Effect Of Okadaic Acid On Proliferation And Apoptosis Pathway Mechanism In Hela Cells

The most common genital malignant tumor in women is cervical cancer.Due to various biological factors and behavioral factors,the prevalence of cervical cancer remains high.At present,the treatment method of cervical cancer including surgical resection,chemotherapy and radiotherapy.Surgical resection is accepted by people who are have no fertility needs,and those are in the early of cancer.Both chemotherapy and radiotherapy can cause a great killing effects on tumors,but the side effects are strong.As a result,some people decide to give up therapy.Therefore,the task of finding low-toxic,high-efficiency anti-tumor drugs is urgent.Okadaic acid,because it can efficiently select and inhibit the phosphoproteinases PP1 and PP2 A,scientists have used it as a powerful tool for studying cell signals.And many studies have shown that okadaic acid can inhibit the proliferation of tumor cells and induce apoptosis of tumor cells in a certain concentration range.In this paper,we selected the maritime algae toxin-Okadaic acid.We researched the mechanism of Okadaic acid on HeLa cell proliferation,cell morphology and cell cycle.We studied the effect of Okadaic acid on apoptosis of HeLa cell.Including nuclear morphology and DNA,reactive oxygen species,mitochondrial depolarization,apoptosis and mitochondrial pathwayrelated enzymes,protein expression.In addition,we explored the pathway by which Okadaic acid induces apoptosis in HeLa cell.In order to explored new theoretical foundation for treating cervical cancer and using of Okadaic acid.Results:MTT assay results showed that within a certain concentration range of 0-120ng/ml,Okadaic acid inhibited the proliferation of HeLa cells,showing a dose and time dependent.The results of trypan blue exclusion and phase contrast microscopy showed that compared with the control group,the number of living cells decreased with the increase of drug concentration the cell morphology gradually became round.The cell cycle flow pattern showed that the proportion of G0/G1 phase decreases with the increase of drug concentration.The proportion of S phase was basically unchanged.The proportion of G2 /M phase increased from 16.39% to 37.61%.The result is that the Hela cells treated with Okadaic acid were arrested at G2/M phase.The results of Hoechst 33258 staining showed that the cells in the control group had clear outlines.The cells connected with each other.The nucleus was clear and complete.After dosing treatment,the nucleus is marginalized and distributed along the cell membrane.Some cell nucleus was crescent-shaped.With the appearance of apoptosis body following Okadaic acid treatment.The results of agarose gel electrophoresis showed that trapezoidal strips appeared in the dosing group,compared with the control group.With the increasing of drug concentration,the band intensity and the number of bands increased.This indicated that DNA is fragmented after Okadaic acid was treated with HeLa cells.After drug treatment,ROS content increased significant and mitochondrial depolarization was severe.It was initially shown that Okadaic acid can cause oxidative stress and destroy mitochondrial membrane potential in HeLa cells.The Annexin-V/PI double staining result showed that apoptosis was observed after Okadaic acid treatment of cell.Most of the cells were in late apoptosis,and a few were in early apoptosis.The apoptosis rate was increased gradually.The apoptosis rate was reached 56.97% and 82.16% at 50 ng/ml and 100 ng/ml.The results showed that Okadaic acid can induce Hela cells apoptosis.The results of detecting Caspase-3 and Caspase-9 enzyme activities showed that the expression of Caspase-3 and Caspase-9 was increased in the dosing group.It indicated that drug treatment can activate the activity of Caspase-3 and 9 enzymes and induce the apoptosis by Caspase-3 and 9.The mitochondrial pathway-associated proteins Bax,Bcl-2,Bid and Caspase-3 were detected by Western Blot.The results showed that the expression level of Bax,Bid,and Caspase-3 were increased and expression level of bcl-2 was falling.It was shown that after Okadaic acid acts on HeLa cells,apoptosis can be induced by a mitochondrial pathway dependent on Caspase.Conclusion:After the action of Okadaic acid on Hela cells,it can inhibit cell proliferation.It can arrest the Hela cell cycle in the G2/M phase.Okadaic acid can induce apoptosis in Hela cells,and this apoptosis may be mediated through the mitochondrial pathway.