اثرات سمیت سلولی و آپوپتوزیسی نانوذرات سلنیوم بر روی رده سلولی سرطان کولون (ht29)

هدف: امروزه نانوذرات اهمیت کاربردی بالایی را در تشخیص و درمان سرطان پیدا کرده‫اند. هدف از این مطالعه ارزیابی اثرات سایتوتوکسیک و ضد سرطانی نانوذرات سلنیوم بر روی رده سلولی سرطان کولون و آنالیز بیان ژن  cad  caspase-activated dnaseمی‫باشد. مواد و روش‫ها: در این مطالعه، ابتدا اثرات سمیت سلولی نانوذرات سلنیوم در مدت زمان 24 ساعت بر روی رده سلولی سرطان کولون ht-29 و نرمال hek293 توسط روش mtt مورد بررسی قرار گرفت. به‫دنبال آن، پس از تیمار سلول‫ها با غلظت 50 درصد کشندگی،rna  سلول استخراج شده و به cdna تبدیل شد. میزان بیان ژن cad با استفاده از روش real time pcr مطالعه شد. در نهایت، مطالعه آپوپتوزیس و نکروزیس با کمک روش فلوسایتومتری مورد ارزیابی قرار گرفت. نتایج: نتایج نشان داد که نانوذره سلنیوم در غلظت‫های µg/ml25/31 تا µg/ml500 بیشترین مهار تکثیر سلولی را داشته که از لحاظ آماری معنی‫دار بوده است (p<0.001). میزان ic50 برای نانوذرات سلنیوم در مدت زمان 24 ساعت µg/ml 75 محاسبه شد. بیان ژن cad نسبت به ژن مرجع در رده سلولی ht29 تیمار شده با نانوذرات سلنیوم طی 24ساعت به‫میزان 0/125±4/04  برابر افزایش یافت. همچنین داده‫های فلوسایتومتری بیانگر افزایش به‫میزان 34/71 درصدی مرگ آپوپتوزیسی در رده سلولی سرطانی کولون بود. نتیجه گیری: با توجه به داده‫های افزایش بیان ژنی درگیردر آپوپتوزیس (cad) و فعال نمودن آپوپتوزیس می توان از نانوذرات سلنیوم به‫عنوان کاندیدای دارویی در درمان سرطان کولون استفاده نمود، که نیاز به مطالعات بیشتری در خصوص اهمیت دارویی نانوذرات دارد.

cytotoxicity and apoptotic effects of selenium nanoparticles toward ht29 colon cancer cells

aims: nanoparticles due to their wide applications in medicine,industry,and biotechnology, have attracted many scientists’ attentions. recently, nanoparticles especially selenium nanoparticles are widely used to diagnosis and cancer treatment. the aim of this study was to evaluate the cytotoxic and anticancer effects of selenium nanoparticles on colon cancer cell line and analysis of cad (caspase activated dnase) gene expression.material and methods: in this study, colon cancer ht29 and normal hek293 cell lines were purchased from the pasteur institute cell bank of tehran and treated with selenium nanoparticles overnight. the cells were cultured in dulbecco’s modified eagle’s medium (dmem) (gibco, scotland) medium with 10% fbs serum and 1% streptomycin antibiotic (gibco, scotland). the cells were then stored at 37 ° c. in this study, cytotoxic effect of selenium nps was evaluated on ht29 and hek293 cells using mtt (3-(4, 5-dimethyltetrazollium bromide) assay. subsequently, they were treated with selenium nanoparticles in different concentrations (0, 7.81, 15.62, 31.25, 62.5, 125, 250 and 500 mg/ml) for 24 hours. to solubilize the viable cells formazan crystals production, we added 100 μl/well of dimethyl sulfoxide (dmso) to them. after treatment of ht29 cells with ic50 concentration, the total rna was extracted and cdna synthesized. moreover, cad gene expression was evaluated using real time pcr method. the data was evaluated by abi stepone utilizing the applied biosystems qrt-pcr (abi 7300 system, applied biosystems). the quantification of the mode of selenium nps -induced cell death in the ht29 cells were ascertained using flow cytometry followed by staining with fluorescein isothiocyanate (fitc)‐annexin v and propidium iodide (pi) staining. finally, the study of apoptosis and necrosis of selenium nps was evaluated using flow cytometry method. data analysis was statistically determined by using one-way analysis of variance (anova) with spss/22 software followed by a tukey test.results: the result showed that the treatment of selenium nps at 31.25 to 500 µg/ml concentration had maximum cytotoxic effect, revealed statistically significant (p˂0.001). the ic50 value for selenium nps were measured at 75 µg/ml after 24 hours. in order to determine the effect of selenium nps on cancerous cells, alterations in the mrna expression levels of cad gene in ht29 cells were done by qrt-pcr technique followed by the exposure to nanoparticle. the cad gene expression comparing to reference gene was up-regulated 4.04±0.125 fold. to determine the mechanism of cell death in the cancer cells, annexin v/pi flow cytometry was carried out. in the treatment of ht29 cells by ic50 of selenium nps, 10.43%, and, 24.28% of early and late stages’ apoptosis were observed, respectivelyconclusion: our results suggest that selenium nps can display some promising cytotoxic properties through inducing apoptosis pathway. based on the results, up-regulated gene expression involved in apoptosis (cad) and activating apoptosis, it can be concluded that the selenium nps can be used as drug candidate in colon cancer treatment, but more studies are needed regarding the medicinal importance of nanoparticles.