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Background: b-AP15/VLX1570 are small molecule inhibitors of the ubiquitin specific peptidase 14 (USP14) and ubiquitin carboxyl-terminal hydrolase 5 (UCHL5) deubiquitinases (DUBs) of the 19S proteasome. b-AP15/VLX1570 have been shown to be cytotoxic to cells resistant to bortezomib, raising the possibility that this class of drugs can be used as a second-line therapy for treatment-resistant multiple myeloma. Limited information is available with regard to potential resistance mechanisms to b-AP15/VLX1570., Results: We found that b-AP15-induced cell death is cell-cycle dependent and that non-cycling tumor cells may evade b-AP15-induced cell death. Such non-cycling cells may re-enter the proliferative state to form colonies of drug-sensitive cells. Long-term selection of cells with b-AP15 resulted in limited drug resistance (~2-fold) that could be reversed by buthionine sulphoximine, implying altered glutathione (GSH) metabolism as a resistance mechanism. In contrast, drug uptake and overexpression of drug efflux transporters were found not to be associated with b-AP15 resistance., Conclusions: The proteasome DUB inhibitors b-AP15/VLX1570 are cell cycle-active. The slow and incomplete development of resistance towards these compounds is an attractive feature in view of future clinical use., Competing Interests: The authors have declared that no competing interests exist.

The machinery that maintains cellular and tissue homeostasis in a healthy individual is recruited and hijacked by cancer cells to support tumor growth and progression. Activation of often unpredictable alternative or complementary signaling pathways allows cancer cells to bypass the intrinsic self-destructive machinery and the limited replicative potential present in every cell for correct homeostasis maintenance. Therefore, evasion/resistance to apoptosis/cell death, self-sufficiency in growth/survival signals, and limitless replicative potential remain undoubted hallmarks of cancer, contributing to drug resistance. Herein, we specifically review antitumor strategies involving agents targeting deregulated receptor tyrosine kinases, selected epigenetic modifications, the ubiquitin-proteasome system, the unfolded protein response, the apoptotic pathway, and peculiar nucleic acid structures, with the aim of highlighting the mechanisms underlying favorable drug interaction. In this context, we focus on preclinical studies that have already been translated into clinical investigation. Particular emphasis is devoted to strategies effective in solid tumors because of the peculiarity and distinctive features of solid tumors in terms of drug delivery and relative to the impact of the tumor microenvironment.

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.

Treatment of ovarian carcinoma often fails to be curative because of drug resistance, and many efforts are directed to overcome tumor cell resistance by increasing apoptosis induction. The potential of second mitochondria-derived activator of caspases (SMAC) mimetics (SMACm) has appeared in preclinical studies, but novel proapoptotic agents of this class with improved pharmacological profile are needed. To identify novel treatment options for ovarian carcinoma by interfering with antiapoptotic factors, in the present study a novel homodimeric SMACm (SM83) was employed in preclinical models both in vitro and in vivo. An investigation of the structural features of dimeric SM83 as compared to a closely related reference compound indicated slight differences, likely because of the interaction between one of the terminal phenyl groups and triazole rings of SM83 with the BIR2 domain. Although SM83 per se did not inhibit cell proliferation, it displayed a synergistic effect in combination with TNF-related apoptosis inducing ligand (TRAIL) in cell sensitivity assays. Because the tumor microenvironment is a reservoir of cytokines that may act in conjunction with SMACm to affect tumor growth, the activity of the novel compound was tested in vivo in ovarian carcinoma cells subcutaneously xenografted into immunodeficient mice. A significant tumor volume inhibition was observed together with activation of caspase 3 and apoptotic cell death. A biochemical analysis of tumor necrosis factor (TNF) and TRAIL content in specimens from xenografted mice indicated that SM83 downmodulated the levels of human TNF in plasma samples and tended to upmodulate human TRAIL levels in tumors. Thus, TRAIL appears to contribute to the antitumor activity of novel SMACm SM83 in subcutaneously grown ovarian carcinoma. Overall, our results indicate that SM83 is an attractive candidate for further development.

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.

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.

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.

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.

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.)

Stable transfection of human ovarian carcinoma cells with survivin cDNA caused a four- to sixfold increase in cell resistance to taxotere and taxol (two-sided Student's t test, p <0.05), with a concomitant reduction in the apoptotic response to taxol, but did not affect cell sensitivity to cisplatin or oxaliplatin. Such findings were indirectly supported by similar observations obtained with clinical tumours. In fact, high levels of survivin protein expression (>30% positive cells), detected by immunohistochemistry in 90/124 (73%) advanced ovarian carcinomas, were significantly associated with clinical resistance to a taxol/platinum-based regimen but unrelated to tumour shrinkage following cisplatin-including combinations (non-taxol based). In the 95 patients receiving a taxol/platinum-based regimen, survivin overexpression correlated with a lower clinical or pathologic complete remission rate than absent/low protein expression (43 vs 75%, p = 0.0058 by logistic regression adjusted for tumour stage, histological grade and p53 expression). Conversely, in the 29 cases treated with cisplatin-containing regimens (not taxol based), survivin expression was unrelated to tumour response. Cellular studies and clinical data suggest a direct link between survivin expression and tumour cell susceptibility to taxol. [ABSTRACT FROM AUTHOR]

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