Inhibition of cyclin D1 by new biguanide 4C increases the sensitivity of proficient homologous recombination repair bladder cancer cells to Olaparib via causing G0 / G1 arrest

Abstract Background Results from recent clinical trials do not support PARP inhibitors as monotherapy in urological tumor. Interestingly, biguanides inhibiting homologous recombination repair (HRR) are thought to increase the sensitivity of proficient HRR (HRR-proficient) cancers to Olaparib, but the mechanism of which is not yet clear. New biguanide derivative 4C in our laboratory inhibited significantly proliferation of BC, given that the effects of biguanides and PARP inhibitors on cell cycle are generally opposite, we explored the anti-BC mechanism of 4C and the efficacy and cause of the combination of 4C and Olaparib in HRR-proficient BC from the cell cycle perspective. Materials and methods In vitro, RT4 and T24 were treated with 4C, Olaparib and combination of the two, MTT for cell viability, RT-PCR for Cyclin mRNA levels , flow cytometry for cell cycle and HRR efficiency, Western Blot (WB) for cyclin and HRR protein expression, immunofluorescence for HRR protein localization and expression, and Comet assay for DNA damage degree. In vivo, we explored the effects of 4C, Olaparib and the combination on tumor growth using the T24 xenograft nude mice model, H&E for the hepatorenal toxicity, and WB and immunohistochemistry for the effects of different treatments on HRR proteins . Results In vitro, 4C induced G0/G1 phase arrest suppressed HRR protein, causing sustained DNA damage, while Olaparib induced S and G2/M phase arrest of HRR-proficient BC and increased HRR protein, causing reversible DNA damage. The two had good combined effects, and the effects on cell cycle and HRR of Olaparib were reversed by 4C when combining the two. Mechanistically, the trend of CCND1 (Cyclin D1) mRNA was consistent with HRR efficiency after different treatments, which is high expression in the two BC cells. silencing CCND1 decreased HRR, and increaed the sensitivity of the two cells to Olaparib, exacerbating DNA damage. The effects of different drugs in vivo were consistent with that in vitro. Conclusions Inhibition of cyclin D1 by new biguanide 4C increases the sensitivity of HRR-proficient BC cells to Olaparib via causing G0 / G1 arrest