Expression Assay of PTEN, CDK4, and mTOR Genes and Cell Proliferation in Breast Cancer Cell Line (T47D) under Grape Seed Extract Treatment

Document Type : Original Article (s)

Authors

1 Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Assistant Professor, Department of Anatomical Sciences, School of Medicine, Qom Branch, Islamic Azad university, Qom, Iran

3 Professor, Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Assistant Professor, Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: Breast cancer is a highly heterogeneous disease and the most common malignancy in women. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene that reduces in breast cancer. This gene is a phosphatase, and by its lipid phosphatase capacity separates a phosphate from PIP3, and produces PIP2. So, it tends to inhibition of phosphatidylinositol-3-kinase/AKT (PI3K/AKT) pathway and a break in its cellular cycle in phase G1, increase in apoptosis, and decrease in proliferation of cancer cells. The mammalian target of rapamycin (mTOR) gene is activated by phospho/AKT, and its expression increases in many cancers. The cyclin-dependent kinase 4 (CDK4) gene also acts as an oncogene and its expression increases in various types of human cancers, including breast cancer. Grape seed extract (GSE) is a reach source of natural antioxidants, and treatment with it can tend to inhibition of cell cycle and finally, inhibition of cancer progression. This study aimed to assess the expression of PTEN, CDK4, and mTOR genes and cell proliferation in breast cancer cell line (T47D) under GSE treatment.Methods: T47D cells were purchased and cultured and treated with GSE of a concentration of 60 μg/ml. MTT and real time-polymerase chain reaction (PCR) methods were used to evaluate cell viability and gene expression. Colony survival assay method was used to assess colony formation.Findings: The amount of cell life in the treatment group was significantly lower than the control group. Moreover, the results of real time PCR showed that the relative expression of PTEN gene was significantly higher than the control group, and also the expression of mTOR and CDK4 genes reduced.Conclusion: The use of GSE can significantly increase the expression of PTEN tumor suppressor gene, and ultimately cause the death of T47D cancer cells.

Keywords


  1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61(2): 69-90.
  2. Yu S, Kim T, Yoo KH, Kang K. The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Biochem Biophys Res Commun 2017; 486(3): 752-8.
  3. Vestey SB, Sen C, Calder CJ, Perks CM, Pignatelli M, Winters ZE. Activated Akt expression in breast cancer: Correlation with p53, Hdm2 and patient outcome. Eur J Cancer 2005; 41(7): 1017-25.
  4. Easton JB, Houghton PJ. mTOR and cancer therapy. Oncogene 2006; 25(48): 6436-46.
  5. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway: Effective combinations and clinical considerations. Drug Resist Updat 2008; 11(1-2): 32-50.
  6. Moschetta M, Reale A, Marasco C, Vacca A, Carratu MR. Therapeutic targeting of the mTOR-signalling pathway in cancer: Benefits and limitations. Br J Pharmacol 2014; 171(16): 3801-13.
  7. Santo L, Siu KT, Raje N. Targeting cyclin-dependent kinases and cell cycle progression in human cancers. Semin Oncol 2015; 42(6): 788-800.
  8. Tyagi A, Tyagi A, Chan D, Agarwal C, Agarwal R. Grape seed extract sensitizes human breast carcinoma cells to chemotherapy agents-induced growth inhibition and apoptotic death via cytochrome c release and caspase activation pathway. Cancer Res 2006; 66(8 Suppl): 1283.
  9. Dinicola S, Cucina A, Antonacci D, Bizzarri M. Anticancer effects of grape seed extract on human cancers: A review. J Carcinog Mutag 2014; S8: 005.
  10. He Z, Chen AY, Rojanasakul Y, Rankin GO, Chen YC. Gallic acid, a phenolic compound, exerts anti-angiogenic effects via the PTEN/AKT/HIF-1alpha/VEGF signaling pathway in ovarian cancer cells. Oncol Rep 2016; 35(1): 291-7.
  11. Yu S, Kim T, Yoo KH, Kang K. The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Biochem Biophys Res Commun 2017; 486(3): 752-8.
  12. Dinicola S, Pasqualato A, Cucina A, Coluccia P, Ferranti F, Canipari R, et al. Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion. Eur J Nutr 2014; 53(2): 421-31.
  13. Kaur M, Mandair R, Agarwal R, Agarwal C. Grape seed extract induces cell cycle arrest and apoptosis in human colon carcinoma cells. Nutr Cancer 2008; 60(Suppl 1): 2-11.
  14. Mao JT, Lu QY, Xue B, Neis P, Zamora FD, Lundmark L, et al. A pilot study of a grape seed procyanidin extract for lung cancer chemoprevention. Cancer Prev Res (Phila) 2019; 12(8): 557-66.