| Epithelial ovarian cancer(EOC)continues to be the most frequent gynaecologic malignancy,and it ranks as the first leading cause of gynaecologic cancer-related mortality among women worldwide.More than 70% of affected women are diagnosed at an advanced stage because early-stage disease is usually asymptomatic and symptoms of late-stage disease are nonspecific.so the EOC is also known as the "silent killer." Although treatment for EOC,including surgery and platinum/paclitaxel-based chemotherapy,has improved,the overall survival rate of patients remains at ～40%,with a devastating diagnosis,and 80% of these patients who receive standard treatment will relapse and die due to chemoresistance.Acquired chemoresistance remains a major obstacle for the cure of EOC.Therefore,more effective drugs or novel effective treatments are still urgently needed.Autophagy,also known as typeⅡprogrammed cell death(PCD),is a key intracellular degradative process that is generally characterized beginning with autophagosome formation,vesicle fusion and autolysosome formation and ultimately participates in recycling to sustain cellular metabolism and cellular homeostasis.Tumor cells can use autophagy to adapt to various stresses such as hypoxia,hunger,ischemia,etc.to promote cell survival and proliferation.Current genetic and pharmacological studies have also demonstrated that autophagy exerts an important role in antineoplastic therapy such as radiotherapy,chemotherapy and targeted treatment processes,to help tumor cells adapt to the treatment pressure and produce resistance.Numerous studies have shown that to inhibit autophagy by the use of autophagy inhibitors(3-MA,chloroquine,hydroxychloroquine and so on)or the knockout of autophagy key genes(inhibition of ATG5,ATG7,etc.)can enhance the therapeutic effect of many anti-tumor drugs on ovarian cancer,suggesting that autophagy caused by anti-tumor drugs is closely related to ovarian cancer prognosis.Therefore,when looking for new anti-tumor drugs for ovarian cancer,it is important to focus on whether the drugs could enhance the autophagy activity and to investigate the molecular mechanism to induce autophagy.Natural active ingredients originating from Chinese herbal medicines have been indicated to be beneficial in the prevention and treatment of cancer for hundreds of years.To develop new drugs by use of active ingredients from Chinese medicine for cancer treatment has been favored by researchers,because of its ideal effect,multi-target role,high biosecurity and easy to clinical transformation.Recent studies have shown that a variety of active ingredients of traditional Chinese medicine with anti-cancer activity can activate cell autophagy and play a dual role in tumor cells:most studies suggest that induced autophagy plays a cytoprotective role to antagonize apoptotic cell death,a small number of studies have found that autophagy can lead to cell death,so to study whether Chinese medicine induces autophagy and the role in its anti-tumor effect,which has great significance for the development of new anti-tumor drugs and improve its therapeutic effect.Paeonol(Pae),a phenolic acid compound derived from the root bark of the Moutan Cortex,which can be used clinically to treat cardiovascular disease,diabetes,arthritis,etc.,and show good safety.In recent years,Pae was shown to exhibit favourable anticancer activities in ovarian cancer cells and other types of cancer cell lines.Although the antitumour activity of Pae has been suggested by cumulative evidence,the detailed underlying mechanisms have not been investigated.In particular,the effect of Pae on autophagy activity in tumour cells and the internal connection between autophagy and antineoplastic activity are unclear.This study mainly investigated the above-mentioned problems and the corresponding molecular mechanism.In this study,we found that Pae induced not only antiproliferation activity and apoptosis but also autophagy,and complete autophagic flux was observed in A2780 and SKOV3 cells.Moreover,the test doses of Pae were markedly less cytotoxicity toward human normal ovarian epithelial cell IOSE80.In addition,combination treatment with Pae and an autophagy inhibitor(3-methyladenine and hydroxychloroquine)showed significant synergetic effects on inhibiting cell viability and promoting apoptosis in vitro and in the A2780 xenograft model,without severe side effects which is often had by cisplatin few side effects.These results indicate that autophagy induced by Pae plays a cytoprotective role in both A2780 and SKOV3 cells.Mechanistically,we found that Pae inhibited the protein kinase B(Akt)/mammalian target of rapamycin(m TOR)pathway.Furthermore,when combined with the inhibitors MK2206 and rapamycin to inhibit Akt and m TOR kinase activity,Pae-induced autophagy was increased.Taken together,our results demonstrate that Pae induced cytoprotective autophagy by inhibiting the Akt/m TOR pathway in ovarian cancer cells.Thus,the strategy of combining Pae with an autophagy inhibitor to block Akt/m TOR-dependent autophagy could enhance the antitumour activity of Pae and warrants further application for the treatment of ovarian cancer.Part 1 Effect of paeonol on the growth of ovarian cancer cells and its mechanismsObjective:To explore and further verify the effect and mechanism of paeonol on the growth of ovarian cancer cells,while research on the effect of paeonol on normal epithelial cells.Methods:Human ovarian cancer cell lines A2780 and SKOV3 were treated with paeonol at different concentration gradients for 24 h and 48 h,respectively,and normal ovarian epithelial cells IOSE80 were used as negative controls.CCK-8 method was used to detect the effect of paeonol at different concentrations on the viability of ovarian cancer cells.The cell colony formation assay was used to detect the effect of paeonol at different concentrations on the ovarian cancer cell clone formation rate.Annexin-V PE / 7-AAD double stained cells were detected by flow cytometry for changes in apoptosis rate.Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2 and Bax in ovarian cancer cells.Results:1.Pae exhibits an antiproliferative effect in A2780 and SKOV3 cells.CCK-8 results showed that paeonol significantly inhibited the survival rate of A2780 and SKOV3 cells in a concentration-and time-dependent manners,and the cytotoxicity of human normal ovarian epithelial cells IOSE80 was significantly reduced.The results of colony formation assay showed that colony formation in both cell lines was significantly inhibited by a concentration of 1.2 m M Paeonol.2.Pae-induced ovarian cancer cell apoptosis.The results of flow cytometry showed that an increase in both early and late apoptosis in A2780 and SKOV3 cells was induced in a dose-dependent manner after paeonol treatment.There was no significant increase in early and late apoptotic rates in IOSE80 cells.Western Blot analysis showed that the ratio of Bcl-2 protein / Bax in paeonol-treated group was lower than that in the control group,and it showed a concentration-dependence manner.Part 2 Paeonol induces cytoprotective autophagy via blocking the Akt/m TOR pathway in ovarian cancer cellsObjective:To investigate whether Pae simultaneously induced autophagy and exerted antitumour activity in A2780 and SKOV3 cells,and to explore specific molecular mechanisms.Methods:1.Human ovarian cancer A2780 and SKOV3 cells were treated with paeonol at different concentration gradients and time gradients(optimal concentration),respectively.The expressions of autophagy-related proteins LC3 and p62 were detected by Western Blot.After treatment with Pae(1.2 m M)for 24 h,A2780 and SKOV3 cells were fixed in 4% glutaraldehyde overnight and then fixed with 1%osmium tetroxide.Observe the changes of ultrastructure under electron microscope.A2780 cells were pretransfected with autophagy double-labeled adenovirus m RFP-GFP-LC3.After paeonol was treated with ovarian cancer cells at different time gradients,changes in autophagy flow were observed under a confocal microscope.2.In order to clarify autophagy mediated by Pae plays a cytoprotective role,or induces autophagic cell death,A2780 cells were pretransfected with autophagy double-labeled adenovirus m RFP-GFP-LC3.The autophagy inhibitor 3-MA or HCQ combined with paeonol were applied to A2780 and SKOV3 cells,respectively.Western Blot was used to detect the expression of autophagy-related proteins LC3 and p62 in each group.The m RFP and GFP LC3 fluorescence signals were observed under a confocal microscope;CCK-8 method was used to detect the effect of cell viability;Annexin V-PE / 7-AAD kit was used to detect the change of apoptosis rate by flow cytometry.3.To ascertain whether the Akt/m TOR pathway plays an important role in Pae-induced autophagy,Western Blot was used to detect expression changes in Akt,m TOR,and p70S6 K proteins,as well as p-AKT,p-m TOR,and p-p70S6 K proteins,which are key proteins of the Akt / m TOR signaling pathway in ovarian cancer cells treated with paeonol at different concentrations.Akt inhibitor MK2206 or m TOR inhibitor Rapamycin was combined with paeonol to treat ovarian cancer cells.Western Blot was used to detect the expression of Akt / m TOR signaling pathway related proteins and autophagy-related proteins in the cells for verification.Results:1.Pae induces autophagy and activates autophagy flux in ovarian cancer cells.To investigate whether Pae simultaneously induced autophagy and exerted antitumour activity in A2780 and SKOV3 cells,Western Blot analysis showed that LC3-Ⅱ expression increased in a dose-and time-dependent manner in A2780 and SKOV3 cells after Paeonol treatment,and the increased level of LC3-Ⅱ conversion in Paeonol-treated cells were significantly increased not only following treatment with0.6 m M and 1.2 m M Paeonol but also after treatment for 24 h and 48 h.Furthermore,in A2780 and SKOV3 cells,the expression of p62 decreased after Paeonol treatment in a dose-dependent manner.Transmission electron microscopy(TEM)results showed that multiple autophagosomes or autolysosomes appeared in both A2780 and SKOV3 cells in the paeonol-treated group.To dynamically visualize LC3-labelled cytoplasmic vacuolation to further clarify whether the complete progression of autophagy was affected by paeonol,a tandem m RFP-GFP-LC3 adenovirus was transfected into A2780 and SKOV3 cells.This result suggests that Paeonol treatment can accumulate both autophagosomes and autolysosomes,consistent with the Western Blot results.The tandem fluorescent markers observed and quantified by confocal microscopy strongly confirmed that Pae activated complete autophagic flux.2.Autophagy inhibitors inhibit autophagy induced by paeonol in ovarian cancer cells.Western Blot data showed that 3-MA attenuated autophagy induced by Pae,decreased LC3-Ⅱ protein expression and increased p62 protein levels.HCQ induced LC3-Ⅱ and p62 protein accumulation,consistent with previous studies.Furthermore,we used confocal microscopy to analyse m RFP and GFP LC3 puncta.3-MA could significantly inhibit autolysosome accumulation in A2780 cells after exposure to Pae,and HCQ could accumulate yellow autophagic LC3 puncta(m RFP+/GFP+).3.The combination treatment of Pae with 3-MA and HCQ enhanced the antitumour effects in ovarian cancer cells.Compared with paeonol treatment alone,CCK-8 assays showed that the combination of 3-MA and HCQ with paeonol could strengthen the inhibitory effect of paeonol on cell viability.Annexin V-PE/7-AAD assays showed that the combination of Pae with 3-MA or HCQ also significantly increased the number of apoptotic ovarian cancer cells.4.Inhibition of the Akt/m TOR signalling pathway is required for Pae-induced cell autophagy.To ascertain whether the Akt/m TOR pathway has an important role in Paeonol-induced autophagy,we investigated key proteins related to the Akt/m TOR pathway in Paeonol-treated ovarian cancer cells by Western Blot analysis.First,the phosphorylated levels of Akt,m TOR,and p70S6 K were analysed.Compared with the untreated control,treatment with Paeonol did not significantly change total Akt or m TOR expression,whereas it significantly decreased p-Akt,p-m TOR,and p-p70S6 K protein expression in A2780 and SKOV3 cells in a dosedependent manner.Furthermore,to further investigate the inhibition of the Akt/m TOR signalling pathway in paeonol-induced autophagy,we rescued paeonol-induced Akt/ m TOR inhibition by decreasing p-Akt with MK2206 and analysed LC3-Ⅱ conversion in paeonol-treated ovarian cancer cells.Our finding shows that LC3-Ⅱ conversion was significantly increased in paeonol-treated cells.Moreover,rapamycin(an m TOR inhibitor)significantly reduced p-m TOR expression and increased LC3-Ⅱ conversion in Paeonol-treated cells.Part 3 Combination treatment with Pae and hydroxychloroquine enhances antitumour activities in a xenograft animalObjective:To investigate the effects of paeonol,combined paeonol and autophagy inhibitors on the growth of transplanted tumors in ovarian cancer animal models.Methods:Ovarian cancer cell transplantation tumor models were constructed and randomly divided into 3 groups based on 7 animals in each group: DMSO group,paeonol group,paeonol + HCQ combined group.Draw the growth curve according to the tumor volume.Observe the toxicity of paeonol group or paeonol + HCQ group to animals in vivo.collect the transplanted tumor tissue for HE staining.IHC method to detect the expression of the apoptosis related protein and the autophagy related protein in the ovarian cancer nude mice transplanted with paeonol or paeonol and HCQ treatment.Observe the changes of important organs of heart,liver,lung and kidney.Results:We generated a xenograft tumour model to determine whether Pae induced anticancer activity in vivo.The xenograft mice were treated with DMSO control(without Pae),Pae(40 mg/kg)or a combination of Pae(40 mg/kg)and HCQ(60mg/kg).Tumour volume and weight were reduced in the Pae and Pae+HCQ groups,especially in the Pae+HCQ group,and there were significant differences between each group.Our data confirmed the in vitro results and strongly indicate that combination treatment with the autophagy inhibitor HCQ enhances the antitumour activities of Pae.Compared with the untreated control,H&Esections of tumor tissue showed large areas of necrosis.Immunohistochemistry(IHC)staining of tumor tissue revealed decrease in levels of Ki-67 and Bcl-2 protein expression as well as increase of in level of LC3 protein in Pae-treated and Pae+HCQ-treated tumour tissues,whereas the protein of p62 expression was decreased in Pae treated tumour tissues but increased in Pae+ HCQ-treated tumour tissues compared with the control group.In addition,the protein expression level of bcl-2,Ki-67,LC3 and p62 expression between Pae and Pae+HCQ groups was significant differences.Furthermore,Body body weight of Mice mice in the Pae group and Pae+HCQ group maintained normal gain throughout during the treatment.And H&E staining of organs revealed no significant major organ-related toxicity in the combined groups compared with the untreated control.These data suggest that combination therapy of Pae and the autophagy inhibitor HCQ enhances antitumour activity in vivo and has low levels of organ-related toxicity,and further confirmed the in vitro results related autophagy.Conclusion:1.Paeonol can inhibit ovarian cancer cell growth by inhibiting cell proliferation and selectively inducing apoptosis in human ovarian cancer cell lines.2.Pae induces autophagy and activates autophagy flux in ovarian cancer cells,and the autophagy play a cytoprotective role.3.Inhibition of the Akt/m TOR pathway is required for paeonol-induced autophagy in ovarian cancer cells.4.Combination therapy of Pae and the autophagy inhibitor HCQ enhances antitumour activity in vivo and has low levels of organ-related toxicity,and further confirmed the in vitro results related autophagy. |
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