosis.
Lung carcinogenesis was induced in AKR mice using N-nitrosodiethylamine (NDEA). Tumors were detected in 46.8% of mice provided with 100 ppm NDEA in drinking water. The incidence of tumors was increased to 64.2% when the same carcinogenesis was promoted by phenobarbitone (PB). Lung tumor bearing mice showed no tumors in other organs. Characteristic features of these lung tumors are: (i) appearance of tumors within a short period of time i.e. less than 75 days; (ii) no increase in the number and size of tumors with the increase in dose and duration of treatment of carcinogen; (iii) the same histological type was maintained in more than 80% of tumors. Animals that received treatment for 75-125 days showed no significant advancement in the stage of carcinogenesis in comparison to the 50-75 days treatment period. Moreover, mice which received treatment for 125-150 days, did not have any neoplastic lesions in lungs, but they consisted of liver tumors generally. Expression of oncoproteins, c-myc and c-jun, was detected in all lung tumors but the expression of c-myc protein was more than that of c-jun and both of these oncoproteins were enhanced by the promoter, PB. Highest level of expression of c-myc and c-jun was detected within the period of 50-75 days, whereafter it was decreased significantly within the period of 75-125 days and 125-150 days of treatment. Thus, the results indicate that c-myc/c-jun might be involved in the development of lung cancer in AKR mice, but may not have any role in the maintenance of the malignant phenotype of lungs.
Interest has been increasingly focused on all-trans-retinoic acid (tRA) and 13-cis-retinoic acid (13cRA) in cancer chemoprevention and treatment. We have examined the in vitro effects of these 2 retinoic acids (RAs) on human breast-cancer cell lines MCF-7 and ZR-75.1 (both estrogen-receptor-positive, ER+) and MDA-MB-231 (estrogen-receptor-negative, ER-), in terms of inhibition of proliferation and induction of apoptosis. Both retinoic acids exerted an evident dose-dependent growth inhibition, although in the ER- cell line the anti-proliferative effect was obtained only with the highest concentration used; the anti-proliferative activity of tRA was more evident than 13cRA on all 3 tested cell lines. tRA and 13cRA induced apoptosis in MCF-7 and MDA-MB-231 cell lines, but not in ZR-75.1. The apoptotic phenomenon was clearly time-dependent, and in our experience it was not related to the arrest in a specific phase of cell cycle. After treatment with RAs the levels of bcl-2 were reduced in MCF-7, while in ZR-75.1 and in MDA-MB-231 no treatment-related modifications were observed. An analysis of estrogen-receptor status, used as a marker of differentiation, demonstrated that after treatment with RAs the levels of estrogen receptor (ER) decreased in ZR-75.1 only. Our study indicates that the anti-proliferative effects of RAs are sustained by induction of apoptosis in MCF-7 and MDA-MB-231 cells, while in ZR-75.1 cells an induction of differentiation without apoptosis was the prevalent mechanism of growth inhibition. Our results encourage further studies on in vivo effects of these retinoids in breast cancer.
The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 microM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 microM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 microM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 microM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.
We have used the cDNA differential display technique to isolate genes regulated by the synthetic retinoid N-(4-hydroxyphenyl)-all-trans-retinamide (HPR), a cancer chemopreventive agent in vivo and a powerful inducer of apoptotic cell death in vitro. Here we report the identification of a novel gene, the expression of which is markedly up-regulated in tumor cells after treatment for 30-60 min with HPR. The full-length cDNA of this gene, determined by screening of a human placenta cDNA, is 3.5 kb long and contains an open reading frame of 2037 nt. The gene is > 90% homologous to the mouse KIF2, a gene belonging to the family of kinesin-related motor proteins, and we therefore named it HK2 (human kinesin 2). A shorter form of the HK2 mRNA (HK2s), containing a 57-nt deletion in the open reading frame, has also been detected. Northern analysis revealed that HK2 is widely expressed among hemopoietic and nonhemopoietic cell lines and tissues. By the use of radiation hybrids, HK2 has been localized to chromosome 5q12-q13. Kinesins constitute a superfamily of motor proteins that use energy liberated from ATP hydrolysis to move cargo along microtubules and are implicated in mechanisms of mitosis or meiosis. The role of HK2 in the growth-inhibitory and apoptotic responses elicited by HPR remains to be established.
Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.
Natural and synthetic vitamin A metabolites and analogs (retinoids) were found to suppress head and neck and lung carcinogenesis in animal models and inhibit carcinogenesis in individuals with premalignant lesions and a high risk to develop cancer of the aerodigestive tract. Likewise, retinoids prevent the development of second primary cancers in head and neck and lung cancer patients who had been treated for the first primary. These effects are thought to result from changes in the expression of genes that regulate cell growth and differentiation. Most of the effects of retinoids on gene expression are mediated by nuclear retinoic acid receptors RARs (alpha, beta, and gamma) and retinoid X receptors (RXR alpha, beta, and gamma), which function as retinoid-activated transcription factors. Like vitamin A deficiency, alterations in receptor expression or function could interfere with the retinoid signaling pathway and thereby enhance cancer development even in vitamin A sufficient individuals. We found that the expression of RAR beta was suppressed in more than 50% of oral and lung premalignant lesions in individuals without cancer (e.g., oral leukoplakia and squamous metaplasia), in dysplastic lesions adjacent to cancer, and in malignant oral and lung carcinomas. The expression of the other receptors was not different among normal, dysplastic, and malignant oral tissues. However, the expression of RAR gamma and RXR beta was somewhat decreased in lung cancers. These results show that RAR beta expression is lost at early stages of carcinogenesis in the aerodigestive tract and support the hypothesis that the loss of RAR beta expression may facilitate the development of some of these cancers.
We have used conformationally restricted retinoids to investigate the role of individual RAR subtypes and RXR in mediating the growth response of ovarian tumor cells to retinoids. Our results show that treatment of all-trans-RA-sensitive CAOV-3 cells with retinoids that bind and activate a single RAR or RXR led to a partial inhibition of growth. Treatment of all-trans-RA- resistant SKOV-3 cells did not alter growth. Maximum inhibition of growth, comparable to that observed following treatment with natural retinoids such as all-trans-RA and 9-cis-RA, was obtained only following treatment with a combination of an RAR-selective compound and an RXR-selective one. These results suggest that activation of both RAR and RXR classes is required in order to obtain maximum inhibition of ovarian tumor cell growth by retinoids. In addition, one compound, AHPN, was found to inhibit both RA-sensitive CAOV-3 and RA-resistant SKOV-3 cells. Further study of the effects of this retinoid showed that AHPN acts through an apoptotic pathway. Taken together, our results suggest that retinoids may serve as effective anti-proliferative agents in the treatment of ovarian cancer.
Induction of apoptosis in MCF-7 breast carcinoma cell line by various retinoids was measured by cytofluorimetry and DNA fragmentation assay. Retinoids with marked or high selectivity for RAR alpha, RAR beta, RAR gamma or RXR alpha were tested. All these retinoids were capable of inducing apoptosis, in a dose- and time-dependent way. MCF-7 cell line expressed RAR alpha, RAR gamma and RXRs, but not RAR beta. Compared to untreated MCF-7 cells, after 2 days of incubation with each of the selective retinoids, a substantial increase in apoptotic cells was observed, even at the lowest concentration of 10(-8) M. Among the various analysed selective retinoids only slight differences were observed. All-trans retinoic acid and 13-cis retinoic acid induced apoptosis only after 6 days and 9-cis-retinoic acid after 4 days of incubation. Since all receptor selective retinoids substantially inducedapoptosis, it may be concluded that RAR alpha, RAR gamma and RXR alpha are able to mediate programmed cell death in the tested tumor cell line. Highly selective retinoid receptor agonists and antagonists may be useful for clarifying the function of retinoid receptors and for further progress in the field of cancer prevention and therapy by retinoids.
It is increasingly clear that apoptosis plays a crucial role in the promotional phase of cancer development. Initiated pre-neoplastic clones in rat liver experience a high rate of apoptosis, and this rate has an important impact on the survival and growth of these clones. Suppression of apoptosis appears to be a universal property of cancer promoters, suggesting conversely that agents which inhibit cancer induction during the promotional phase increase the rate of apoptosis in initiated cells. Modulation of apoptosis is a likely explanation for recent striking evidence that use of calcium channel blockers substantially increases, whereas supplemental selenium substantially decreases, human cancer incidence. Non-genotoxic measures which are likely to upregulate apoptosis in pre-neoplastic/neoplastic cells--and thus may be useful in prevention and/or therapy--include selenium, retinoids/carotenoids, green tea polyphenols, caloric restriction, downregulation of IGF-I activity, high-dose tamoxifen and other protein kinase C antagonists, withdrawal or blockade of trophic hormones, isoflavones, limonene, vitamin D and cholecalciferol analogs, dietary fiber/sodium butyrate, hyperthermia, benzaldehyde derivatives, and creatine.
Experimental studies of N-(4-hydroxyphenyl)retinamide, a potential cancer chemopreventive agent, have primarily involved breast cancer and neuroblastoma cell populations together with an investigation of myeloid leukemia cells and have principally been concerned with the induction of apoptosis. This investigation of N-(4-hydroxyphenyl)retinamide-induced apoptosis using T-cell-derived human lymphoblastoid lines extends these studies by indicating distinctive features associated with this drug. The induction of apoptosis is restricted to a limited concentration range, which, if exceeded, results in cell death by necrosis. While morphological changes typical of apoptosis induced by many agents are readily demonstrable after treatment of lymphoblastoid cells with 3 microM N-(4-hydroxyphenyl)retinamide, distinctive features evident using the retinoid include the absence of cell cycle arrest along with the mode and pattern of DNA breakage. Analysis by conventional gel electrophoresis indicated that internucleosomal fragmentation of DNA was an unreliable indicator of apoptosis. On the other hand, higher order DNA breakage was consistently detected during drug-induced apoptosis, but not as a result of treatment causing necrosis.
The dysregulation of both myc gene expression and retinoid signaling pathways commonly occurs in small cell lung carcinoma (SCLC). Because preclinical data showed that all-trans-retinoic acid (RA) inhibited SCLC growth, altered myc expression, and blocked transition to a treatment-resistant phenotype, a Phase II trial was designed to determine the effects of the combination of RA, cisplatin, and etoposide in patients with SCLC.
Patients with untreated, extensive stage SCLC were treated with up to 8 cycles of cisplatin, 60 mg/m2, intravenously (i.v.) on Day 1 and etoposide, 120 mg/m2, i.v. on Days 1-3 in addition to up to 1 year of oral RA, 150 mg/m2/day.
Of 22 assessable patients 1 had a complete response and 9 had a partial response, for an overall response rate of 45% (95% confidence interval, 24-68%). The median survival was 10.9 months and the 1-year survival was 41%. The median duration of chemotherapy was 6 cycles and the median duration of RA treatment was 2.8 months. Thirteen patients discontinued RA prematurely due to toxicity and only 4 responders were receiving RA at the time of recurrence. Toxicity-limiting RA treatment mainly was comprised of mucocutaneous changes and headaches.
RA at a dose of 150 mg/m2/day was tolerated poorly in combination with cisplatin plus etoposide, leading to early discontinuation of RA in the majority of patients. The hematologic toxicity, response rate, and survival were similar to those associated with cisplatin and etoposide in prior trials. Further studies with more active and less toxic agents will be required to determine the role of retinoids in the treatment of SCLC.
The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
Concurrent with a phase-II trial of 4HPR in patients with various cancers, we studied the plasma pharmacokinetics of both 4HPR and its major metabolite 4MPR as well as the effect of 4HPR administration on plasma retinol concentrations using a simple, specific and sensitive HPLC procedure. Initial estimates of plasma pharmacokinetic parameters after oral administration of 4HPR (300 mg/day) [corrected] in 3 cancer patients were the following: 4HPR, t beta 1/2 = 13.7 hr, AUC = 3.49 micrograms.hr/ml, CL = 56.57 L/hr/m2; 4MPR, t beta 1/2 = 23.0 hr, AUC = 1.15 micrograms.hr/ml, CL = 239.29 L/hr/m2. We also found that oral administration of 4HPR resulted in a rapid, profound and significant reduction in plasma retinol concentrations. The mean plasma retinol concentrations for 9 patients decreased 60% from baseline to below 200 ng/ml within 1-2 weeks of 4HPR dosing initiation. In addition, there was a concurrent, significant reduction in plasma retinol-binding protein levels in these patients. The mechanism whereby 4HPR reduces plasma retinol levels in vivo has not been determined. The addition of 4HPR to pooled human plasma at 37 degrees C in vitro did not reduce endogenous retinol levels, suggesting no direct chemical interaction between these 2 retinoids.
Fenretinide, N-(4-hydroxyphenyl)retinamide (HPR), is a synthetic retinoid which has been proven effective in inducing cell differentiation and in inhibiting carcinogen induced mammary tumors in rodents. Because of its efficacy and low toxicity in animals, HPR has been proposed for chemopreventive evaluation in humans. Thus, a randomized trial has been conducted to select a dose which can be administered over a lengthy period of time and with acceptable toxicity. The retinoid was administered orally to patients already operated on for breast cancer in daily doses of 100, 200 and 300 mg for 6 months and subsequently at 200 mg for another 6 months. No acute toxicity was found. Dermatological toxicity was minimal and no liver function abnormalities were observed. Nausea and headaches were infrequent and always mild. Menstrual irregularities were recorded with similar frequency in the treatment and placebo groups and appeared to be more age related than drug dependent. After 6 months of treatment one of 25 patients taking 300 mg HPR daily experienced impaired night vision, confirmed by the electroretinogram, and resolved by interruption of treatment. Because the 300 mg daily dose is possibly associated with impaired dark adaptation, the recommended dose for chemoprevention trials of HPR is 200 mg per day.
Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes
3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations
in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined
with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors,
4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but
were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and
75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in
90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary
carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.
We studied the effects of 13-cis-retinoic acid (13cRA) and 4-hydroxyphenyl-all-trans-retinamide (4HPR) on the proliferation of clonogenic human tumor colony-forming units (TCFU) from patient biopsies in soft agar. Continuous exposure (5 X 10(-6)M) to 13cRA and 4HPR reduced colony formation to less than 50% of control in 49% and 31% of cases, respectively. This suggests that these retinoids have antiproliferative activity against TCFU. Continuous exposure to 13cRA reduced the number of TCFU to 30% of control in 22.6% of cases while 4HPR reduced colony formation to less than 30% of control in only 10% of patients tested. Prospective in vitro/in vivo correlations for 13cRA indicated that clinical resistance was correctly predicted in 4 of 4 cases treated while clinical response was predicted in 2 of 2 cases (one minor and one partial response). These data suggest that retinoids may have modest antitumor effects and that inhibition of TCFU growth in vitro may be used to screen for such activity.
ALTHOUGH laboratory workers have known for decades that tumor incidence in animals can be affected by nutritional manipulation,1 the possibility that diet may be important in the cause and prevention of cancer in human beings has received major attention only recently. Since knowledge in this area is developing rapidly, we will outline processes by which hypotheses relating dietary factors to cancer are formulated and tested. In addition, we will discuss the limited available data that are relevant to some hypotheses that have attracted particular interest. An exhaustive review of the literature is contained in Diet. Nutrition, and Cancer, published . . .
The American Cancer Society's Department of Epidemiology and Statistics reports its 29th annual compilation of cancer incidence, survival and mortality data for the United States and around the world.
The predominant mode of either spontaneous or drug-induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis-associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twenty-two tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymph node squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors. Extremely high variability in the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs.(ABSTRACT TRUNCATED AT 250 WORDS)