Non-Conventional Metal-Based Anticancer Complexes Targeting DNA Damage Repair and Combinatorial Photodynamic-Chemotherapy to Overcome Cisplatin Resistance

Abstract

Since the serendipitous discovery of the anti-proliferative activity of cisplatin by Barnett Rosenberg in 1965, cisplatin has been widely used in clinics for the treatment of various cancers including bladder, head and neck, lung, ovarian, and testicular cancers. In particular, the cure rate of testicular cancer with cisplatin exceeds 90%. However, the treatment is limited by severe side effects including nephrotoxicity, emetogenesis, and neurotoxicity. The clinical performance of cisplatin is significantly hindered by inherent and acquired resistance. The later approved platinum-based drugs carboplatin and oxaliplatin show better clinical activity than cisplatin, with carboplatin inducing less systemic toxicities and oxaliplatin exhibiting distinct pharmacological properties than cisplatin. Nevertheless, carboplatin is cross-resistant to cisplatin-resistant tumors, and oxaliplatin is moderately active in cisplatin-refractory cancers. Thus, the development of new anticancer agents to overcome cisplatin resistance is a critical goal for anticancer therapy. This thesis aims to develop platinum- and ruthenium-based anticancer complexes to achieve lower cross-resistance and higher cytotoxicity.

Cationic monofunctional platinum(II)-based anticancer agents with a general formula of cis-[Pt(NH3)2(N-donor)Cl]+ have recently drawn much attention due to their unique mode of action and anticancer spectrum as well as promising antitumor activity both in vitro and in vivo. A monofunctional platinum-based anticancer agent SA-Pt containing a bulky salicylanilide moiety was synthesized and evaluated in the first part of this study for understanding of the mechanism of action of the novel monofunctional platinum compound. The compound showed the same level of cytotoxicity against a panel of cancer and normal cells compared with cisplatin. Mechanistic studies revealed that SA-Pt entered cells more efficiently than cisplatin, blocked the cell cycle at the S-phase, and induced apoptosis. SA-Pt bound to DNA as effectively as cisplatin, but did not block RNA polymerase II-mediated transcription as strongly as cisplatin, suggesting that once the compound formed Pt-DNA lesions, the bulky group was more easily recognized and removed.

Ruthenium-based anticancer agents have a more distinct anticancer profile and pharmacological activity than cisplatin. Thus, they are thought to be potential chemotherapeutics to conquer the limitations of cisplatin. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have emerged as anticancer drugs alone or in combination with other chemotherapies. For the purpose of developing ruthenium agents with higher cytotoxicity and more active PARP inhibitors, organometallic ruthenium(II)-arene compounds conjugated with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors were synthesized and characterized spectroscopically in the second part of this study. A systematic investigation was conducted to reveal the mechanism of action of these ruthenium-PARP-1 inhibitor conjugates. The improved antiproliferative activity from the as-prepared complexes in four human cancer cell lines indicates their potential for further development as antitumor drugs. ELISA showed that the complexes inhibited PARP-1 slightly better than the original PARP inhibitors. The most active complex 3 arrested the cell cycle at the G0/G1 phase, bound to DNA efficiently, and inhibited RNA polymerase II-mediated transcription.

Co-treatment of cisplatin with other anticancer agents is an effective strategy to combat cisplatin resistance. In the third part of this thesis, layered double hydroxide (LDH) nanoparticles (NPs) are employed to co-deliver a cisplatin prodrug c,c,t-[diamminedichloro-disuccinatoplatinum(IV)] (DSCP) and a photosensitizer chlorin e6 (Ce6) to cancer cells to achieve combinational therapy. The two agents were ratiometrically co-encapsulated into LDH NPs through an ion-exchange process. The as-prepared nanohybrid showed synergistic cell-killing effects and was significantly active against the proliferation of cisplatin-resistant cancer cells with nanomolar IC50 values. Mechanistic investigations confirmed its action mode of combined chemo- and photodynamic therapy. Also, LDH NPs were able to enhance the cellular accumulation of Pt and Ce6 in both cisplatin-sensitive and -resistant cancer cells.

Increased repair of DNA lesions generated by cisplatin is one of the vital reasons for cisplatin resistance. The heterodimeric ERCC1-XPF complex is a key structure-specific endonuclease in the nucleotide excision repair (NER) pathway that is involved in the repair of cisplatin-induced DNA damage. A Pt(IV) prodrug [Pt(IV)(NH3)2Cl2(OH)L, L = (E)-2-(((8a,9a-dihydro-9H-fluoren-9-ylidene)hydrazono)methyl)benzoate] containing an inhibitor of ERCC1-XPF interaction at its axial position was prepared and characterized in the last part of the thesis. In vitro cytotoxicity assay showed that the prodrug had a significantly elevated cytotoxicity compared with cisplatin in both cisplatin-sensitive and -resistant cell lines. Mechanism studies on the elevated cytotoxicity suggested that the prodrug entered cells more efficiently, arrested the cell cycle at the S phase, and induced apoptosis. An immunoslot-blot assay proved that the prodrug-induced more Pt-DNA intrastrand cross-links (Pt-GG) compared with cisplatin, and the rate of removing Pt-GG adducts was lower in cisplatin-resistant cells treated with the prodrug compared with that of cisplatin. These results imply that the enhanced cytotoxicity of the prodrug can be attributed to the synergetic effects from elevated uptake and repair inhibition. The outcome of this study may help to develop more potent platinum-based anticancer drugs to conquer cisplatin resistance.

Date of Award	8 Jun 2017
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Guangyu ZHU (Supervisor)