Abstract
Survival rate of patients affected with anaplastic thyroid carcinoma (ATC) is less than 5% with current treatment. In ATC, BRAFV600E mutation is the major mutation that results in the transformation of normal cells in to an undifferentiated cancer cells via aberrant molecular signaling mechanisms. Although vemurufenib is a selective oral drug for the BRAFV600E mutant kinase with a response rate of nearly 50% in metastatic melanoma, our study has showed resistance to this drug in ATC. Hence the rationale of the study is to explore combinational therapeutic effect to improve the efficacy of vemurafenib along with metformin. Metformin, a diabetic drug is an AMPK activator and has recently proved to be involved in preventing or treating several types of cancer. Using iGEMDock software, a protein-ligand interaction was successful between Metformin and TSHR (receptor present in the thyroid follicular cells). Our study demonstrates that combination of vemurufenib with metformin has synergistic anti-cancer effects which was evaluated through MTT assay (cytotoxicity), colony formation assay (antiproliferation evaluation) and suppressed the progression of ATC cells growth by inducing significant apoptosis, proven by Annexin V-FITC assay (Early Apoptosis Detection). Downregulation of ERK signaling, upregulation of AMPK pathway and precision in epithelial-mesenchymal transition (EMT) pathway which were assessed by RT-PCR and Western blot provide the evidence that the combination of drugs involved in the precision of altered molecular signaling Further our results suggest that Metformin act as a demethylating agent in anaplastic thyroid cancer cells by inducing the expression of NIS and TSHR. Our study for the first time explored cAMP signaling in ATC wherein cAMP signaling is downregulated due to decrease in intracellular cAMP level upon metformin treatment. To conclude, our findings demonstrate novel therapeutic targets and treatment strategies for undifferentiated ATC.
Highlights
Thyroid cancer is one of the most common endocrine malignancies and its incidence is rapidly increasing worldwide
Using iGEMDock software, a protein-ligand interaction was successful between Metformin and Thyroid Stimulating Hormone Receptor (TSHR) in our study and demonstrated that combination of Vemurufenib with metformin has synergistic anti-cancer effects and provide the evidence that the combination of drugs suppressed the progression of anaplastic thyroid carcinoma (ATC) cells growth by inducing significant apoptosis through downregulation of ERK signaling, upregulation of AMPK pathway and by reversing epithelial-mesenchymal transition (EMT) markers - E-cadherin and Vimentin expression
In order to analyze the drug targets at the TSHR protein; drugs such as Metformin, Vemurafenib and 5-Azacytididne were docked with TSHR using iGEMDOCK software and docked scores of those molecules were represented in Table 1, with their binding energy, Vanderwaal energy, electrostatic and hydrogen bond profiles
Summary
Thyroid cancer is one of the most common endocrine malignancies and its incidence is rapidly increasing worldwide. Majority of differentiated thyroid tumors respond well to therapy, anaplastic thyroid cancer is untreatable due to high malignant potential where patients succumb within six months of diagnosis despite intensive treatment approaches including surgery, chemotherapy and radiation [1], [2]. Identifying a novel therapeutic approach and understanding the underlying molecular mechanisms of undifferentiated thyroid cancer is highly desirable to improve patient survival. The proto-oncogene BRAF is situated on 7q24 encodes a serine/threonine kinase, transduces regulatory signals via growth factors, hormones and cytokines and are involved in the regulation of cell proliferation, differentiation and apoptosis through the RAS/RAF/MEK/ERK cascade. Mutation in the BRAF gene results in aberrant activation of ERK signaling and induce tumorigenesis in several human cancers including, melanoma, thyroid and colon carcinoma [7,8,9]
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