Sinha, Saptarshi, Chatterjee, Subhajit, Paul, Subarno, Das, Biswajit, Dash, Somya Ranjan, Das, Chinmay, and Kundu, Chanakya Nath
Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Here, we aim to investigate whether a bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES + OLA combination in breast cancer has been evaluated using in vitro , ex vivo , and in vivo preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR pathway proteins, caused a late S/G2 cell cycle arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES + OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR pathway proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES + OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES + OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also simultaneously inhibiting the HR pathway. (1) Exposure of cancer cells to the DNA damaging agent, Resveratrol (RES), causes a double-stranded break (DSB) within the DNA molecule. (2) Activation of PARP1 and PARP1-mediated PARylation take place at the DNA damage site. (3) PARylated PARP1 interacts with BRCA1 and induces activation of HR pathway proteins (MRN complex, RPA70, BRCA2, RAD51, etc.) by forming a PAR chain around the HR protein complex. (4) Auto-PARylation of PARP-1 results in dissociation of PARP-1 from the HR complex resulting in full progression of HR repair. (5) Successful and error-free repair of the DSB by HR cascade leads to cancer cell survival. (6) When RES-treated breast cancer cells are exposed to Olaparib (OLA), the functions of PARP1 are inhibited by PARP1-trapping at the DNA damage site. Inhibition of PARP1-mediated PARylation terminates the interaction between PARP1 and BRCA1. (7) This causes inactivation of HR pathway proteins and cessation of the HR pathway. (8) HR pathway inhibition leads to accumulation of DSBs in and around the DNA damage site. (9) This results in cell cycle arrest, apoptosis, and cancer cell death (Created with BioRender.com). Olaparib enhances Resveratrol-mediated apoptosis in breast cancer cells by inhibiting the homologous recombination repair pathway. [Display omitted] • Olaparib enhanced the resveratrol-induced cytotoxicity in breast cancer cells. • Olaparib inhibited PARylation, and blocked PARP1-BRCA1 interaction. • HR pathway inhibition led to cell cycle arrest and apoptosis of the cancer cells. • Results were confirmed in patient-derived breast cancer cells and xenograft mice. [ABSTRACT FROM AUTHOR]