A Novel Biological Activity of the STAT3 Inhibitor Stattic in Inhibiting Glutathione Reductase and Suppressing the Tumorigenicity of Human Cervical Cancer Cells via a ROS-Dependent Pathway

INTRODUCTION: Glutathione reductase (GSR) provides reduced glutathione (GSH) to maintain redox homeostasis. Inhibition of GSR disrupts this balance, resulting in cell damage, which benefits cancer therapy. However, the effect of GSR inhibition on the tumorigenicity of human cervical cancer is not fully understood. MATERIALS AND METHODS: Tissue microarray analysis was employed to determine GSR expression in cervical cancer tissues by immunohistochemical staining. Cell death was measured with PI/FITC-annexin V staining. mRNA levels were measured via quantitative RT-PCR. Protein expression was measured by Western blotting and flow cytometry. STAT3 deletion was performed with CRISPR/Cas9 technology. GSR knockdown was achieved by RNA interference. Reactive oxygen species (ROS) levels were measured by DCF staining. GSR enzymatic activity was measured with a GSR assay kit. The effect of GSR inhibition on the growth of tumors formed by cervical cancer cells was investigated using a xenograft model. RESULTS: The expression of GSR was increased in human cervical cancer tissues, as shown by immunohistochemical staining. GSR knockdown by RNA interference in human cervical cancer cell lines resulted in cell death, suggesting the ability of GSR to maintain cancer cell survival. The STAT3 inhibitor 6-nitrobenzo[b]thiophene 1,1-dioxide (Stattic) also inhibited the enzymatic activity of GSR and induced the death of cervical cancer cells. More importantly, Stattic decreased the growth of xenograft tumors formed by cervical cancer cells in nude mice. Mechanistically, tumor cell death induced by Stattic-mediated GSR inhibition was ROS-dependent, since the ROS scavengers GSH and N-acetyl cysteine (NAC) reversed the effect of Stattic. In contrast, pharmacological and molecular inhibition of STAT3 did not induce the death of cervical cancer cells, suggesting a STAT3-independent activity of Stattic. CONCLUSION: Stattic inhibits the enzymatic activity of GSR and induces STAT3-independent but ROS-dependent death of cervical cancer cells, suggesting its potential application as a therapeutic agent for human cervical cancers.