Effect Of Activin A On NS-1 Cell Death

Activin A is a double-stranded glycoprotein that was first extracted in the follicular fluid in 1986,it promotes the release of follicle-stimulating(FS)hormone and belongs to the growth factor-?(TGF-?)superfamily.Activin A has been shown to have multipotent biological effects,such as affecting early embryonic development,regulating the proliferation and apoptosis of tumor cells and so on.Besides the two classic methods of apoptosis and necrosis,there are also many ways of cell death,such as autophagy,phroptosis,and ferroptosis.Thus,in this study we selected mouse myeloma cell line NS-1 cells to investigate the inductive effect of Activin A on the dealth of NS-1 cells,the results of the study showed that:1.The effect of Activin A on the viability of NS-1 cells.The viability was detected by MTT assay.The results showed that Activin A inhibited the viability of NS-1 cells.Giemsa staining further showed that the number of viable cells decreased significantly and small fragments of cells were detected after treated with Activin A for 24 h,suggesting that Activin A may inhibit the viability of NS-1 cells and induce cell death.2.The effect of Activin A on the apoptosis of NS-1 cells.In order to determine whether Activin A can induce death of NS-1 cells by inducing apoptosis,flow cytometry was adopted to detect the percentage of apoptosis cells by using Annexin-V/7-AAD double staining.The results showed that the proportions of late apoptosis increased after treated with Activin A.In order to investigate the apoptosis mechanism of NS-1 cells induced by Activin A,we use western blotting and RT-PCR at the same time to detect the expression of the signal transduction molecules and related apoptotic proteins of Activin A,it showed that Activin A can up-regulate the expression of ActRIIA,Smad3 and p-Smad3,and also up-regulate the expressions of CHOP,caspase-3,caspase-12 and GADD34 proteins;overexpression of Smad3 increased CHOP,caspase-3,caspase-12 and GADD34 apoptosis related protein expression.We constructed the NS-1 cell line which high expresses Smad3 genes by transient transfection with Lipofectamine 2000,and we found that the over-expression of Smad3 increased the expression of CHOP and caspase-3.The results suggested that Activin A may induce the apoptosis in NS-1 cells via the endoplasmic reticulum(ER)stress pathway and Smad3 signal transduction pathway mediates the effect of Activin A on the apoptosis of NS-1 cells.3.The effect of Activin A on the ferrroptosis of NS-1 cells.To explore the effect of Activin A on ferroptosis in NS-1 cells,we measure the level of intracellular ROS by DCFH-DA probe and detecting the level of glutathione peroxidase(GPX)and divalent metal transporter 1(DMT1)by using the GPX kit and western blotting.The results showed that Activin A promotes the production of cytoplasmic ROS in NS-1,inhibits the level of intracellular GPX activity and GPX4 and up-regulates the level of DMT1 protein,while Ferrostatin-1,the ferroptosis inhibitor can attenuate the effects of Activin A on the level of GPX and GPX4,suggesting that Activin A may also induces the dealth by promoting the ferroptosis in NS-1 cells.4.The effect of Activin A on the growth of transplanted tumor of NS-1 cells in mice.To determine the effect of Activin A on the growth of myeloma in vivo,we established the transplantation model of NS-1 cells in Balb/c mice.After the tumor formation of NS-1 cells,the tumor was injected with Activin A,and we observed the growth of tumors everyday.The results showed that the tumor growth volume of the intradermal injection group of exogenous Activin A was significantly lower than that of the intradermal injection group of normal saline,suggesting that Activin A can also inhibit the growth of NS-1 cell solid tumor in vivo.In conclusion,Activin A may induce the apoptosis of NS-1 cells through endoplasmic reticulum stress pathway and may promote the ferroptosis in NS-1 cells,result in inducing NS-1 cell death,suggesting that Activin A has the potential to be used as a drug to inhibit the proliferation of myeloma.