Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression

We previously reported that fenretinide (4HPR) is effective against a human ovarian carcinoma xenografted in nude mice. The effects of 4HPR on ovarian tumors have been further studied in in vitro ovarian carcinoma cell lines A2780, IGROV-I, SW626 and OVCA432. A2780 was the most sensitive line: 50% growth inhibition was obtained after 3 days of exposure to 1 μM 4HPR, a pharmacologically achievable concentration, whereas approx. 10 μM 4HPR gave a similar inhibition in the other cell lines. All-trans retinoic acid (RA), at doses up to 10 μM, did not inhibit cell proliferation. Gel electrophoresis of DNA from either detached or attached A2780 cells treated with 4HPR revealed DNA ladders in detached cells. Apoptosis was also evidenced in detached 4HPR-treated cells by flow cytometry and microscopic observation. The difference in cell line sensitivity to the anti-proliferative effect of 4HPR was not related to drug uptake or efflux. Only A2780 cells, the most sensitive to 4HPR, expressed constitutive levels of RARβ; moreover, the levels of RARα and RARγ expression in these cells were higher than in the other cell lines. In A2780 cells, the association of an IC₂₀ of 4HPR to cisplatin resulted in a strong potentiation of the anti-proliferative effect. These data show (i) that 4HPR, in contrast to RA, has an anti-proliferative effect in human ovarian carcinoma cells which is related to induction of apoptosis and (ii) that among the tested lines, the most responsive to the drug expressed RARβ and the highest levels of RARα and RARγ. The results also suggest that 4HPR can potentiate the effects of cisplatin in ovarian carcinoma. © 1996 Wiley-Liss, Inc.     

N-(4-hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.     

Inhibition of cancer cell growth by all- trans retinoic acid and its analog N-(4-hydroxyphenyl) retinamide: a possible mechanism of action via regulation of retinoid receptors expression

In order to better understand the mechanisms that underlie the antiproliferative effect of retinoids, we have examined the response of human carcinoma cell lines to all-trans retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4HPR) in terms of cell growth, apoptosis and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mRNA. GLC82 (lung adenocarcinoma), BGC823 (stomach adenocarcinoma) and EC109 (esophageal squamous carcinoma) cells were treated with 10 μM of RA or 4HPR for various length of time and analyzed. The results show that growth inhibition by RA and 4HPR in GLC82 and BGC823 cells correlates with the induction of RARβ2 gene, whereas RA resistance in EC109 cells parallels loss of RARβ2 induction. Exogenous RARβ2 expression did not restore RA responsiveness in EC109 cells, but potentiated 4HPR-induced growth inhibition, suggesting that 4HPR acts at least in part via the RARβ receptor. We speculate that the loss of RARβ2 inducibility in EC109 cells may be due to an unknown repressor. Int. J. Cancer 78:248–254, 1998.© 1998 Wiley-Liss, Inc.     

Fenretinide inhibits myeloma cell growth,osteoclastogenesis and osteoclast viability

Fenretinide (4HPR), a nontoxic analog of ATRA, has been investigated in various malignancies but not in multiple myeloma (MM), a plasma cell malignancy associated with induction of osteolytic bone disease. Here we show that 4HPR induces apoptosis through increased level of ROS and activation of caspase-8, 9 and 3, and inhibits growth of several MM cell lines in a dose-dependent manner. Serum or co-culture with the supportive osteoclasts partially protects MM cells from 4HPR-induced growth inhibition. Sphingosine-1 phosphate (S1P) significantly protects MM cells from 4HPR-induced apoptosis suggesting that as in other malignancies, this drug up-regulates ceramide in MM cells. 4HPR has no toxic effects on non-malignant cells such as blood mononucleated cells, mesenchymal stem cells and osteoblasts, but markedly reduces viability of endothelial cells and mature osteoclasts and inhibits differentiation of osteoclasts and MM-induced tube formation. 4HPR is a potential anti-MM agent, affecting MM cells and MM-induced bone disease and angiogenesis.     

Reactive oxygen species mediate N-(4-hydroxyphenyl)retinamide-induced cell death in malignant T cells and are inhibited by the HTLV-I oncoprotein Tax.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth of many human tumor cells, including those resistant to natural retinoids. HPR is an effective chemopreventive agent for prostate, cervix, breast, bladder, skin and lung cancers, and has shown promise for the treatment of neuroblastomas. We have previously shown that HPR inhibits proliferation and induces apoptosis of human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia (ATL) and HTLV-I-negative malignant T cells, whereas no effect is observed on normal lymphocytes. In this report, we identified HPR-induced reactive oxygen species (ROS) generation as the key mediator of cell cycle arrest and apoptosis of malignant T cells. HPR treatment of HTLV-I-negative malignant T cells was associated with a rapid and progressive ROS accumulation. Pre-treatment with the antioxidants vitamin C and dithiothreitol inhibited ROS generation, prevented HPR-induced ceramide accumulation, cell cycle arrest, cytochrome c release, caspase-activation and apoptosis. Therefore, anti-oxidants protected malignant T cells from HPR-induced growth inhibition. The expression of the HTLV-I oncoprotein Tax abrogated HPR-induced ROS accumulation in HTLV-I-infected cells, which explains their lower sensitivity to HPR. Defining the mechanism of free radical induction by HPR may support a potential therapeutic role for this synthetic retinoid in ATL and HTLV-I-negative T-cell lymphomas.     

Mediation of N-(4-hydoxyphenyl)retinamide-induced apoptosis in human cancer cells by different mechanisms.

The induction of apoptosis by the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been documented in vitro in various cancer types. A role for reactive oxygen species (ROS) in apoptosis induced by 4HPR in some cancer cells has been demonstrated recently. We studied five different human head and neck and five lung cancer cell lines to determine whether the ROS play a general role in 4HPR-induced apoptosis. We found that 4HPR induced apoptosis in all of the cell lines; however, this effect was blocked by antioxidants in only 2 of the 10 cell lines. 4HPR induced a greater than 4-fold increase in the generation of intracellular ROS in these two cell lines compared with a much lower effect in other cell lines. Furthermore, these two cell lines were most sensitive to the induction of apoptosis by 4HPR. The level of the cellular antioxidant thiol and superoxide dismutase activity were relatively lower in cells, which responded to 4HPR with a high level of ROS generation. These results indicate that although ROS can mediate 4HPR-induced apoptosis in some cells, which may have a low endogenous cellular antioxidant levels, other mechanisms exist for 4HPR-induced apoptosis. One such mechanism may involve retinoic acid receptors (RARs) because an RAR antagonist was able to block partially 4HPR-induced apoptosis. In conclusion, 4HPR-induced apoptosis involves at least three different mechanisms, which are complex and can overlap in the same cell line: (a) one mechanism involving 4HPR-induced ROS; (b) one involving RARs; and (c) at least one that does not involve ROS or RARs and remains unclear.     

Increased level of the p67phox subunit of NADPH oxidase by 4HPR in head and neck squamous carcinoma cells

N-(4-hydroxyphenyl)retinamide (4HPR) is a synthetic retinoid that has shown efficacy in cancer chemoprevention and therapy possibly due to its ability to induce growth inhibition and apoptosis in cancer cells. Reactive oxygen species (ROS) have been implicated in mediating 4HPR-induced apoptosis in various cancer cells, including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR increased the level of p67phox, which is a subunit of the NADPH oxidase complex that is involved in the generation of ROS. The increase in the level of p67phox protein may be a downstream effect of the activation of c-Jun N-terminal kinase (JNK) induced by 4HPR. Suppression of endogenous and 4HPR-induced levels of p67phox using small interfering RNA did not result in a change in ROS generation or apoptosis. These results suggest that p67phox is not a critical component or a limiting factor in the 4HPR-induced apoptosis pathway in HNSCC cells.     

N-(4-hydroxyphenyl)retinamide induces growth arrest and apoptosis in HTLV-I-transformed cells.

N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid that inhibits growth and induces apoptosis in many human cell lines. We explored the effects of HPR on human T-cell lymphotropic virus type I (HTLV-I)-positive and HTLV-I-negative malignant T-cell lines, most of which are resistant to all-trans retinoic acid. Clinically achievable concentrations of HPR caused a dramatic inhibition of cell proliferation, G(0)/G(1) arrest, and massive apoptosis in all tested malignant T cells, while no effect was observed on resting or activated normal lymphocytes. Interestingly, HTLV-I-negative cell lines were significantly more sensitive to HPR compared to HTLV-I-positive and Tax-transfected cells. In HTLV-I-negative cells only, HPR-induced apoptosis was associated with ceramide accumulation, sharp decrease in mitochondrial membrane potential, and activation of caspases 8, 9 and 3, and could be partially reverted by the caspase inhibitor z-VAD suggesting that Tax protects infected cells from ceramide accumulation and caspase-mediated apoptosis. In HTLV-I-positive cells, HPR treatment rapidly induced proteasomal-mediated degradation of p21, downregulated cyclin D(1), and upregulated bax protein levels. These findings support a potential therapeutic role for HPR in both HTLV-I-associated adult T-cell leukemia/lymphoma (ATL) and HTLV-I-negative peripheral T-cell lymphomas.     

Mitochondrial permeability transition is a central coordinating event in N-(4-hydroxyphenyl)retinamide-induced apoptosis.

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on the process of carcinogenesis are not fully understood and may result from its ability to induce apoptosis in transformed cells. This study investigated the apoptotic properties of 4HPR in four human cutaneous squamous cell carcinoma cell lines. Apoptosis induction, detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling method, occurred in a dose- and time-dependent fashion after treatment with 4HPR. 4HPR promoted reactive oxygen species (ROS) determined by oxidation of 2',7'-dichlorofluorescin. 4HPR-induced ROS, and apoptosis could be inhibited by L-ascorbic acid. Rhodamine 123 retention revealed that 4HPR treatment promoted a gradual dissipation of mitochondrial inner transmembrane potential, and this could be inhibited by L-ascorbic acid, implying that mitochondrial permeability transition was involved in apoptosis induction. Cyclosporin A and bongkrekic acid inhibited dissipation of mitochondrial inner transmembrane potential, ROS production, and DNA fragmentation after exposure to 4HPR, demonstrating that mitochondrial permeability transition was a central coordinating feature of 4HPR-induced apoptosis.     

N-(4-hydroxyphenyl) retinamide is cytotoxic to melanoma cells in vitro through induction of programmed cell death

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 microM, while IC90 values were between 10 and 45 microM. 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.     

Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-hydroxyphenyl)retinamide-induced apoptosis in cervical carcinoma cells

N-(4-Hydroxyphenyl)retinamide (4HPR) is currently used in cancer prevention and therapy trials. It is thought that its effects result from induction of apoptosis. 4HPR-induced apoptosis in human cervical carcinoma C33A cells involves enhanced generation of reactive oxygen species (ROS). In this study we explored the mechanism by which 4HPR increases ROS and induces apoptosis in these cells. 4HPR induced cytochrome c release from mitochondria to cytoplasm, activated caspase-3, and caused a membrane permeability transition (MPT). All these 4HPR's effects, as well as the induction of apoptosis, were inhibited by antioxidants, which decrease ROS. Thenoyltrifluoroacetone, a mitochondrial respiratory chain (MRC) complex II inhibitor, and carbonylcyanide m-chlorophenyl hydrazone, which uncouples electron transfer and ATP synthesis and inhibits ROS generation by MRC, inhibited 4HPR-induced ROS generation very effectively. Rotenone, an MRC complex I inhibitor was less effective and azide, an MRC complex IV inhibitor, exhibited a marginal effect. In contrast, antimycin A, an MRC complex III inhibitor, enhanced 4HPR-induced ROS generation. These findings suggest that 4HPR enhances ROS generation by affecting a target between complex II and complex III, presumably coenzyme Q. This effect is followed by release of cytochrome c, increased caspase-3 activity, induction of MPT and eventual DNA fragmentation and cell death.     

Role of antioxidants and intracellular free radicals in retinamide- induced cell death

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.      

Inhibition of N-(4-hydroxyphenyl)retinamide-induced apoptosis in breast cancer cells by galectin-3

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in a variety of human cancer cells including breast carcinoma and this property may be important for its chemopreventive and therapeutic effects. Resistance to 4HPR has been described, however, the molecular mechanisms underlying sensitivity or resistance to this retinoid are not clear. Recently, it has been shown that the carbohydrate-binding protein galectin-3, which has been implicated in tumor progression, contains the anti-death motif NWGR present in the anti-apoptotic protein Bcl-2. To determine whether galectin-3 expression can abrogate the effect of 4HPR, we tested the effects of 4HPR on apoptosis of cell clones derived from the galectin-3 deficient human BT549 breast carcinoma cells after transfection with either wild type galectin-3 (BT549Gal-3Wt), galectin-3 inactivated by a point mutation in the NWGR motif (BT549Gal-3Mu), or empty vector control (BT549Vec). Both BT549Vec and BT549Gal-3Mu cells showed a marked decrease in survival after treatment with 4HPR principally due to induction of apoptosis. 4HPR-induced apoptosis in these cells was associated with stimulation of reactive oxygen species generation, decreased levels of Bcl-2 protein, release of cytochrome c into the cytosol, increased caspase-3 activity, and poly(ADP-ribose) polymerase cleavage. In contrast, 4HPR failed to exert any of these effects in the BT549Gal-3Wt cells. The demonstration that galectin-3 suppresses 4HPR-induced apoptosis in human breast carcinoma cells suggests that the increased expression of galectin-3 during cancer progression may be associated with 4HPR resistance.     

N-(4-hydroxyphenyl)retinamide induces apoptosis in human leukemia hl-60 cells and mediates vimentin down-regulation

N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid with anticancer properties and lower toxicity than all-trans retinoic acid (RA). We have studied the effects of HPR on apoptosis and differentiation in the human promyelocytic leukemia HL-60 cell line. In addition, we have tested the hypothesis that vimentin expression after HPR and RA, taken as indirect evidence of the mechanisms of action of the two retinoids, may be different. Quantitative evaluation of the percentage of apoptotic cells was carried out on a cell by cell basis by the flow cytometric DNA-content in situ-terminal-deoxynucleotydil-transferase (TdT assay). HPR was found to clearly induce apoptosis, while RA: instead, induced differentiation without apoptosis. These data confirm previous observations. Vimentin protein content was evaluated by flow cytometry with use of monoclonal antibodies simultaneously with DNA content. We found that HPR treated apoptotic cells were characterized by negative vimentin expression, while the HPR treated non apoptotic cells had about the same level of vimentin as the RA treated cells. These latter findings suggest that HPR may induce a functional effect (apoptosis) by a mechanism of action different from that of RA. Further work is necessary to clarify this finding.     

Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity

The synthetic retinoid N-(4-hydroxphenyl) retinamide (4HPR) has manifold actions, which may contribute to its chemopreventive effects on breast cancer cell growth and progression. A role for ceramide as a stress-response signal is investigated here during the cytotoxic action of 4HPR in MCF-7 cells. N-(4-hydroxphenyl) retinamide induced a dose-dependent decline in cell growth and survival associated with a maximal 10-fold increase in ceramide production at 10 microM. N-(4-hydroxphenyl) retinamide exhibited a greater potency than all-trans retinoic acid (ATRA) on growth inhibition and ceramide production. The synthetic peroxisome proliferator-activated receptors agonist troglitazone (TGZ), but not the native ligand 15-deoxy-delta 12,14-prostaglandin J2, abrogated both these actions of 4HPR but not that of ATRA. The antioxidant N-acetylcysteine mimicked the abrogative effect of TGZ on 4HPR action, while the exogenous oxidant H2O2 also stimulated ceramide production. The inhibitors of de novo ceramide synthesis, fumonisin B1 and myriocin, blocked the ceramide response to 4HPR and partially reversed the apoptotic response, but did not prevent the overall decline in cell survival. The pancaspase inhibitor Z-VAD fmk reduced the decrease in cell survival caused by 4HPR, but did not affect the ceramide response. These findings describe a novel redox-sensitive elevation of ceramide levels associated with the cytotoxic response of breast cancer cells to 4HPR. However, a major mediatory role for this sphingolipid in this context remains equivocal.     

Sphingosine and its methylated derivative N,N-dimethylsphingosine (DMS) induce apoptosis in a variety of human cancer cell lines

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 μM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylysphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C₂₋, C₃₋ or C₈₋ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C₂Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway. © 1996 Wiley-Liss, Inc.     

Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-hydroxyphenyl)retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells

N-(4-hydroxyphenyl)retinamide (4HPR), which has shown efficacy in cancer chemopreventionand therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous cell carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the upregulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by cotreatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.     

Cell cycle-independent induction of apoptosis by the anti-tumor drug flavopiridol in endothelial cells

The anti-tumor drug Flavopiridol is a potent inhibitor of cyclin-dependent kinases (cdks). As a consequence, Flavopiridol-treated cells arrest in both G₁ and G₂, but Flavopiridol has also been shown to be cytotoxic for some tumor cell lines. The underlying molecular events are, however, unclear. We now show that Flavopiridol induces apoptosis in human umbilical vein endothelial cells (HUVECs), as judged by the occurrence of classical apoptotic markers, including chromatin condensation, internucleosomal cleavage, DNA fragmentation (TUNEL assay), annexin V binding and poly(ADP-ribose) polymerase (PARP)-cleavage. Such induction of apoptosis occurs with equal efficiency in both proliferating and G₀/G₁-arrested cells. Because growth-arrested HUVECs lack cdk2 activity and contain high levels of the cdk inhibitor p27, our observations suggest that cell cycle regulated cdks may not be the only critical target for Flavopiridol-induced apoptosis. Surprisingly, A549 lung carcinoma cells were clearly dependent on cell proliferation for the induction of cell death, pointing to cell type-related differences in the mechanism of Flavopiridol action. Int. J. Cancer 77:146–152, 1998.© 1998 Wiley-Liss, Inc.     

Activity of fenretinide plus chemotherapeutic agents in small-cell lung cancer cell lines

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide, 4HPR], a synthetic retinoid, is a potent inducer of apoptosis in small-cell lung cancer (SCLC) cell lines that may act through the generation of reactive oxygen species, suggesting that it may enhance the activity of other cytotoxic agents. In light of 4HPR's clinical potential and potent activity against SCLC cells, we evaluated the in vitro activity of 4HPR in combination with cisplatin, etoposide or paclitaxel. Methods: The growth-inhibitory activities of single-agent 4HPR, cisplatin, etoposide or paclitaxel, and combinations of 4HPR and individual chemotherapeutic agents, were evaluated using an MTT assay in two SCLC cell lines. Each two-drug combination was studied over a range of concentrations at a fixed ratio corresponding to the ratio of the IC₅₀ values of the individual agents. Data were analyzed by median-effect analysis as previously applied to drug combination studies. Results: All four agents inhibited growth in a dose-dependent manner in the NCI-H82 and NCI-H446 SCLC cell lines. At clinically reported drug concentrations that resulted in over 50% growth inhibition, the activities of the combinations 4HPR and cisplatin and 4HPR and etoposide were more than additive in both cell lines, and the activity of 4HPR plus paclitaxel was more than additive in NCI-H446 cells. Conclusion: 4HPR's potent single-agent activity, minimal toxicity, and potential synergy with standard cytotoxic drugs will allow for the development of promising investigational regimens for the treatment of patients with SCLC. Received: 17 February 1998 / Accepted: 20 May 1998