APPROACHES OF MODULATION OF THERAPEUTIC TARGETS RELEVANT TO DRUG RESISTANCE

An emerging anticancer strategy is to identify molecular targets that when inhibited pharmacologically, result in pleiotropic downstream effects on pathways relevant to the malignant phenotype, with particular reference to the development of resistance. In recent years, the molecular chaperone Hsp90 has emerged as leading example of such a target-specific therapy. Heat-shock proteins are found at increased levels in many solid tumors and haematological malignancies. Their expression may account for the ability of malignant cells to maintain protein homeostasis even in the hostile hypoxic microenvironment of the tumor. Particularly, Hsp90 has emerged as a target for cancer therapy due to its critical roles in retaining the conformation and the function of its client proteins, many of which are associated with cancer pathology. There are 12 HSP90 inhibitors in clinical trials; all have potencies on the order of 7-35 nM in a cell-based assay and half-lives of 1-3 h in the plasma of rodents. These inhibitors may distribute preferentially to the tumors and may have longer half-lives in the tumors than in the plasma, as in the case of 17-AAG and its active metabolite. However, side effects and poor formulability of 17-AAG restricted its potential of clinical application and motivated many groups to synthesize new compounds. The aim of this project is to identify new Hsp90 inhibitors which could overcome the limitations of available inhibitors and exhibit therapeutic advantages in terms of specificity, therapeutic index and antitumor efficacy. Several compounds have been tested during these years, especially a series of mold metabolites of Ascomycetes, structurally belonging to the class of azaphilones, were found to inhibit the heat shock protein Hsp90. In particular, bulgarialactone B was tested for its binding to Hsp90 using surface plasmon resonance and limited proteolysis assays and for its effects on Hsp90 client proteins expression in a series of human tumor cell lines. This compound showed high affinity for Hsp90, interacting with the 90–280 region of the N-terminal domain and down-regulated the Hsp90 clientproteins Raf-1, survivin, Cdk4, Akt, and EGFR. Bulgarialactone B and other natural azaphilones showed antiproliferative activity in a panel of human tumor cell lines; their conversion into semisyntheticderivatives by reaction with primary amines increased the antiproliferative activity. Preliminary results indicated in vivo activity of bulgarialactone B against an ascitic ovarian carcinoma xenograft, thus supporting the therapeutic potential of this novel series of Hsp90 inhibitors. Moreover, to identify favourable interactions between Hsp90 inhibitors and target-specific agents, combinations between curcumin and two well-known HDAC inhibitors (vorinostat and panobinostat), have been tested. 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, the 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. Another new therapeutic target is GGT (gamma-glutamyl transferase), the interest in this enzyme is related to recent evidence supporting that it is implicated in tumor progression and in drug resistance to stress-inducing agents (i.e. platinum compounds). The extracellular -glutamyltransferase-mediated metabolism of glutathione has been implicated in prooxidant events which may have impact on cellular functions including drug resistance. Therefore the objective of this approach was to investigate the role of GGT in response to various stress-inducing agents. The study was performed in two GGT-transfected melanoma clones to explore the hypothesis that GGT expression in tumour cells is implicated in modulation of cell behaviour under stress conditions. Our results show that GGT-overexpression in melanoma cells was associated with resistance to oxidative stress produced by prooxidant agents such as hydrogen peroxide and ascorbic acid. In GGT-overexpressing cells, ability to tolerate oxidative stress was evidenced by the presence of a moderate level of ROS and lack of DNA damage response following treatment with H2O2. Cellular response to oxidative stress induced by ascorbic acid was detectable only in the clone with low GGT activity which also exhibited an increased susceptibility to apoptosis. The increased resistance of the GGT-overexpressing clone was not related to intracellular GSH content but rather to the increased expression of catalase and to a reduced efficiency of iron-mediated formation of toxic free radicals. Taken together, these findings are consistent with a contribution of GGT in the mechanisms of drug resistance, because induction of oxidative stress is a relevant event in the apoptotic response to cytotoxic agents. Again, in an attempt to develop novel strategies for overcoming the mechanisms of cellular protection against oxidative stress, we have explored the efficacy of the combination of two prooxidant agents in the two human melanoma cell clones differently expressing GGT. The γ-glutamyltransferase-overexpressing clone exhibited a low susceptibility to arsenic trioxide-induced apoptosis, associated with low reactive oxygen species induction and increased catalase activity. The combination of arsenic trioxide with subtoxic concentrations of ascorbic acid resulted in a sensitization to apoptotic cell death. The expression of protective mechanisms, in particular catalase activity, accounted for the behavior of the resistant clone. The sensitization achieved by the combination was associated with a cellular response involving the ASK1/p38 axis, which is implicated in the regulation of catalase expression and the activation of apoptotic signals. In conclusion, the results of our study provide evidence that a rational combination of prooxidant agents may be effective in overcoming cellular tolerance to oxidative stress. Moreover, as a number of recent observations have suggested a potential role for membrane-bound gammaglutamyltransferase (GGT) in tumor progression through redox interactions leading to production of reactive oxygen species, we performed a study to evaluate whether such pro-oxidant activity of GGT can promote oxidative DNA damage, thus contributing to cancer genomic instability. Human GGT-transfected melanoma cells were studied, and DNA damage was measured by using the alkaline comet assay. Our results indicate that higher levels of GGT activity are associated with higher levels of background DNA damage and oxidized bases. This association cannot be explained by differences in cell cycle distribution or apoptotic rates. GGT-over-expressing cells also presented with a markedly higher glucose uptake, a phenomenon potentially leading to higher metabolic rate and oxidative DNA damage. Anyway, when GGT-over-expressing cells were incubated in the presence of GGT substrates and a source of catalytic iron, increased levels of DNA damage and oxidized bases were observed, an effect completely prevented in the presence of GGT inhibitors or various antioxidants. The findings reported indicate that GGT activity is able to promote iron-dependent DNA oxidative damage, thus potentially representing an important mechanism in initiation/progression of neoplastic transformation.