Introduction Withaferin-A(WFA) is a natural compound extracted from Wihania somnifera plant which showed tumour growth inhibiting effects in both in vitro and in vivo models.of various cancers such as colorectal cancer, ovarian cancer, breast cancer, gastric cancers, glioma and hepatocellular carcinoma. Current commonly used therapeutic agents such as cisplatin (CDDP) are effective on cancer cells however there is a lack of efficiency on cancer stem cells (CSCs) which are considered to be responsible of recurrence and metastasis. Although anti-proliferative effect of WFA on non-small cell lunger cancer (NSCLC) cells was shown in previous studies, its effect on NSCLC CSCs remains unclear. Based on this fact, the aim of this study was to evaluate the in vitro effect of WFA on CD133 +NSCLC CSCs.
Material and methods A549 NSCLC cell line was incubated in DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Penicilline/Streptomycin at 5% CO2 and 37°C. Cells were incubated with WFA 24, 48, 72 hours of 2.5,10,20 and 50 µM WFA and 75,100 µM CDDP and their combinations. Cell viability was determined by the WST-1 assay.CD133+ A549 cells were isolated with magnetic beads. The purity of CD133 positivity was assessed by flow cytometry. A549 cells and CD133+ A549 cells were treated with 50 µM WFA, 100 µM CDDP and incombination for 24 hours. Apoptosis was evaluated by flow cytometry with annexin V and PI staining. Furthermore DNA damage and cell proliferation of CD133+ A549 cells were determined by flow cytomerty with BrdU and H2AX stainings.
Results and discussions WFA caused 18,6% early apoptosis (EA), 36,2% late apoptosis (LA), 56% anti-proliferation while CDDP caused 34,3% EA, 2,5% LA,%31,3% anti-proliferation and WFA+CDDP caused 45,6% EA, 34,5% LA 63,8% anti-proliferation on A549 cells. Also WFA 21,6% EA 38,3% LA, 6,6% DNA damage while CDDP caused 30% EA, 4,4% LA, 7,0% DNA damage and WFA+CDDP caused 21,9% EA, 42,9% LA,6,1% anti-proliferation on A549 CSCs.
Conclusion WFA showed anti-proliferative and apoptosis inducing effects of both NSCLCs and their CD133 +CSCs. In addition WFA did not change anti-tumour effect of CDDP and their combination showed more anti-proliferative and apoptosis inducing effect than their administration alone. In addition, WFA caused DNA damage in NSCLC CSCs. Although CDDP has critical anti-tumour effects, it might lead to adverse effects such as ototocity, neprotoxicity, peripheral neuropathy. Therefore, systemic effects of the natural compound WFA are suggested to be evaluated in animal models in future studies.
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