Mechanism Of Rosoloactone Inducing Apoptosis In Human Cercival Cancer Cells Through Endoplasmic Reticulum Stress/autophagy Pathway

Background:Cervical cancer is one of the most common gynecological malignancies.Currently,the commonly used clinical treatments are surgery,radiotherapy and chemotherapy.With the continuous improvement of surgical and radiotheraputic techniques,the 5-year survival rate of cervical cancer has not improve significantly.In addition,the incidence has shown a trend of a younger onset age.Radiotherapy has non-neglectful damage to ovarian function in young patients,and the commonly used chemotherapeutic drugs,such as platinum drugs,are often unsatisfactory and inapplicable on cervical cancer due to their side effects and drug resistance issues.Therefore,more and more researchers try to develop low toxic and high efficient tumor suppressing drugs by studying the antitumorigenic mechanism of chemotherapeutic drugs.It is believed that tumor cells could induce endoplasmic reticulum stress(ERS)by interfering with the protein folding process in endoplasmic reticulum under stress conditions such as administrating chemotherapy drugs.ERS activates a complex intracellular signaling pathway called the unfolded protein response(UPR).When the external stimulation is minor or short,the degree of UPR is limited to reconstruct the homeostasis of the endoplasmic reticulum,which stimulate the occurrence of autophagy and trigger the cell adaptive responses,therefore can protect the cell survival.However,when the external stimulation was significant or prolonged,ERS could convert the cytoprotective effects of UPR and autophagy into a pro-cell-death mechanism.Autophagy is a double-edged sword and its mechanism is particularly complex.The inhibition of autophagy can induce apoptosis,meanwhile,the induction of apoptosis can also inhibit autophagy.The interaction between the two may be through ERS or other pathways.Therefore,we expect to use drugs,radiation and other methods to stimulate cells to achieve the pro-death effects or to block the pro-survival strategies,thereby improving the tumor treatment.In order to restore cell homeostasis,anti-tumor drugs can induce ERS through oxidative stress to activate UPR receptors PERK,IRE1 and ATF6 as well as downstream transcription factors XBP-1,ATF4,CHOP,etc.Moreover,the anti-tumor drugs may also lead to apoptosis of mitochondrial pathways by regulating Bcl-2 family proteins such as Bcl-2,Bim,Noxa etc.Therefore,ROS play an important role in apoptosis mediated by ATF4/CHOP axis.Conventional chemotherapy drugs mostly kill tumor cells by increasing intracellular ROS levels.Compared with the disadvantages of traditional chemotherapeutic drugs,natural compounds derived from plants and microorganisms may have more extensive application prospects.Recent studies have shown that diterpenoids are widely found in various organisms in nature and diterpenoids have been proved to have antitumor,antibacterial,antiviral and anti-inflammatory effects.Most studies have suggested that the anti-tumor mechanism of diterpenoid is mainly medicated by inducing oxidative stress and apoptosis in tumor cells.These findings suggest that diterpenoid may also induce endoplasmic reticulum stress.Objective:In this study,we used human cervical cancer He La cells to analysis natural diterpenoids for antitumor activity.Rosoloactone was identified and its antitumor effect and mechanism in inducing apoptosis of human cervical cancer cells via endoplasmic reticulum stress/autophagy pathway were further investigate.Further in vivo experiments were conducted to observe its tumor inhibition effect.We expect to acquire new,promising natural anti-tumor drugs while providing novel directions for the clinical treatment of human cervical cancer.Method:(1)MTT assay was used to screen the diterpenoid that was isolated from the endophytic fungus Trichothecium roseum.(2)Cell viability of He La cells was detected by MTT assay.The apoptotic He La cell nuclear changes were assessed with Hoechst 33258.Expression of Cleaved Caspase-3 was detected by Western Blot.Apoptotic He La cell populations were quantified by flow cytometry analysis.(3)The expression level of ER stress related proteins including Grp78,p-e IF2α,ATF4,GADD153/CHOP,Cleaved Caspase-4 and ubiquitinated proteins were detected by Western Blot.The distribution of ubiquitinated proteins in cells was observed by indirect immunofluorescence and laser confocal microscopy.(4)Expression of p62,LC3,Beclin-1 and Cleaved Caspase-3 in cells was detected by Western Blot assay.Colocalization of LC3 and p62 was detected by indirect immunofluorescence.(5)DCFH-DA method detects the level of ROS in He La cells.(6)Expression of cytochrome C,Cleaved Caspase-9,Bcl-2 and Bax in He La cells was detected by Western Blot assay.(7)A mouse model of human cervical cancer was established,the tumor volume and body weight of mice were observed and measured.The expression levels of Bcl-2,Bax,and Cleaved Caspase-3 proteins in tumor tissues of tumor-carrying mice were detected by Western Blot.Result:1.Rosoloactone was obtained,a natural diterpenoid with significant antitumor activity,by screening the metabolites of the endophytic fungus Trichothecium roseum.Rosoloactone significantly inhibits the proliferation of human cervical cancer He La cells in a time-dependent and dose-dependent manner.Compared with the cisplatin positive control group,the Rosoloactone group can induce apoptosis in He La cells.2.Rosoloactone increased the expression of ER stress-related proteins Grp78,p-e IF2α,ATF4 and ubiquitinated proteins in He La cells.Meanwhile,the expression levels of apoptosis related proteins induced by ERS were significantly up-regulated with the prolongation of Rosoloactone including GADD153/CHOP and Cleaved Caspase-4.These results suggest that Rosoloactone can induce endoplasmic reticulum stress-mediated apoptosis in He La cells.3.In He La cells treated with Rosoloactone for 4h and 8h,the expression of Beclin-1 increased significantly,the ratio of LC3-II/LC3-I increased,and the expression of p62 gradually decreased.Meanwhile,indirect immunofluorescence results demonstrated the colocalization of p62 and LC3-II,the protein expressions mentioned above were down-regulated in Rosoloactone treated cells for 16 h.These findings indicated that Rosoloactone activated autophagy,and the degree of endoplasmic reticulum stress was further intensified with the prolongation of the treatment,which lead to reduced autophagy activity of cells and eventually result in apoptosis.4.Rosoloactone caused a continuous accumulation of ROS in He La cells and an increase in the ratio of Bax/Bcl-2.Meanwhile,cytochrome C in the mitochondria was released into the cytoplasm and the expression level of Cleaved Caspase-9 was increased.These results suggest that Rosoloactone,like other diterpenoids,can induce apoptosis in tumor cells through mitochondrial pathways.5.The results showed that Rosoloactone could reduce tumor volume,but had no significant effect on body weight of mice compared with the control group in vivo experiments.Bax/Bcl-2 ratio and Cleaved Caspase-3 expression levels were increased in tumor tissues.Conclusion:In this study,human cervical cancer He La cells were used as the cell model.We found that the natural diterpenoid Rosoloactone induces the mitochondrial apoptosis in tumor cells,it also induces the apoptosis of human cervical cancer cells through endoplasmic reticulum stress/autophagy pathways.At the same time,the vivo experiments proved that it has a good anti-tumor effect.1.Rosoloactone,like other diterpenoids,can induce the mitochondrial apoptosis in tumor cells.Rosoloactone up-regulates Bax/Bcl-2 and Cleaved Caspase-3 by increasing the generation of reactive oxygen species,which leads to the release of cytochrome C into the cytoplasm causing mitochondrial dysfunction and inducing mitochondrial-mediated apoptosis.2.Rosoloactone can induce ERS,and lead to accumulation of misfolded proteins in He La cells,then up-regulate the expression of CHOP and Cleaved Caspase-4,and finally induce ERS mediated apoptosis in He La cells.3.At the initial stage of Rosoloactone treatment on He La cells,endoplasmic reticulum stress activates autophagy.With the prolongation of treatment,the stress level of endoplasmic reticulum is further intensified,and the autophagy activity of cells is reduced,then the cells undergo apoptosis mediated by endoplasmic reticulum stress/autophagy pathways.4.Rosoloactone could inhibit tumor growth and had no significant effect on body weight of mice in vivo experiments.