Fangchinoline diminishes STAT3 activation by stimulating oxidative stress and targeting SHP-1 protein in multiple myeloma model

Abstract

Introduction: The development of cancer generally occurs as a result of various deregulated molecular mechanisms affecting the genes that can control normal cellular growth. Signal transducer and activator of transcription 3 (STAT3) pathway, once aberrantly activated can promote carcinogenesis by regulating the transcription of a number of oncogenic genes.

Objectives: Here, we evaluated the impact of fangchinoline (FCN) to attenuate tumor growth and survival through modulation of oncogenic STAT3 signaling pathway using diverse tumor cell lines and a xenograft mouse model.

Methods: To evaluate the action of FCN on STAT3 cascade, protein levels were analyzed by Western blot analysis and electrophoretic mobility shift assay (EMSA). Translocation of STAT3 was detected by immunocytochemistry. Thereafter, FCN-induced ROS was measured by GSH/GSSG assay and H2DCF-DA. FCN-induced apoptosis was analyzed using Western blot analysis and flow cytometry for various assays. Finally, anti-cancer effects of FCN in vivo was evaluated in a myeloma model.

Results: We noted that FCN abrogated protein expression levels of STAT3 and upstream signals (JAK1/2 and Src). In addition, FCN also attenuated DNA binding ability of STAT3 and its translocation into the nucleus. It altered the levels of upstream signaling proteins, increased SHP-1 levels, and induced substantial apoptosis in U266 cells. FCN also promoted an increased production of reactive oxygen species (ROS) and altered GSSG/GSH ratio in tumor cells. Moreover, FCN effectively abrogated tumor progression and STAT3 activation in a preclinical myeloma model.

Conclusion: Overall, this study suggests that FCN may have a tremendous potential to alter abnormal STAT3 activation and induce cell death in malignant cells along with causing the suppression of pathogenesis and growth of cancer through a pro-oxidant dependent molecular mechanism.

Keywords: Apoptosis; DAPI, 4′,6-Diamidino-2-Phenylindole, Dihydrochloride; DMEM, Dulbecco’s Modified Eagle Medium; FBS, Fetal bovine serum; FCN, Fangchinoline; Fangchinoline; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GSH; HRP, Horseradish peroxidase; ICC, Immunocytochemistry; IHC, Immunohistochemistry; JAK, Janus kinase; MMP, Matrix metalloproteinase; Multiple myeloma; NT, Non treat; P/S, Penicillin-streptomycin; PARP, Poly (ADP-ribose) polymerase; ROS; RT-PCR, Reverse transcription polymerase chain reaction; RTCA, Real-time cell analysis; SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase-1; STAT3; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor; c/w, Cell per well; ip, Intraperitoneal injection.