Hexyl-pentynoic acid serves as a novel radiosensitizer for breast cancer by inhibiting UCHL3-dependent Rad51 deubiquitination

Objective: To investigate the effects and underlying mechanism of 2-hexyl-4-pentynoic acid (HPTA), a derivative of valproic acid (VPA), on radiotherapy in breast cancer. Methods: MCF7 cells and 7,12-dimethylbenz-[α]-anthracene (DMBA)-induced transformed human normal breast cells (MCF10A–DMBA cells) were irradiated with 8 ​Gy X-rays. For both cells there were four groups: control, valproic acid (VPA)/HPTA, IR, and VPA/HPTA+IR groups. MTT and clonogenic survival assays were performed to assess cell proliferation, and comet assay was performed to evaluate DNA damage. Protein expression of γH2AX, 53BP1, Rad51, and BRCA1 was examined via immunofluorescence and immunoblotting. Cycloheximide chase and ubiquitination experiments were conducted to determine Rad51 ubiquitination. In vivo experiments involved a rat model of DMBA-induced breast cancer, with four fractionated doses of 2 ​Gy. Tumor tissue pathological changes and γH2AX, Rad51, and UCHL3 expression levels were measured by hematoxylin-eosin staining, immunohistochemistry, and immunoblotting. Results: Compared with the IR group, 15 ​μmol/L HPTA reduced the cell proliferation ability of irradiated MCF7 cells (t=2.16, P<0.05). The VPA/HPTA+IR group exhibited significantly increased DNA double-strand breaks relative to those in the IR group (VPA+IR vs. IR, t=13.37, P<0.05; HPTA+IR vs. IR, t=8.48, P<0.05). Immunofluorescence and immunoblotting experiments demonstrated that the VPA/HPTA+IR group displayed significantly increased cell foci formation, γH2AX and 53BP1 protein expression levels compared to the IR group [(γH2AX: VPA+IR vs. IR, t=8.88, P< 0.05; HPTA+IR vs. IR, t=8.90, P< 0.05), (53BP1, VPA+IR vs. IR, t=5.73, P< 0.05; HPTA+IR vs. IR, t=6.40, P< 0.05)]. Further, Rad51 expression was downregulated (VPA+IR vs. IR, t=3.12, P< 0.05; HPTA+IR vs. IR, t ​= ​2.70, P<0.05), and Rad51 inhibition effectively counteracted HPTA-induced radiosensitization. Ubiquitination detection further verified that HPTA inhibits Rad51 expression via UCHL3-dependent Rad51 deubiquitination. In vivo study results showed that 20 ​mg/kg HPTA significantly enhanced the radiosensitivity of breast tumors in rats by inhibiting Rad51 expression. Conclusions: HPTA is a highly effective radiosensitizer that enhances the radiotherapeutic efficacy of breast cancer treatment through UCHL3-dependent deubiquitination of Rad51.