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Since response to platinum-based therapy in non-small-cell lung cancer (NSCLC) is poor, the present study was designed to rationally identify novel drug combinations in cell models including the A549 cell line and the cisplatin-resistant subline A549/Pt, characterized by reduced sensitivity to cisplatin-induced apoptosis and by upregulation of efflux transporters of the ATP binding cassette (ABC) superfamily. Given the molecular features of these cells, we focused on compounds triggering apoptosis through different mechanisms, such as the mitochondria-targeting drug arsenic trioxide and the phenanthridine analog sanguinarine, which induce apoptosis through the extrinsic pathway. Sanguinarine, not recognized by ABC transporters, could overcome cisplatin resistance and, when used in combination with arsenic trioxide, was synergistic in A549 and A549/Pt cells. The arsenic trioxide/sanguinarine cotreatment upregulated genes implicated in apoptosis activation through the extrinsic pathway. Drug combination experiments indicated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment improved arsenic trioxide/sanguinarine efficacy, a feature associated with a striking apoptosis induction, particularly in the cisplatin-resistant variant. Thus, a synergistic interaction between sanguinarine and arsenic trioxide could be obtained independent of relative cell sensitivity to arsenic trioxide, and an enhanced apoptosis induction could be achieved in combination with TRAIL through modulation of the extrinsic apoptotic pathway. Antitumor activity studies supported the interest of drug combinations including TRAIL in NSCLC, indicating that drug-resistant NSCLC cells can efficiently be killed by the combination of proapoptotic agents. Our results suggest that the molecular changes occurring in treated cells may be exploited to rationally hit surviving cells.

Emerging evidence suggests that Ret oncoproteins expressed in medullary thyroid cancer (MTC) might evade the pro-apoptotic function of the dependence receptor proto-Ret by directly impacting the apoptosis machinery. Identification of the molecular determinants of the interplay between Ret signaling and apoptosis might provide a relevant contribution to the optimization of Ret-targeted therapies. Here, we describe the cross-talk between Ret-M918T oncogenic mutant responsible for type 2B multiple endocrine syndrome (MEN2B), and components of death receptor-mediated extrinsic apoptosis pathway. In the human MEN2B-type MTC cell line MZ-CRC-1 expressing Ret-M918T, Ret was found associated with Fap-1, known as inhibitor of the CD95 death receptor trafficking to the cell membrane, and with procaspase-8, the initiator pro-form caspase in the extrinsic apoptosis pathway. Cell treatment with the anti-tumor Ret kinase inhibitor RPI-1 inhibited tyrosine phosphorylation of procaspase-8, likely inducing its local activation, followed by downregulation of both Ret and Fap-1, and translocation of CD95 into lipid rafts. According to the resulting increase of CD95 cell surface expression, the CD95 agonist antibody CH11 enhanced RPI-1-induced cell growth inhibition and apoptosis. RET RNA interference downregulated Fap-1 protein in MZ-CRC-1 cells, whereas exogenous RET-M918T upregulated Fap-1 in HEK293 cells. Overall, these data indicate that the Ret oncoprotein exerts opposing controls on Fap-1 and CD95, increasing Fap-1 expression and decreasing CD95 cell surface expression. The functional interplay of the Ret mutant with the extrinsic apoptosis pathway provides a mechanism possibly contributing to MTC malignant phenotype and a rational basis for novel therapeutic strategies combining Ret inhibitors and CD95 agonists., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action.

The advent of drugs targeting tumor-associated prosurvival alterations of cancer cells has changed the interest of antitumor drug development from cytotoxic drugs to target-specific agents. Although single-agent therapy with molecularly targeted agents has shown limited success in tumor growth control, a promising strategy is represented by the development of rational combinations of target-specific agents and conventional antitumor drugs. Activation of survival/antiapoptotic pathways is a common feature of cancer cells that converge in the development of cellular resistance to cytotoxic agents. The survival pathways implicated in cellular response to drug treatment are primarily PI3K/Akt and Ras/MAPK, which also mediate the signalling activated by growth factors and play a role in the regulation of critical processes including cell proliferation, metabolism, apoptosis and angiogenesis. Inhibitors of PI3K, Akt and mTOR have been shown to sensitize selected tumor cells to cytotoxic drugs through multiple downstream effects. Moreover, the MAPK pathway, also implicated in the regulation of gene expression in response to stress stimuli, can interfere with the chemotherapy-induced proapoptotic signals. Targeting Hsp90, which acts as a molecular chaperone for survival factors including Akt, may have the potential advantage to simultaneously block multiple oncogenic pathways. Overall, the available evidence supports the interest of rationally designed approaches to enhance the efficacy of conventional antitumor treatments through the inhibition of survival pathways and the notion that the concomitant targeting of multiple pathways may be a successful strategy to deal with tumor heterogeneity and to overcome drug resistance of tumor cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The synthetic atypical retinoids containing an adamantyl group exhibit antiproliferative or proapoptotic activities. Apoptosis induction is a dose-dependent effect independent of retinoid receptors. We have reported that induction of apoptosis by the atypical retinoid, ST1926, is associated with early manifestations of genotoxic stress. Indeed, in this study performed in ovarian carcinoma cells, we show that exposure to ST1926 resulted in an increase of early markers of DNA damage, including ATM and H2AX phosphorylation. In addition, we found that a novel histone deacetylase (HDAC) inhibitor (RC307) was able to enhance sensitivity of ovarian carcinoma cells to ST1926. Under conditions where single-agent treatment caused only antiproliferative effects, the combination of the atypical retinoid and HDAC inhibitor resulted in marked apoptotic cell death with a more rapid onset in wild-type p53 ovarian carcinoma cells. The sensitization to ST1926-induced apoptosis was associated with an enhanced DNA damage response, because a prolonged expression of DNA damage markers (e.g., H2AX, p53 and RPA-2 phosphorylation) and a marked activation of DNA damage checkpoint kinases (in particular, phosphorylation of Chk1) were observed indicating an accumulation of DNA damage by the ST1926/HDAC inhibitor combination. The study provides additional support to the role of DNA damage as a primary event leading to the activation of apoptosis in ovarian carcinoma cells by adamantyl retinoids and documents the potential therapeutic efficacy of the combination of ST1926 and HDAC inhibitors of the novel series.

Curcumin, a natural polyphenol, has been described to exhibit effects on signaling pathways, leading to induction of apoptosis. In this study, we observed that curcumin inhibited Hsp90 activity causing depletion of client proteins implicated in survival pathways. Based on this observation, this study was designed to investigate the cellular effects of curcumin combination with the pan-HDAC inhibitors, vorinostat and panobinostat, which induce hyperacetylation of Hsp90, resulting in inhibition of its chaperone function. The results showed that, at subtoxic concentrations, curcumin markedly sensitized tumor cells to vorinostat- and panobinostat-induced growth inhibition and apoptosis. The sensitization was associated with persistent depletion of Hsp90 client proteins (EGFR, Raf-1, Akt, and survivin). In conclusion, our findings document a novel mechanism of action of curcumin and support the therapeutic potential of curcumin/HDAC inhibitors combination, because the synergistic interaction was observed at pharmacologically achievable concentrations, which were ineffective when each drug was used alone.

As a consequence of multiple functions of p53, its activation in response to cytotoxic stress may have proapoptotic or protective effects depending on the nature of lesions. We have previously shown that mutational inactivation of p53 results in sensitization to paclitaxel. In this study, we used cyclic pifithrin-alpha, a transcriptional inhibitor of p53, to further investigate the relevance of p53 function in the response of tumor cells to microtubule inhibitors. Using drug concentrations causing only antiproliferative effects, the combination of antimicrotubule agents with subtoxic pifithrin-alpha doses resulted in increase of sensitivity of two wild type p53 cell lines, associated with a substantial M phase cell accumulation and marked sensitization to apoptosis. Pifithrin-alpha had no sensitizing effect in p53 defective cells or a marginal effect in normal human fibroblasts. The apoptotic response to the combination was concomitant with p21 down-regulation, Polo-like kinase 1 up-regulation, p34(cdc2) kinase dephosphorylation, and cdc25C phosphatase phosphorylation, supporting mitotic arrest. Sensitization to paclitaxel-induced apoptosis was also achieved by p53-siRNA transfection in wild type p53 H460 cells. Pifithrin-alpha did not enhance the apoptotic response after p53 down-regulation. The results support a protective role of the transcriptional activity of p53 in response to mitotic spindle damage. The inhibition of transcriptional activity of p53 may have therapeutic implications in the treatment of p53 wild type tumors with antimitotic agents.

The present study was designed to explore the role of p73 in response to cisplatin. In contrast to cisplatin-resistant ovarian carcinoma cells, pharmacological drug concentrations, which caused induction of p53, induced a marginal increase of p73 in sensitive cells. The effect was more marked at high concentrations, with no evidence of p21(WAF1) induction. This behaviour, associated with substantial apoptosis, suggests cell inability to activate DNA damage checkpoint following extensive stress. Although p73 appears to be implicated mainly in response to high stress conditions, the available results support a cooperation between p53 and p73 in cisplatin-induced apoptosis in sensitive cells.

Atypical retinoids, or retinoid-related molecules (RRMs), represent a class of proapoptotic agents with a promising potential in the treatment of neoplastic diseases. In the present work, the synthesis and structure-activity relationship of a series of 3'-adamantan-1-yl-biphenyl-4-yl-acrylic acids substituted in ring A were studied. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocitic leukemia cell line (NB4), and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of at least one oxygenated substituent in positions 4' or 5' appears determinant for the antiproliferative activity. With two substituents of this kind the activity increases, particularly in the case of alkylenedioxy compounds. The activation of DNA damage response as indicated by phosphorylation of H2AX histone, RPA-2 protein, and p53 at serine 15 by the most apoptotic compounds provides additional support to the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this group.

Recent evidence indicates that the success of molecular targeted therapies may depend on the identification of drug targets which are essential for the survival of subsets of tumors. RET oncogenes that have been implicated in the development of thyroid carcinomas are emerging as potential therapeutic targets. In the present study, we investigated the efficacy and the cellular bases of antitumor activity of the indolinone Ret tyrosine kinase inhibitor RPI-1 against large established s.c. TT tumor xenograft, a human medullary thyroid carcinoma (MTC) harboring oncogenic MEN-2A-type RET mutation. Oral treatment with RPI-1 caused growth arrest or regression in 81% treated tumors. Following treatment suspension, tumor inhibition was maintained (51%, P<0.05, 100 days) and cures were achieved in 2/11 mice. In treated tumors, Ret was tyrosine dephosphorylated. Moreover, compared to control tumors, a significant increase in apoptotic cells (210%, P<0.0001), loss of cellularity (47%, P<0.0001) and reduction of microvessel density (36%, P<0.0005) were detected. In vivo effects of RPI-1 were reflected in activation of BAD, cleavage of caspases, apoptotic DNA fragmentation and inhibition of VEGF production observed in in vitro RPI-1-treated TT cells. These findings thus indicate that RPI-1 antitumor effect on the MTC was characterized by apoptosis induction and angiogenesis inhibition. The results, consistent with a dependence on RET oncogene activation for maintenance and survival of MEN2A-type MTC, provide further preclinical rationale for a pharmacological RET-targeted intervention in thyroid cancer.

The aim of this study was to investigate cellular response to several ruthenium(III), chromium(III) and rhodium(III) compounds carrying bidentate beta-diketonato ligands: [(acac)--acetylacetonate ligand, (tfac)--trifluoroacetylacetonate ligand]. Cell sensitivity studies were performed on several cell lines (A2780, cisplatin-sensitive and -resistant U2-OS and U2-OS/Pt, HeLa, B16) using growth-inhibition assay. Effect of intracellular GSH depletion on cell sensitivity to the agents was analyzed in A2780 cells. Flow cytometry was used to assess apoptosis by Annexin-V-FITC/PI staining, and to analyze induction of caspase-3 activity. Possible DNA binding/damaging affinity was investigated, by inductively coupled mass spectrometry, and by 14C-thymidine / 3H-uridine incorporation assay. Cell sensitivity studies showed that the pattern of sensitivity to Ru(tfac)3 complex of the two cisplatin-sensitive/-resistant osteosarcoma cell lines, U2-OS and U2-OS/Pt, was similar to that of A2780 cells (72 h exposure), with the IC50 being around 40 microM. The growth-inhibitory effect of Ru(acac)3 ranged over 100 microM, while Cr(III) and Rh(III) complexes were completely devoid of antitumor action in vitro. Ru(tfac)3 exhibited strong potential for apoptosis induction on A2780 cells (up to 40%) and caused cell cycle arrest in the S phase as well as decrease of the percent of G1 and G2 cells. Ru(acac)3-induced apoptosis was slightly higher than 10%, whereas activation of caspase-3 in HeLa cells was moderate. DNA binding study revealed that only Cr(acac)3 was capable of binding DNA, while Cr(III) and Ru(III) compounds possess potential to inhibit DNA/RNA synthesis. In conclusion, only Ru(III) complexes showed potential for antitumor action.

Since the intrinsic resistance of prostate carcinoma likely reflects a low susceptibility to drug-induced apoptosis, in this study we explored the possibility of sensitizing prostate carcinoma cells to apoptosis by combination of TRAIL with camptothecins. Indeed, these agents are known to activate different pathways of apoptosis. Topotecan- and gimatecan induced moderate up-regulation of TRAIL-R1 and -R2 which resulted in a different cell response to the combination in androgen-independent cells (DU-145 and PC-3). In DU-145 cells apoptosis was increased by lower TRAIL concentrations and was earlier than in PC-3 cells, as shown using Annexin V-binding assay. The relative resistance of PC-3 cells to drug-induced apoptosis was associated with constitutive Akt activation, higher levels of cFLIP-L and Bcl-2, and lower levels of Bax. The different expression/activation of apoptosis-related factors appears to influence the sensitization of prostate carcinoma cells by TRAIL. Potentiation of camptothecin-induced apoptosis by TRAIL appears dependent on cooperation between extrinsic and intrinsic pathways, as documented by loss of the sensitization to apoptosis following reduction of caspase 8 after small interfering RNA transfection. The efficacy of the approach may be critically dependent on the intrinsic susceptibility to apoptosis of different tumors. These observations support that the activation of multiple signals could enhance apoptotic response and suggest the therapeutic interest of the TRAIL/camptothecin combination.

Atypical retinoids (AR) represent a class of proapoptotic agents with promising potential in the treatment of neoplastic diseases. In the present work 4'-hydroxybiphenyl-4-ylacrylic acids were studied as a novel series of AR. The synthesized compounds were evaluated for their antiproliferative activity in a human promyelocytic leukemia cell line (NB4) and in an ovarian carcinoma cell system including IGROV-1, carrying a functional wild-type p53, and a cisplatin-resistant subline, IGROV-1/Pt-1. The presence of a bulky lipophilic group at position 3' (adamantan-1-yl being the best) and the E configuration of the acrylic moiety appear essential for activity below 1 muM. No substitution on the rings or on the double bond improved the activity. A qualitative correlation between the log P and molecular volume of the 3'-substituent and the antiproliferative activity was found. From the study of a few selected compounds, it appears that the presence of the carboxylic group is an essential requirement for apoptogenic properties but not for antiproliferative activity, this being maintained in amide derivatives. On the other hand, compounds able to induce apoptosis produced a detectable level of genotoxic damage. This observation supports the hypothesis that the genotoxic stress is a critical event mediating apoptosis induction by compounds of this class. Among the compounds investigated, E-3-(3'-adamantan-1-yl-4'-hydroxybiphenyl-4-yl)acrylic acid (2) was chosen for further investigation.

Amplification of the c-MYC proto-oncogene is a frequent alteration in hormone refractory prostate carcinomas (HRPC). In an attempt to investigate the role of c-myc in the cellular response to paclitaxel (PTX), we used two HRPC cell lines, DU145 and PC3, characterised by different levels of the protein and by different behaviour in response to taxane. In both cell lines, PTX-induced cell death was a caspase-mediated apoptosis. In DU145 cells, PTX induced an early apoptotic response associated with upregulation of c-myc restricted to the G2/M cell population. This event appeared delayed in the presence of c-myc antisense (AS-c-myc), suggesting an upstream regulation of the protein expression. In addition, the antisense approach provided evidence of an involvement of c-myc in the apoptotic response to the taxane. In contrast, in PC3 cells, the overexpressed c-myc was not modulated by drug-treatment and the addition of AS-c-myc did not affect the cell growth inhibition of PTX. In both cell lines, PTX-induced c-myc phosphorylation was concomitant with the mitotic arrest and not related to the modulation of the activation state of AKT and MAPK kinases. Our data indicate that the cellular response to PTX of HRPC cells can involve c-myc and suggest that its pro-apoptotic role is affected by the genetic background, thus supporting a complex and differentiated HRPC cell response to taxanes.

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.

ST1481, a lead compound of a novel potent 7-substituted lipophilic camptothecin series, is able to overcome several mechanisms of drug resistance and was selected for clinical development. This study was designed to examine the antitumor activity of ST1481 in the treatment of preclinical models of human p53-defective hormone-refractory prostate carcinoma (DU145, PC3, and JCA-1) and to explore the cellular bases of the efficacy of camptothecins. A cellular pharmacology study (cytotoxicity, apoptosis, cellular drug accumulation, DNA damage, and cell cycle perturbation) was performed in DU145 and PC3 cells, characterized by a different cell cycle checkpoint status. The introduction of wild-type p53 in PC3 cells appreciably decreased the drug sensitivity. The 7-substituted camptothecins exhibited a high cytotoxic potency that paralleled their relative ability to induce DNA damage and a substantially increased cellular accumulation as compared to topotecan. The cytotoxic effect of camptothecins in DU145 cells was associated with arrest in S phase and early activation of apoptosis, whereas PC3 cells responded to drugs by a persistent block in G2 phase with a cytostatic effect and a late apoptosis. The efficiency of S phase checkpoint in DU145 cells was supported by a time-dependent decrease of DNA synthesis following treatment. In spite of an apparent cytostatic response and apoptosis resistance, the PC3 tumor was more responsive to in vivo treatment with camptothecins than the DU145 model. Indeed, the therapeutic outcome did not reflect the cell susceptibility to early activation of apoptosis. We suggest that cell death in PC3 cells is a delayed event consequent to persistent arrest in G2 and insufficient repair of DNA damage. ST1481 was appreciably more effective than topotecan in all tested tumors. In conclusion, the results indicated a relevant efficacy of camptothecins against human prostate carcinoma models, in spite of p53 alterations. Although p53 status could influence DNA damage and cell cycle checkpoints, p53 mutation was not a determinant of resistance. The results support that, in addition to the extent and persistence of topoisomerase I-mediated DNA damage, cell cycle checkpoints and DNA damage signaling pathways are critical determinants of tumor responsiveness to camptothecins. A role of cell cycle checkpoints activated by DNA damage in cell response is supported by the modulation of transcriptional profile.

The novel atypical retinoid E-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid (ST1926, 4) exhibited a potent antiproliferative activity on a large panel of human tumor cells. Despite almost complete loss of ability to activate RARs, the compound was an effective apoptosis inducer and surprisingly produced DNA damage, that likely contributes to the proapoptotic activity. Following oral administration, 4 was well tolerated and caused tumor growth inhibition in the ovarian carcinoma, A2780/DX, and in the human melanoma, MeWo, growing in nude mice, thus supporting the therapeutic interest of the novel agent.

Mononuclear and multinuclear platinum complexes are known to induce distinct types of DNA lesions and exhibit different profiles of antitumor activity, in relation to p53 mutational status. In this study, we investigated the cellular effects of exposure to two platinum compounds (cisplatin and the multinuclear platinum complex BBR 3464), in the osteosarcoma cell line, U2-OS, carrying the wild-type p53 gene and capable of undergoing apoptosis or cell cycle arrest in response to diverse genotoxic stresses. In spite of the ability of both compounds to up-regulate p53 at cytotoxic concentrations, exposure to BBR 3464 resulted in cell cycle arrest but only cisplatin was capable of inducing significant levels of apoptosis and phosphorylation at the Ser15 residue of p53. The cisplatin-induced protein phosphorylation, not detectable in cells treated with BBR 3464, was associated with RPA phosphorylation, a specific up-regulation of Bax and down-regulation of p21(WAF1). Cells treated with BBR 3464 displayed a different cellular response with evidence of cytostasis associated with a high induction of p21(WAF1). The regulation of p21(WAF1) after cisplatin or BBR 3464 exposure required a p53 signal, as documented using stable transfectants expressing a dominant-negative form of p53 (175(his)). Taken together, these results indicate that cellular response to different genotoxic lesions (i.e. apoptosis or growth arrest) is associated with a specific recognition of DNA damage and a different p53-mediated signaling pathway. Multinuclear platinum complexes could be regarded as useful tools for investigating the p53-mediated process of cell cycle arrest in response to DNA damage.

The expression of the death receptor Fas/CD95 is cell type-specific and can be modulated by different cytotoxic treatments. In spite of a frequent expression of Fas/CD95 in high-grade gliomas, these tumours are typically refractory to conventional therapy. Using a human glioblastoma cell line (GBM), we explored the possibility of modulating susceptibility to Fas/CD95-mediated apoptosis following cytotoxic treatment. GBM cells were sensitive to the antiproliferative effects of topoisomerase I inhibitors (topotecan and a novel lipophilic analog CPT83) and taxol, less sensitive to cisplatin and, in any case, rather resistant to drug-induced apoptosis. This pattern of cellular response was consistent with p53 mutation. GBM cells expressed low levels of Fas/CD95, which was associated with low susceptibility to antibody-stimulated Fas/CD95-mediated apoptosis. A significant up-regulation of Fas/CD95 expression was detected after exposure to topotecan and CPT83, whereas cisplatin induced a low increase and taxol did not modify Fas/CD95 expression. In addition, after treatment with topoisomerase I inhibitors, the up-regulation of Fas/CD95 resulted in an increased susceptibility of GBM cells to antibody-stimulated Fas/CD95-mediated apoptosis, while no synergistic effects were detected after treatment with cisplatin or taxol. Our data suggest that Fas/CD95 up-regulation can be a common response to DNA damage, whereas sensitisation to Fas/CD95-mediated apoptosis appears to be dependent on the type of DNA damage and on the pathway of cellular response. The observed effects might have important therapeutic implications for the design of novel therapeutic strategies in the treatment of malignant gliomas.

Based on the role of p53 in the control of apoptosis following DNA damage, the status of the TP53 gene has been implicated as a major determinant of tumour responsiveness to cytotoxic therapies. In spite of the high frequency of TP53 mutations, small-cell lung cancer (SCLC) is recognised as one of the most chemoresponsive solid tumours. Since the relevance of the TP53 gene status in the modulation of tumour responsiveness is dependent on the molecular/biological context, in the present study, we have examined the relationship between chemosensitivity and susceptibility to apoptosis of a TP53-mutant human SCLC cell line. The cell line, in spite of TP53 mutation, retained an efficient response to genotoxic stress as documented by cells ability to modulate the p53 protein, arrest in the G1 and G2 phases of the cell cycle and its marked susceptibility to apoptosis following treatment with DNA damaging agents. Exposure to DNA-damaging agents caused an increase of c-Myc, a DNA damage-responsive transcription factor. An analysis of damage-induced apoptosis in the presence of an anti-Fas/CD95 inhibitory antibody indicated that Fas/CD95 was not required for the apoptotic response. The results support an implication of c-myc in sensitising cells to apoptosis, since inhibition of c-Myc expression with an antisense oligodeoxynucleotide (AS-ODN) almost abolished the drug-induced apoptotic response. In conclusion, the present results support a role for c-myc in the induction of apoptosis by genotoxic stress in the absence of a functional p53 and provide new insights into the mechanisms that may influence apoptosis in TP53-mutant cells. Elucidation of this pathway and of the possible cooperation with p53-dependent mechanisms may provide a basis for therapeutic intervention.

Although the anti-apoptotic effect of Bcl-2 is well established, the role of Bcl-2 in tumour response to therapy and drug resistance is still unclear. The post-translational modifications of Bcl-2 are likely involved in the control of the apoptotic pathway. In the present study we have investigated the role of Bcl-2 in cellular response to oxidative stress (hydrogen peroxide) and cisplatin using a clone of human metastatic melanoma, which, in spite of Bcl-2 (over)expression, exhibited a moderate chemosensitivity. With both treatments melanoma cells died through an apoptotic process, associated with detachment of cells from the monolayer. In the floating apoptotic cells generated by either hydrogen peroxide or cisplatin, along with morphological and biochemical features of apoptosis, we detected a significant Bcl-2 cleavage, yielding the Bax-like fragment of 23 kDa. Preincubation of cells with the caspase-3/-7 inhibitor DEVD-CHO completely suppressed Bcl-2 cleavage, thus confirming that such a specific proteolysis requires activation of caspase-3/-7. The oxidant- and cisplatin-induced processing of Bcl-2 documented in the present study may represent a regulatory mechanism to circumvent the survival function of Bcl-2 upon apoptosis triggering and to enhance apoptotic response. Since the Bcl-2 cleavage should be regarded as a pro-apoptotic event, Bcl-2 expression is expected to increase susceptibility to apoptosis. Thus, such a pathway could be exploited to improve the efficacy of cytotoxic therapy of melanomas expressing Bcl-2.

Cellular resistance to anthracyclines is a major limitation of their clinical use in the treatment of human tumors. Resistance to doxorubicin is described as a multifactorial phenomenon involving the overexpression of defense factors and alterations in drug-target interactions. Such changes do not account for all manifestations of drug resistance, in particular intrinsic resistance of solid tumors. Since anthracyclines can induce apoptotic cell death, an alternative promising approach to drug resistance has focused on the study of cellular response to drug-induced DNA damage, with particular reference to the relationship between cytotoxicity/antitumor efficacy and apoptotic response. The evidence that a novel disaccharide analog (MEN 10755), endowed with an improved preclinical activity over doxorubicin, was also more effective as an inducer of apoptosis provided additional insights to better understand the cellular processes that confer sensitivity to anthracyclines. Although the presence or alteration of a single apoptosis-related factor (e.g., p53, bcl-2) is not predictive of the sensitivity/resistance status, the complex interplay among DNA damage-activated pathways is likely an important determinant of tumor cell sensitivity to anthracyclines

The biochemical basis of the anti-proliferative effect of exogenous glutathione was investigated in A2780 ovarian carcinoma cells. Previous observations have implicated gamma-glutamyl transpeptidase-mediated pro-oxidant reactions as a primary mechanism of the extracellular effects of glutathione. In 2 cell lines (A2780 and IGROV-1), glutathione led to H(2)O(2) production, but only A2780 cells, characterized by low expression of detoxification enzymes, were sensitive to the thiol compound. In A2780 cells, glutathione exposure resulted in DNA single-strand break formation, as measured by alkaline elution. Glutathione-induced DNA damage generated significant levels of apoptosis in A2780 cells, but not in IGROV-1 cells. The capability of glutathione to induce apoptosis was associated with cleavage of poly(ADP-ribose)polymerase and with generation of a low-molecular-weight form of the pro-apoptotic protein bax. In A2780 cells, glutathione exposure was followed by p21 and Bax induction and p53 up-regulation, as expected for genotoxic stress. Consistently, analysis of cell-cycle perturbations demonstrated the occurrence of G(2)M accumulation after exposure to glutathione, similar to what was observed for H(2)O(2). Taken together, these results indicate that the cytotoxic effect of extracellular glutathione, related to membrane metabolism, is mediated by production of H(2)O(2) leading to DNA damage and a cellular response involving p53. These findings might also provide insights into the cellular and molecular determinants of chemosensitivity to DNA damaging agents, as oxidative stress is implicated in p53-dependent apoptosis., (Copyright 2000 Wiley-Liss, Inc.)

The pharmacological interest in lonidamine is related to its ability to enhance the cytotoxic effects of several DNA-damaging anti-tumour agents. This study was undertaken to better understand the in vivo interaction between lonidamine and cisplatin in the treatment of human tumour xenografts, including three carcinoma models characterized by a different responsiveness to cisplatin, in spite of the presence of a wild-type p53 gene in all tumours. The drug combination was more effective in tumour growth inhibition than cisplatin alone against MX-1 breast carcinoma and A2780 ovarian carcinoma, both highly responsive to cisplatin, whereas no influence of ionidamine was observed on anti-tumour activity of cisplatin in the treatment of the relatively resistant IGROV-1 ovarian carcinoma. As cisplatin activity is related to induction of apoptosis, the modulation of drug-induced apoptosis by lonidamine was investigated. Under conditions in which lonidamine itself had negligible effects on tumour growth and apoptosis, the modulating agent stimulated the apoptotic response induced by cisplatin in the responsive but not in the resistant tumours. Tumour response was dependent not only on the drug activation of apoptosis, but mainly on the persistence over time of the event. In the breast carcinoma MX-1, hypersensitive to cisplatin and to the lonidamine+cisplatin combination, the efficacy of drug treatment was associated with phosphorylation of bcl-2 followed by down-regulation of the protein. Lonidamine itself caused a delayed phosphorylation of bcl-2. These results are consistent with the interpretation that lonidamine is effective in modulating biochemical factors involved in regulation of apoptosis.

The development of drug resistance is a major obstacle to effectiveness of chemotherapeutic treatment of human tumors with cytotoxic agents. Drug resistance is described as a multifactorial phenomenon, involving the expression of defense factors and/or detoxification mechanisms, alterations in drug-target interactions, and cellular response to specific cytotoxic lesions (in particular, DNA damage). Although the proposed mechanisms may contribute to the development of a variable degree of cellular resistance, it is possible that the cell response (i.e., DNA repair or apoptosis) following DNA damage plays a critical role in determining cellular chemosensitivity. The preclinical observations that tumor response to effective drug treatments is associated with induction of apoptosis support the possibility that a decreased susceptibility to apoptosis (apoptosis resistance) is relevant to clinical resistance. A number of molecular alterations associated with transformation and/or tumor progression may also be implicated in regulation of cell death pathways and in the development of drug resistance. There is evidence that the wild-type p53 is involved in cellular response to DNA damage, including cell cycle regulation, DNA repair, and activation of the pathway leading to apoptosis. Loss of wild-type p53 function could cause resistance to DNA-damaging agents, as a consequence of abrogation of p53-dependent apoptosis. The identification of new agents able to trigger p53-independent apoptosis and the search for biochemical modulators downstream of p53 may be of clinical relevance because many tumors are deficient in p53 function due to mutation or deletion. An overview of the resistance mechanisms is presented, with particular reference to the role of p53 mutations in clinical resistance and of apoptosis-related genes in cellular chemosensitivity.

Background: Although doxorubicin remains one of the most effective agents for the treatment of solid tumors, there is an intensive effort to synthesize doxorubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties. We have synthesized a novel doxorubicin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, and pharmacologic properties., Methods: The antitumor activity of this compound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice., Results: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxorubicin-resistant breast carcinoma cell line MX-1., Conclusions: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubicin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

Preclinical and clinical studies have documented the pharmacological interest in camptothecin derivatives in the treatment of resistant tumors. In particular, topotecan, a water-soluble derivative, exhibited promising activity in pretreated ovarian carcinoma. The present study investigated the pattern of tumor response in two human ovarian carcinoma xenografts and in their cisplatin-resistant sublines characterized by different mechanisms of drug resistance. In IGROV-1/Pt1 cells, cisplatin resistance has been ascribed to a reduced susceptibility to apoptosis as a consequence of p53 mutation and inactivation of its function. In the A2780 cisplatin-resistant subline, which retained the wild-type p53 gene status, the development of resistance has been possibly related to increased cell ability to repair drug-induced DNA damage. The in vivo results of the present study showed that topotecan could overcome the resistance in A2780/CP but not in IGROV-1/Pt1 tumor xenografts. The pattern of tumor response following in vivo topotecan treatment correlated with drug ability to induce apoptosis but not with its in vitro antiproliferative activity. The antitumor efficacy of topotecan in the four tumors reflected a different cell response to drug-induced DNA damage, as suggested by different perturbations of cell cycle progression. Indeed, only in the subline refractory to topotecan in vivo, IGROV-1/Pt1, did we observe a persistent arrest of the cells in the S-phase, resulting in a cytostatic and not a cytotoxic effect, since a low level of apoptosis was induced by the drug. In conclusion, the current results support that determination of drug-induced apoptosis is a useful predictor of tumor response to topotecan in ovarian carcinomas and suggest that p53 gene status may be a critical determinant of cell response to topoisomerase inhibitors.

Background: The novel adamantyl retinoid ST1926 is a potent inducer of apoptosis in ovarian carcinoma cells. Since the pro-apoptotic effect is associated with activation of p53, in this study we have investigated the efficacy of combination of ST1926 with cisplatin, a DNA-damaging agent that is known to induce p53-dependent apoptosis. Materials and methods: The efficacy of ST1926 and its combination with cisplatin was evaluated in human ovarian carcinoma models, including resistant tumors. Results: Oral treatment with ST1926 alone caused a marginal tumor growth inhibition (Conclusions: The present results document the efficacy of the combination of cisplatin with ST1926 and provide a rational basis for the design of novel, well-tolerated platinum-based treatment approaches in human ovarian carcinoma. [ABSTRACT FROM AUTHOR]