Curcumin Inhibits Proliferation And Induces Apoptosis In Malignant Pleural Mesothelioma RN5 Cells

Background and ObjectiveMalignant pleural mesothelioma is an aggressive cancer that develops from pleural mesothelial cells and is usually associated with previous asbestos exposure.The incubation period from asbestos exposure to mesothelioma is approximately 40 years.Asbestos is an important fire prevention,insulation and heat preservation material,but the asbestos fiber can cause diseases such as asbestosis,pleural mesothelioma.It has gained a high degree of attention abroad,most European Union and other developed countries have a total ban on all the use of asbestos.But the incidence of mesothelioma has been increasing,and it is estimated that by 2020,the mortality rate of MPM in most industrialized countries will increase by 5-10%annually.China as a superpower in the production and use of chrysotile.The problem of asbestos security and malignant pleural mesothelioma induced by asbestos needs to be taken seriously.The prognosis of patients with malignant pleural mesothelioma is poor and the efficacy of single treatment is not ideal.Combined surgery,radiotherapy and chemotherapy can improve the symptoms and prolong the survival.The standard first-line chemotherapy regimen is cisplatin plus pemetrexed.But the course of chemotherapy is short and is not effective for some patients.The resistance of tumor cells to apoptosis caused by anticancer drugs is the main cause of failure in chemotherapy.Therefore,it is an important subject involving the study of the anti-apoptosis related pathways and mechanisms in malignant pleural mesothelioma,as well as finding for drugs inhibiting anti-apoptotic pathway to improve the success rate of chemotherapy.Curcumin is a polyphenolic compound extracted from the rhizome of Zingiberaceae and has therapeutic effects on a variety of human tumors.Studies have shown that its anti-tumor effect is mainly to induce tumor cell apoptosis by acting on multiple targets.Apoptosis as one of the main forms of cell death,can be induced by exogenous and endogenous pathways.The exogenous approach is caused by interaction of external death signal with apoptotic receptors on the cell surface which activates the intracellular Caspase cascade;the endogenous approach is due to intracellular signaling leading to changes ofmitochondrial membrane permeability,resulting in the release of various apoptotic factors into the cytoplasm,and eventually apoptosis.The focal point of apoptosis is the change of mitochondrial membrane permeability and the activation of Caspase.Apoptosis-related factors and various signaling pathways form an extremely complex and interactive network in the cell to regulate the occurrence of apoptosis.In the past 30 years,PI3K/Akt/mTOR,as a downstream pathway of receptor tyrosine kinase,has been gaining great attention for its important role in maintaining cellular protein synthesis,survival,growth,metastasis,resistance to apoptosis,and angiogenesis.It has become one of the most important pathways in oncology.Abnormal activation of the PI3K/Akt/mTOR pathway due to oncogenic mutations or inactivation of tumor suppressor factors is a common phenomenon seen in almost all kinds of cancers.Mutations often occur at sites of the inhibitory genes including PTEN and TSC1/2,oncogenes as PIK3CA,PIK3R1 and Akt.It has become an important target for tumor treatment research.It has been reported that curcumin plays an anti-tumor role by inhibiting the PI3K-Akt-mTOR pathway,which is reported to be active in malignant pleural mesothelioma specimens.There were few reports focus on the curcumin treatment of MPM.The mechanism and related signal pathways still need to be further investigated.This study was designed to evaluate the anticancer effect of curcumin in malignant pleural mesothelioma RN5 cell,and to investigate the mechanism by detecting apoptosis factors as well as the proteins involved in the PI3K-Akt-mTOR pathway for providing a better theoretical basis for the application of curcumin in malignant pleural mesothelioma.Part I Effects of curcumin on proliferation and apoptosis of pleural mesothelioma RN5 cells.Objective:The effects of curcumin on proliferation and apoptosis of pleural mesothelioma RN5 cells were investigated in this part.Methods:1.After incubating in different concentrations of curcumin and cisplatin for 72 hours,the cell viability of RN5 cells was analyzed by SRB method,and the semi-inhibitory concentration(EC50)was calculated.2.The RN5 cells were seeded in a 6-well plate and then treated with various concentrations of curcumin for 24 h,whereas cisplatin was used as a positive control,DMSO as a blank control.The effect of drugs on cell cycle was detected by PI labeling combined with flow cytometry;apoptosis of cells was detected by Annexin V-FITC/PI double staining combined with flow cytometry.3.The effects of curcumin in vivo were investigated on tumor-bearing mice.When the largest diameter of tumors reached 6mm,the animals were randomly assigned into four groups:the solvent control group,low-dose curcumin group,high-dose curcumin group and cisplatin group.The mice were treated with intraperitoneal administration of drugs every 5 days.Tumor volumes were determined by measurement fo the diameters.Body weight was measured in order to monitor the toxicity of the drugs.The mice were euthanized when the largest diameter of tumor in the solvent-treated group reached 15 mm.The tumor samples were then collected and the weight was recorded.4.The animal specimens were fixed in 10%formalin and embedded in paraffin.After treatment,immunohistochemical staining of CD31 and Ki67 was performed,and tumor samples were also stained by end-notch in situ labeling method(TUNEL).The staining results were observed under microscope.The H-score was used to evaluate the expression of Ki67 and the proportion of TUNEL-positive cells for semi-quantitative analysis of cell proliferation and apoptosis in tumor sections,and CD31-positive areas were used to assess angiogenesis.Results:1.The results demonstrated that curcumin and cisplatin induced a significant inhibition in RN5 cell viability,which was associated with the concentration of the curcumin and cisplatin,as measured by the SRB assay after 72h of incubation.The EC50 value of curcumin at 72h was 19.1±1.3?M,whereas the EC50 value of cisplatin was 7.09±1.14?M.2.The results of cell cycle experiments showed that the proportion of cells in the G2/M phase increased significantly after 24 hours of curcumin and cisplatin treatment of RN5 cells,compared with the negative control DMSO group.The apoptosis experiment showed that after curcumin and cisplatin treatment,the proportion of early and late apoptotic cells increased,compared with the negative control group.3.Tumor formation was well after RN5 cell inoculated into mice.After injection of 5 times,Tumor volume was significantly reduced following five times injections of cisplatin and high dose curcumin,compared with the solvent control group.The mice were sacrificed on day 25 after tumor cell injection.The mean tumor weight of cisplatin and curcumin group was decreased compared with that of the control group,and the tumor weight of cisplatin group and high-dose curcumin group decreased more significantly.The average weight of the mice in the curcumin group was not significantly reduced compared with the control group,while the body weight of the cisplatin group was significantly lower than that of the control group.4.Immunohistochemistry of tumor specimens showed that the rate of Ki67 positive cells of the animal specimens treated with curcumin and cisplatin were less than that of the control group,and the H-score method was used for semi-quantitative evaluation.In the groups of high-dose curcumin and the cisplatin,the H-score of Ki67 was significantly lower than that of the control group.The positive rate of TUNEL staining of the curcumin and cisplatin groups was higher than that of the control group,and the H scores of TUNEL of the high-dose curcumin group and cisplatin group were significantly higher than that of the control group.CD31 was stained positive in vascular endothelial cells.The CD31 positive areas of curcumin group and cisplatin group were lower than that of the control group,and the positive area of CD31 in high dose curcumin group was statistically decreased compared with the control group.Conclusions:1.Curcumin can inhibit the proliferation of RN5 cells in malignant pleural mesothelioma.2.Curcumin can induce G2/M phase cell arrest,and induce cell apoptosis in RN5 cells.3.Curcumin can inhibit the growth of transplanted tumors in mice,and the toxicity is not obvious.4.Curcumin can inhibit cell proliferation,induce apoptosis and inhibit angiogenesis in animals.Part II Investigation of the apoptosis inducing mechanism of curcumin in RN5 cells.Objective:This section was designed to explore the role of apoptotic related factors as well as the PI3K/Akt/mTOR signaling pathway in curcumin-induced apoptosis of RN5 cells.Methods:1.Cells were seeded in a 10-cm dish and were incubated for 24 h.Cells were then treated with vehicle control(DMSO 0.1%),various doses of curcumin or cisplatinfor 24 h.The level of apoptosis associated proteins such as Caspase-3/cleaved Caspase-3,Caspase-8/cleaved Caspase-8,Caspase-9/cleaved Caspase-9,Bax,Bcl-xL,cleaved PARP,were detected by Western Blot,for a further assessment of the role of endogenous and exogenous apoptotic pathways in the apoptosis induced by curcumin.2.Western blot was performed for the nuclear fraction of AIF protein levels.The nuclear transfer of AIF was evaluated by immunofluorescence for assessment of the role of AIF in the apoptosis induced by curcumin in RN5 cells.3.RN5 cells were inoculated in dishs,the negative control DMSO group and the curcumin group with different concentrations were set,and the cells were incubated with the drug for 24 hours.Western blot was used to detect the level of PI3K/Akt/mTOR pathway-related proteins such as PI3K,Akt/p-Akt,mTOR/p-mTOR,p70S6K/p-p70S6K,for assessment of the role of PI3K/Akt/mTOR pathway in the apoptosis induced by curcumin in RN5 cells.4.RN5 cells were respectively pretreated with PI3K,Akt,and mTOR inhibitors for 2 hours,then incubated with curcumin for 24 hours.Western blot was used to detect apoptosis-related proteins and the level of PI3K/Akt/mTOR pathway-related proteins,for assessment of the key target protein in PI3K/Akt/mTOR pathway with the application of curcumin.Results:1.After incubation of RN5 cells with different concentrations of curcumin,there was no significant change in the expression of Caspase-3 in the cells of each group.The level of cleaved Caspase-3 fragment was increased after cisplatin treatment,but none was detected in the curcumin group or the DMSO group.The expression of Caspase-3,Caspase-8 and Caspase-9 was similar in different group,and no obvious fragment of cleaved Caspase-8 or cleaved Caspase-9 was detected.After curcumin treatment,the level of Bax,an intracellular pro-apoptotic protein was increased in RN5 cells,while the level of Bcl-xL as an intracellular apoptotic inhibitory protein was decreased,and the protein level of cleaved PARP was also increased.The levels of Bax,Bcl-xL,and cleaved PARP in curcumin groups were statistically different compared with DMSO group.2.After the incubation of RN5 cells with curcumin,the nuclear AIF expression was significantly increased after 24 h curcumin treatment compared with DMSO group.Immunofluorescence method was used to detect the nuclear translocation of AIF.and the results indicated that the immunofluorescence intensity of AIF in the nucleus increased with the concentration of curcumin,and the AIF positive immunofluorescence was mainly concentrated in cytoplasm of DMSO group.3.After RN5 cells were incubated with different concentrations of curcumin,the protein levels of PI3K,p-Akt,p-mTOR and p-p70S6K were down-regulated in each experimental group,with a statistically difference compared with the DMSO group.There was no significant difference in the total expression levels of Akt,mTOR and p70S6K.4.RN5 cells were respectively pretreated with PI3K inhibitor LY294002,Akt inhibitor MK 2206 and mTOR inhibitor Rapamycin,,and then incubated with curcumin.After application of the Akt inhibitor MK 2206,we observed that the effect of curcumin on cleaved-PARP?Bax?Bcl-xL and p-Akt was interfered;but for LY294002 and Rapamycin,there was no significant change in the curcumin inhibitory effect on pathway-related proteins or apoptosis-related proteins.Conclusions:1.Curcumin can induce apoptosis in RN5 cells the by a Caspase-independent endogenous mitochondrial apoptosis pathway mediated by AIF nuclear translocation.2.Curcumin can induce apoptosis in RN5 cells by inhibiting the PI3K-Akt-mTOR signal pathway.3.The target protein of curcumin may be Akt,which means curcumin might inhibits tumor proliferation and induces apoptosis by inhibiting Akt phosphorylation.