No abstract

Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin‐resistant subline, A431/Pt, were used as a model system. The experimental set‐up involved not just a two‐way comparison of the control vs. the drug‐resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four‐way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat‐shock proteins HSP60, HSP90, HSC71, heat‐shock cognate 71 kDa protein), Ca2plus;‐binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione‐S‐transferase, GST), anti‐apoptotic proteins (such as 14‐3‐3 switched on in cisplatin‐exposed cells) and ion channels (such as VDAC‐1, voltage‐dependent anion‐selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up‐regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up‐regulated the anti‐apoptotic 14‐3‐3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug‐induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.

Summary Multinuclear platinum compounds were rationally designed to bind to DNA in a different manner from that of cisplatin and its mononuclear analogues. A triplatinum compound of the series (BBR 3464) was selected for preclinical development, since, in spite of its charged nature, it was very potent as cytotoxic agent and effective against cisplatin-resistant tumour cells. Anti-tumour efficacy studies were performed in a panel of human tumour xenografts refractory or poorly responsive to cisplatin. The novel platinum compound exhibited efficacy in all tested tumours and an impressive efficacy (including complete tumour regressions) was displayed in two lung carcinoma models, CaLu-3 and POCS. Surprisingly, BBR 3464 showed a superior activity against p53-mutant tumours as compared to those carrying the wild-type gene. The involvement of p53 in tumour response was investigated in an osteosarcoma cell line, SAOS, which is null for p53 and is highly sensitive to BBR 3464, and in the same cells following introduction of the wild-type p53 gene. Thus the pattern of cellular response was investigated in a panel of human tumour cells with a different p53 gene status. The results showed that the transfer of functional p53 resulted in a marked (tenfold) reduction of cellular chemosensitivity to the multinuclear platinum complex but in a moderate sensitization to cisplatin. In addition, in contrast to cisplatin, the triplatinum complex was very effective as an inducer of apoptosis in a lung carcinoma cell line carrying mutant p53. The peculiar pattern of anti-tumour activity of the triplatinum complex and its ability to induce p53-independent cell death may have relevant pharmacological implications, since p53, a critical protein involved in DNA repair and induction of apoptosis by conventional DNA-damaging agents, is defective in several human tumours. We suggest that the peculiar DNA binding properties of the triplatinum complex may contribute to the striking profile of anti-tumour efficacy. Taken together, the available information supports that anti-tumour activity of the novel compound is mediated by a mechanism different from that of conventional platinum complexes, and compounds of this series could represent a new class of promising anti-tumour agents.

Because cytotoxic stress elicits various signaling pathways that may be implicated in cell survival or cell death, their alterations may have relevance in the development of platinum-resistant phenotype. Thus, in the present study, we investigated cell response to the epidermal growth factor receptor (EGFR) inhibitor gefitinib of ovarian carcinoma cell lines, including cells selected for resistance to cisplatin (IGROV-1/Pt1) and oxaliplatin (IGROV-1/OHP). Resistant sublines exhibited a marked decrease in sensitivity to gefitinib and resistance to apoptosis. Gefitinib was capable of inhibiting the phosphorylation of EGFR in all the studied cell lines. The Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, which act downstream of EGFR, were constitutively active in the three cell lines, but phospho-ERK1/2 levels were increased in the two resistant sublines. This feature was associated with reduced sensitivity to the MEK1/2 inhibitor U0126. Pretreatment of resistant cells with U0126 resulted in restoration of sensitivity to gefitinib. Gefitinib was more effective in inhibiting ERK1/ 2 and Akt phosphorylation in IGROV-1 cells than in IGROV-1/OHP and IGROV-1/Pt1 cells. Phospho-p38 was up-regulated in the resistant sublines, indicating the concomitant activation of distinct mitogen-activated protein kinases. The up-regulation of phospho-p38 was associated with a peculiar localization of EGFR, which, in resistant sublines, was mainly internalized. In conclusion, our results indicate that the development of resistance to platinum drugs is associated with multiple alterations including deregulation of survival pathways activated by EGFR resulting in a reduced cellular response to gefitinib.

Since apoptosis is the primary mode of cell death induced by cisplatin, the role of apoptosis and apoptosis-related gene products in cisplatin resistance was investigated in four human cisplatin-resistant cell lines of different tumour type. A common feature of the resistant sublines was a reduced susceptibility to drug-induced apoptosis compared to parental sensitive lines. Loss of wild-type p53 function was not a general event associated with the development of drug resistance. An increased bcl-2 expression was found in resistant cells characterized by mutant p53 (A431/Pt and IGROV-1/Pt), whereas in osteosarcoma (U2-OS/Pt) and in ovarian carcinoma (A2780/CP) cells with wild-type p53, bcl-2 levels were markedly reduced. U2-OS/Pt cells had a 16-fold increase in the level of Bcl-xL protein. Stable transfection of U2-OS cells with bcl-xL cDNA conferred a low level of drug resistance to cisplatin, suggesting that overexpression of this gene contributes to the cisplatin-resistant phenotype of this osteosarcoma cell system. In conclusion, these observations suggest a variable contribution of apoptosis-related genes to cisplatin resistance depending on the biological background of the cell system and presumably reflecting different pathways of apoptosis.

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21 WAF1 levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21 WAF1 only in parental cells. An inverse relationship with p21 WAF1 modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21 WAF1 at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464. © 2003 Wiley-Liss, Inc. Key words: ovarian carcinoma; platinum drugs; resistancePlatinum compounds are antitumor agents effective in the treatment of human solid tumors, including ovarian carcinomas. 1 Development of resistance, however, is a major limitation of the efficacy of these compounds. [2][3][4] Over the past years, many platinum-containing drugs have been developed. Among these, mononuclear platinum complexes have shown molecular mechanisms similar to those of cisplatin. 5 The development of multinuclear platinum complexes has led recently to the identification of the trinuclear complex BBR3464. 6 -9 An investigation of the antitumor effects of BBR3464 in human tumor xenografts resistant or poorly responsive to cisplatin indicated a higher efficacy than cisplatin and a marked efficacy in p53 mutant carcinoma models. 8 Gene transfer studies documented a relevant role for p53 in regulating sensitivity to BBR3464. In particular, introduction of functional p53 in the SAOS osteosarcoma cell line n...

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.

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 downregulation 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.