Alternative strategies to overcome Cisplatin side effects and resistance

Cisplatin is one of the most effective chemotherapeutic agents used in the treatment of many type of tumours including non-small cell lung cancer, ovarian, testicular, glioblastoma, neuroblastoma and others. Cisplatin is able to form bonds with the N7 atom on guanine and adenine bases and when this damage is not repaired, DNA replication and transcription were stopped, inducing death of the cells. Despite the clinical benefit provided by Cisplatin many patients undergo phenomena of resistance and toxicity (especially ototoxicity, nephrotoxicity and neurotoxicity). Cisplatin resistance is generally multifactorial and arises through different mechanisms; one of this is correlated to high activity of PARP-1, an enzyme that modify proteins by poly-adenosine ribosylation (named PARylation). There are many targets of PARylation, one of these are histones. Moreover, high PAR levels correlate with weak infiltration by CD8+ cytotoxic lymphocytes (CTL) in human NSCLC. To overcome Cisplatin resistance and toxicity, alternative strategies were proposed: 1) the use of alternative platinum compounds, such as [Pt(O,O'-acac)(γ-acac)(DMS)], a platinum (II) complex containing acetylacetonate (acac) and a dimethylsulpide (DMS) in the coordination sphere of the metal and synthetized by the team of Prof. Fanizzi (University of Salento, Lecce), considering its activity and mechanism of action respect to Cisplatin and 2) the study of new mechanisms of resistance linked to a different immune response between Cisplatin sensitive and resistant tumour cells. For the first strategy we used human glioblastoma T98G cell line. Our results suggest that PtAcacDMS is able to induce apoptosis and necrosis with a concentration five-fold lower respect to Cisplatin (10µM respect to 40µM). Furthermore, ultrastructural analysis of TEM revealed an intense process of autophagy takes place in the cells confirm by the analysis of autophagic markers. Different cell death can be regulated by calcium and by oxidative stress. We find that PtAcacDMS induce an acute increase in [Ca2+]i respect to CDDP that was likely to be due to extracellular Ca2+ entry and enhanced both cytosolic and endoplasmic reticulum Ca2+ concentration after 48h of treatment. Increase of oxidative stress, especially the expression of ROS, can also represents a mechanism of cell death of PtAcacDMS and can be modulate the modification of chromatin and thus gene expression. Further experiments are needed to confirm these data, in particular the analysis of specific mechanisms involved in the modulation of intracellular calcium and the analysis of apoptosis and autophagy when cells growing in absence of Ca2+. For the second stage we used mouse LLC (Lewis lung cancer) non-small cell lung cancer cell line. After generating cell lines resistant to Cisplatin, followed by their characterization with respect to PARylation, cells were injected in immunocompetent C57BL/6 mouse to evaluate the immune infiltrated. In vivo results show differences in immune infiltrate, especially in TAMs and Treg, more expressed in resistant cells, but to confirm if PARP-1 is implicated in this modulation it will be necessary to manipulate the expression of PARP-1 in the clones CDDP-resistant, silencing its expression by siRNA, to evaluate whether these injected cells exhibit the same immune infiltrate as the WT cells.