WASH regulates the oxidative stress Nrf2/ARE pathway to inhibit proliferation and promote apoptosis of HeLa cells under the action of Jolkinolide B

Abstract

Jolkinolide B (JB), a diterpenoid compound isolated from the roots of Euphorbia fischeriana, has gained research attention for its antitumor effects. In recent years, JB reportedly displayed anti-tumor activity in solid tumors, such as breast, ovarian, and prostate cancer, and leukemia. In this study, we evaluated the effect of JB on HeLa cells with a focus on cell growth inhibition and related mechanisms. HeLa cells were cultured in vitro and divided into a blank control group, HeLa-Scramble (0, 0.25, 0.5 mM), and Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) protein silenced group, HeLa-shWASH (0, 0.25, 0.5 mM). Morphological changes were observed using an inverted microscope. The inhibition rate of cell proliferation was detected using the WST-1 method. Flow cytometry Brdu+PI double standard method was used to detect cell replication ability and FITC+PI double standard method was used to detect cell apoptosis rate. Western blot was used to verify the expression of Nrf2, HO-1, WASH, Bax, Bcl-2, and PCNA. The mRNA expression of cytokines (IL-1α, IL-6, and IL-8) was detected using RT-qPCR. The results showed that JB induced cell apoptosis and arrested cells at the G2/M phase in HeLa-shWASH cells compared with HeLa-Scramble cells (P < 0.05, P < 0.01, respectively). In addition, JB upregulated IL-1α, IL-6, and IL-8 in HeLa-shWASH cells. We conclude that WASH protein participates in JB-induced regulation of the Nrf2/ARE pathway, aggravates inflammatory responses, and promotes cancer cell apoptosis, thus inhibiting the proliferation and invasion abilities of HeLa cells. JB may have anti-tumor effects and potential clinical value for the treatment of cervical cancer.

Keywords

• JB
• Apoptosis
• Cervical cancer
• Nrf2/ARE pathway
• WASH protein