ANTI-CANCER ACTIVITY OF SANGUINARINE ON HUMAN PAPILLARY THYROID CANCER CELL LINES BY TARGETING JAK/STAT3 PATHWAY

Abstract

Background: Papillary thyroid carcinoma is the furthermost common type of thyroid cancer, representing 85 % of all thyroid cancer cases. It occurs more frequently in women and usually affects 20-55 year age group. Aim: This study intended to explore the potential underlying mechanisms of sanguinarine (SNG) mediated anti-cancer actions in papillary thyroid cancer (PTC) cell line, BCPAP and TPC-1. Methods: PTC cell lines were cultured, and cell viability experiments were performed following treatment with SNG for 24 hours using cell counting kit-8 (CCK-8) assay. Apoptosis was measured using fluorescent AnnexinV/PI stain and investigated by flow cytometry. Western blot was done following treatment with SNG and other compounds to identify different proteins associated with apoptosis and autophagy. Further, LC3 transfection was done using GFP-LC3 plasmid to transfect LC3 protein. To explore the underlying mechanisms and sensitizing potential of SNG, PTC cells were treated with N-Acetyl cysteine ,VAD and cisplatin respectively, alone and in combination with SNG and expression of protein markers were done. Functional assay such as: scratch assay and colony assay were performed to assess the cell's migration and colony formation ability after SNG treatment. Furthermore, effect of SNG on stemness markers were also studied. Results: SNG inhibits proliferation/cell viability of PTC cells dose dependently. Real time cell analyzer (RTCA) findings supports that SNG mediated growth inhibition of PTC cells is time and dose dependent. SNG treatment downregulates constitutive expression of JAK/STAT3 in PTC cells. SNG mediated cell death occurs through activation of caspases and induction of double strand DNA breaks. z-VAD-FMK, a pan-inhibitor, partially prevents SNG mediated cell death suggesting involvement of caspases-cascades in SNG mediated apoptosis. SNG induced autophagy demonstrated by enhanced LC3 expression, and LC3 transfection resulted in cytoplasmic puncta formation. SNG induced molecular changes leading to apoptosis or growth inhibition is due to ROS involvement. Furthermore, our data also indicates that IL6 play important role in SNG mediated downregulation OF STAT3. Interestingly, we also observed that SNG potentially sensitizes PTC cells to anticancer drug cisplatin. Conclusion: SNG activates apoptosis in PTC cells through the inactivation of STAT3 survival pathway. SNG demonstrated caspase dependent apoptosis. In addition, drug combination of SNG with cisplatin enhances anti-cancer effect on PTC, which makes it a potential therapeutic