New mechanisms of platinum-based drug effects as a tool for anticancer therapeutic strategies

Platinum-based drugs, alone or in combination with other compounds, are commonly used chemotherapeutics in the treatment of many type of tumours. Namely, oxaliplatin in combination scheme FOLFOX, FOLFIRI etc. is widely applied in the chemotherapy treatment of colon tumours. However, for more efficient therapy, further knowledge about detail mechanisms of platinum-based drug action is necessary. Our research was focused on novel mechanisms of the effects of platinum-based cytostatics, PPAR (Peroxisome Proliferator – Activated Receptors) ligands and their combinations at both molecular and cellular levels. We used human colon and prostate cancer cell lines as well as samples obtained from cancer patients.

PPAR s are transcription factors which belong to large family of nuclear receptors. PPAR s directly or indirectly modulate expression of many genes involved in lipid metabolism, inflammation, proliferation and apoptosis. PPAR γ ligands, thiazolidinediones, are known to improved insulin sensitivity and lipid metabolism. In addition to their role in lipid and glucose metabolism, inflammation, adipocyte differentiation or atherosclerosis, PPAR s play a role in cancer development and represent promising targets for cancer prevention and treatment. However, the effects of PPAR γ in tumours remain still controversial. Activation of PPAR γ, e.g. by exposure to specific ligands, has been shown to exert antitumour activity through induction of differentiation and inhibition of proliferation in a variety of cancers. Despite of these promising results, the target genes involved in the anticancer activity of PPAR γ ligands are still not well understood.

The outcomes of our in vitro analyses showed that PPAR γ ligands (rosiglitazone and ciglitazone) and platinum-based drugs (cisplatin and oxaliplatin) decreased proliferation of different human colon and prostate cancer cell lines in doseand time- dependent manner. Rosiglitazone and oxaliplatin, which were more effective than ciglitazone and cisplatin, were used for all experiments. Our results demonstrated that rosiglitazone increased PPAR γ activity simultaneously with decreasing its nuclear localization (fluorescent immunostaining) and expression (Western blot analysis). Three different protocols were used for analysis of combined effect of rosiglitazone and oxaliplatin. The most effective approach appeared to be rosiglitazone pre-treatment, which was then used for analyses of combined effect in human colon (HT-29) and prostate (BPH-1 CAFTD 03) cancer cell lines. Combination of rosiglitazone with oxaliplatin arrested cells in G2/M phase of the cell cycle, enhanced apoptosis, and increased cell cycle related (cyclin B, p21^Waf1/Cip1 etc.) and DNA damage protein expression (p-H2AX, Chk2, ATM, ATR, etc). Interestingly, after 24 hours rosiglitazone slightly supported BrdU (5-bromo-2- deoxyuridine) incorporation into the nucleus. We suggest that increased percentage of active BrdU cells by rosiglitazone may support the effects of oxaliplatin.

Next, we evidenced that MDM protein is particularly involved in PPARγ signalling in prostate cancer cells. Importantly, we found positive correlation between protein expression in prostate cell line and patient prostate tumour samples. MDMX and PPARγ protein expression were decreased after combined rosiglitazone and oxaliplatin treatment in prostate but not in colon cancer cells. Protein expression of tumour suppressor p53, which is mutated or nonfunctional in our tested cell lines, was not changed either in prostate or in colon cancer cells after this treatment.

Our results bring new knowledge of molecular mechanisms of platinum-based drugs and specific PPARγ ligand effects in prostate and colon cancer cells. Cooperative effects of oxaliplatin and rosiglitazone particularly on regulation of cell cycle and cell death were described. Moreover, we demonstrated important differences of specific protein expression both in cancer cell lines and clinical samples from cancer patients.

This work was supported by grant of Internal Grant Agency of Ministry of Health of the Czech Republic No. NT 11201-5/2010, grant Czech Science Foundation No. 13-09766S, grant FNUSA-ICRC European Regional Development Found no. CZ.1.05/1.1.00/02.0123, grant CZ.1.07/2.4.00/31.0155 and project MUNI/A/0927/2013