Abstract
Background:Radioresistance remains a challenge for cancer radiotherapy. The present study aims to investigate the role of TMPRSS4 in triple negative breast cancer (TNBC) cell radiosensitivity. Materials and Methods:After transfection of MDA-MD-468 triple negative breast cancer cells line by using the lentivirus vector, the effect of TMPRSS4 down-regulation on TNBC radiosensitivity was evaluated by using cloning assay and CCK-8 assay. The CCK-8 assay was also used for performing cell proliferation analysis. Western blot was carried out to detect the expression of certain proteins related to cell cycle pathways (cyclin D1), cell apoptosis pathways (Bax, Bcl2, and Caspase3), DNA damage and DNA damage repair (TRF2, Ku80 , ˠH2AX) . The cell cycle and cell apoptosis were also investigated using flow cytometer analysis. Results: TMPRSS4 expression was down-regulated in MDA-MB-468 cells which enhanced MDA-MB-468 cells radiosensitivity. TMPRSS4 silencing also improved IR induced cell proliferation ability reduction and promoted cell arrested at G2/M phase mediated by 6 Gy IR associated with cyclin D1 expression inhibition. Moreover, TMPRSS4 inhibition enhanced TNBC apoptosis induced by 6 Gy IR following by over-expression of (Bax, Caspase3) and down-regulation of Bcl2 as the pro-apoptotic and anti-apoptotic proteins, respectively. Otherwise, TMPRSS4 down-regulation increases DNA damage induced by 6 Gy IR and delays DNA damage repair respectively illustrated by downregulation of TRF2 and permanent increase of Ku80 and ˠH2AX expression at 1 h and 10 h post-IR. Conclusion:Down-regulation of TMPRSS4 increases triple negative breast cancer cell radiosensitivity and the use of TMPRSS4 inhibitor can be encouraged for improving radiotherapy effectiveness in TNBC radioresistant patients.
Highlights
Breast cancer is cancer frequently diagnosed in about 84% of countries in the world and the leading cause of cancer death in over 54% of world countries (Bray et al, 2018)
The CCK-8 test indicated that IR decreased the viability of the cell in a dose-dependent manner (Figure 1A) associated with the high decrease at 8 Gy in MDA-MB-468-shTMPRSS4 groups compared to MDA-MB-468-NC [(53.00% ± 4.6% vs 82, 22% ± 2.3%); Figure 1A;***P< 0.001)]
Colony formation assay showed that transmembrane protease serine4 (TMPRSS4) silencing significantly decrease colony number in irradiation dose-dependent manner (0,2 and 4Gy) with a high decrease at 4 Gy in MDA-MB-468-shTMPRSS4 groups compared to MDA-MB-468-NC (Figure 1B,***P
Summary
Breast cancer is cancer frequently diagnosed in about 84% of countries in the world and the leading cause of cancer death in over 54% of world countries (Bray et al, 2018). In 2012, the number of the new cases occur was estimated at 1, 7 million and 520.000 deaths (Torre et al, 2012 and 2016; Lee et al, 2018,Chen et al, 2015) against 2, 1 million and 626.679 death in 2018 (Bray et al, 2018) Based on these data, breast cancer can be considered as an important public health issue and more research have to be encouraged for its preventive and curative treatment. Materials and Methods: After transfection of MDA-MD-468 triple negative breast cancer cells line by using the lentivirus vector, the effect of TMPRSS4 down-regulation on TNBC radiosensitivity was evaluated by using cloning assay and CCK-8 assay. Conclusion: Down-regulation of TMPRSS4 increases triple negative breast cancer cell radiosensitivity and the use of TMPRSS4 inhibitor can be encouraged for improving radiotherapy effectiveness in TNBC radioresistant patients
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