The Inhibitory Effect Of Mullein Combined With Cisplatin On Lung Adenocarcinoma SPC-A1 Cells And Its Molecular Mechanism

Purpose:SPC-A1 cells in human lung adenocarcinoma were selected as our study subjects,with the aid of exploring the effection of cell cycle progression?apoptosis?invasion and metastasis after treated with calycosin combined with the chemotherapy drug of cisplatin,and the possible molecular mechanisms of its inhibition of cell cycle progression?inducing apoptosis and inhibition of invasion and metastasis were also be analyzed,to determine whether the combination of calycosin and cisplatin can increase the sensitivity of cisplatin to the inhibitory effect of lung adenocarcinoma of SPC-A1 cells,it is expected to provide new ideas for reducing the dosage of cisplatin in the treatment of lung adenocarcinoma,improving cisplatin resistance and reducing the toxic and side effects of chemotherapy.Material and methods:1.The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma treated by calycosin combined with cisplatin1.1.The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma by calycosin.The concentration and action time of calycosin were detected by MTT assay.After the SPC-A1 cells were treated by the final concentration?5?15?25?50?75?100?M?of calycosin with 12?24?36 and 48 hours,optical density value?OD value?were measured at the wavelength of 570nm,the cell proliferation rate and the IC₃₀ and IC₅₀ values of 12?24?36 and48 hours after the action of calycosin were calculated.The effect of different concentrations?0?25?50?100?M?of calycosin on the cell cycle distribution of SPC-A1 cells after 24 hours were detected by flow cytometry assay.1.2 The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma by calycosin combined with cisplatin and its molecular mechanism.The effect on the proliferation inhibition rate of SPC-A1 cells after treated by different drugs?50?M of calycosin,20?M of cisplatin,50?M of calycosin combined with 20?M of cisplatin?with 12?24?36 and 48 hours were detected by MTT assay.The effect of different drugs?50?M of calycosin,20?M of cisplatin,50?M of calycosin combined with 20?M of cisplatin?on the cell cycle distribution of SPC-A1 cells with 24 hours were detected by flow cytometry assay.Further,the protein expression levels of CyclinD1 and CDK4 in SPC-A1 cells were detected after treated with each group drug of SPC-A1 cells by Western blot assay,in order to explore the possible molecular mechanism of the cell proliferation inhibition of SPC-A1 cells in human lung adenocarcinoma by calycosin combined with cisplatin.2.The effect on apoptosis the of SPC-A1 in lung adenocarcinoma cells treated by calycosin combined with cisplatin and its molecular mechanism.2.1 The effect on the apoptosis of SPC-A1 cells in human lung adenocarcinoma by calycosin.The nuclear morphology of apoptotic cells were observed by Hoechst33258 staining;the effect on the apoptosis rate of SPC-A1 cells after treated by different concentrations?0?25?50?100?M?of calycosin with 12?24?36 and 48 hours were detected by Annexinv-fitc/PI double staining.2.2 The effect on apoptosis the of SPC-A1 cells in human lung adenocarcinoma treated by calycosin combined with cisplatin and its molecular mechanism..The nuclear morphology of apoptotic cells were observed by Hoechst33258 staining;the effect on the apoptosis rate of SPC-A1 cells after treated by different drugs?50?M of calycosin,20?M of cisplatin,50?M of calycosin combined with 20?M of cisplatin?with12?24?36 and 48 hours were detected by Annexinv-fitc/PI double staining.Further,after the SPC-A1 cells were treated by each group drugs,the protein expression levels of Bcl-2?Bax?Cyt-c and Cleaved-caspase9 in mitochondrial pathway of SPC-A1 cells and PTEN?Akt and p-AKT in PTEN/P13K/Akt signaling pathway were detected by Western blot assay,in order to investigate the possible molecular mechanism of apoptosis induced of SPC-A1 cells in lung adenocarcinoma by calycosin combined with cisplatin.3.The effect on migration and invasion of SPC-A1 in lung adenocarcinoma cells treated by calycosin combined with cisplatin and its molecular mechanism..3.1 The effect on migration and invasion of SPC-A1 cells in lung adenocarcinoma treated by calycosin.The effect on the migration rate of SPC-A1 cells after treated with different concentrations of calycosin were detected by transwell migration assays.The cells injury healing rate were detected after treated with different concentrations?0?25?50?100?M?of calycosin by scratch test;the effect on the invasion rate of SPC-A1 cells after treated with different concentrations of calycosin were detected by transwell invasion assays.3.2 The effect on migration and invasion the of SPC-A1 cells in lung adenocarcinoma treated by calycosin combined with cisplatin and its molecular mechanism.The effect on the migration rate of SPC-A1 cells was detected by transwell migration assays and cell scratch test.The cells injury healing rate was detected after treated with different drugs?50?M of calycosin,20?M of cisplatin,50?M of calycosin combined with 20?M of cisplatin?by scratch test;the effect on the invasion rate of SPC-A1 cells after treated with different drugs were detected by transwell invasion assays.The protein and mRNA expression levels of MMP2and MMP9 were detected by Western blot and real-time fluorescence quantitative PCR after treated with different drugs,in order to investigate the possible molecular mechanism of migration and invasion of SPC-A1 cells in lung adenocarcinoma by calycosin and calycosin combined with cisplatin.Results:1.The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma treated by calycosin combined with cisplatin.1.1 The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma by calycosin.The results of MTT assay showed that,the proliferation of SPC-A1 cells was not significantly inhibited when the concentration of calycosin was lower than 15?M.When the concentration of calycosin was higher than 25?M,with the increasing of the concentration and the extending of time,the proliferation rate of SPC-A1 cells was decreased significantly in a concentration and time dependence;the cell proliferation rate decreased slowly after 36hour,there was no significant difference with that at the 48th hour?p>0.05?,the results indicated that the inhibitory of cell proliferation was concentration dependent but not-time dependent after 36h of SPC-A1 cells.The cell IC30 and IC50 values were calculated by MTT assay.The values of IC₃₀ at 12th?24th?36th and 48th hours were 72.49?M?52.32?M?35.16?M and 32.45?M,;and the values of IC₅₀0 at 12th?24th?36th and 48th hours were 124.34?M?101.30?M?99.02?M?97.47?M.Flow cytometry assay results showed that calycosin can affect the cycle distribution of SPC-A1 cells,with the increasing of concentration,the proportion of cells in S phase decreased significantly,while the proportion of cells in G0/G1phase increased significantly,indicating that calycosin can inhibit the G1/S conversion in G1phase and prevent the cell process of SPC-A1 cells.1.2 The effect on cell cycle progression of SPC-A1 cells in human lung adenocarcinoma by calycosin combined with cisplatin and its potential molecular mechanism.The results of MTT assay showed that the proliferation inhibition rate SPC-A1 cells was significantly increased after treated with the drugs in each experimental group;The inhibitory of cell proliferation of calycosin combined with cisplatin group was significantly higher than that of cisplatin alone group.Flow cytometry assay results showed that,all the drugs in the experimental group could inhibit the G1/S phase transformation of cells,the proportion of G0/G1 cells was significantly increased,while that of S phase cells was significantly decreased;when calycosin combined with cisplatin,the decline of S phase cells was significantly higher than that of cisplatin group.Western blot assay results showed that,the protein expression levels of CyclinD1 and CDK4 of SPC-A1 cells were down-regulated after treated with each experimental group compared with control group;when calycosin combined with cisplatin,the down-regulation levels of the protein of CyclinD1 and CDK4 in cells were significantly higher than those in cisplatin group.These results suggest that,the inhibition of cell cycle progression of SPC-A1 cells was more obvious when calycosin was combined with cisplatin,and the possible molecular mechanism may be downregulated the protein expression levels of Cyclin D1 and CDK4.2.The effect on apoptosis the of SPC-A1 in lung adenocarcinoma cells treated by calycosin combined with cisplatin and its molecular mechanism.2.1 The effect on the apoptosis of SPC-A1 cells by calycosin.The staining results of Hoechst33258 showed that,when the SPC-A1 cells were treated by calycosin for 24h.The normal nuclei showed diffuse uniform blue fluorescence,dense particles and clumps of fluorescence can be seen in the nucleus or cytoplasm,with the color turning white when apoptosis occurs.With the increasing of the concentration of calycosin,the number of cells with apoptotic morphology also increased.Annexinv-fitc/PI double staining results showed that,with the increasing of the concentration and the extending of time,the apoptosis rate of SPC-A1cells was increased significantly,and had the concentration and time dependence;the cell apoptosis rate increased slowly after 36 hour,there was no significant difference with that at the 48th hour?p>0.05?,the results indicated that the effect on apoptosis of SPC-A1 cells by calycosin was concentration dependent but not-time dependent after 36h.The results showed that calycosin could induce apoptosis of SPC-A1 cells in a concentration dependent manner.2.2 The effect on the apoptosis of SPC-A1 cells in human lung adenocarcinoma treated by calycosin combined with cisplatin and its molecular mechanism.The staining results of Hoechst33258 showed that,when the SPC-A1 cells were treated with calycosin combined with cisplatin for 24h,the nuclear morphology changed obviously,the apoptotic cells were bright and obviously smaller,adherent cells were reduced,the nuclei were concentrated,marginalized,and the number of apoptotic bodies was significantly increased.Annexinv-fitc/PI double staining results showed that,all the drugs in the experimental group could increase the apoptosis rate of SPC-A1 cells compared with control group.when calycosin combined with cisplatin,the apoptosis rate of cells was significantly higher than that of cisplatin group.The results showed that calycosin could promote the apoptosis of SPC-A1 cells induced by cisplatin.Western blot assay results showed that,the protein expression levels of Bax?Cyt-c and Cleaved-caspase 9were all up-regulated and the levels of Bcl-2 were down-regulated after treated with each experimental group compared with the control group;the protein expression levels of PTEN were up-regulated and p-AKT were down-regulated,but the levels of Akt showed no significant changes of SPC-A1 cells after treated with each experimental group compared with the control group;the up-regulation levels of the protein of PTEN?Bax?Cyt-c and Cleaved-caspase9 in cells were significantly higher than those in cisplatin group,and the down-regulation levels of the protein of p-AKT and Bcl-2 in cells were significantly lower than those in cisplatin group,but the levels of Akt showed no significant changes.These results suggest that inducing apoptosis of SPC-A1 cells was more obvious treated by calycosin combined with cisplatin,and the possible molecular mechanism is maybe by regulating the protein expression levels of Bcl-2?Bax?Cyt-c and Cleaved-caspase 9 in mitochondrial pathway and PTEN and p-AKT in PTEN/P13K/Akt signaling pathway of SPC-A1 cells.3.The effect on migration and invasion of SPC-A1 cells in lung adenocarcinoma treated by calycosin combined with cisplatin and its molecular mechanism.3.1 The effect on migration and invasion of SPC-A1 cells in lung adenocarcinoma treated by calycosin.Scratch test results showed that,when the SPC-A1 cells were treated with different concentrations of calycosin,the cells scratch injury healing rate was significantly lower compared with the control group in a concentration dependence;results of transwell migration experiments showed that,after treated with different concentrations of calycosin,the number of cells migrating through the polycarbonate membrane was significantly lower than that of the control group,and the inhibition rate of migration was significantly higher than that of the control group with concentration dependence.Results of transwell invasion experiments showed that,after treated with different concentrations of calycosin,the number of cells invading through the polycarbonate membrane was significantly lower than that of the control group,and the inhibition rate of invasion was significantly higher than that of the control group with concentration dependence.The results showed that calycosin could inhibit the migration and invasion of SPC-A1 cells in a concentration dependent manner.3.2 The effect on migration and invasion of SPC-A1 cells in lung adenocarcinoma treated by calycosin combined with cisplatin and its molecular mechanism.The results of scratch test and transwell migration test were showed that,in the experimental group,the cell scratch injury healing rates were all reduced,the number of cells migrating through the polycarbonate membrane were all reduced,the cells migration inhibition rates were were all rised compared with control group;The results of transwell invasion test was showed that,the number of cells invading through the polycarbonate membrane were all lower and the cells invading inhibition rates were all higher than that of control group.When calycosin combined with cisplatin,the results of scratch test and transwell migration test were showed that,the cell scratch injury healing rate was significantly lower than that of cisplatin single drug group,the number of cells migrating through the polycarbonate membrane was significantly lower and the cells migration inhibition rate were significantly higher than those of cisplatin single drug group.Results of transwell invasion experiment showed that,the number of cells invading through the polycarbonate membrane was significantly lower than that of the cisplatin single drug group,and the rate of invasion was significantly lower than that of the cisplatin single drug group.These results suggest that calycosin can enhance the sensitivity of cisplatin to inhibit the invasion and metastasis of SPC-A1cells.Western blot and realtime fluorescence quantitative PCR results showed that,the protein and mRNA expression levels of MMP2 and MMP9 were down-regulated of SPC-A1 cells after treated with each experimental group compared with the control group;when calycosin combine with cisplatin,the down-regulation levels of the protein and mRNA of MMP2 and MMP9 in cells were significantly lower than those in cisplatin group These results suggest that the inhibition of migration and invasion of SPC-A1 cells were more obvious treated by calycosin combined with cisplatin,the possible molecular mechanism is maybe by regulating the protein and mRNA expression levels of MMP2 and MMP9.Conclusion:1.Calycosin can inhibit the rate of cell proliferation,prevent cell cycle progression,induce apoptosis,and inhibit cell invasion and metastasis of SPC-A1 cells in lung adenocarcinoma.2.When calycosin was combined with cisplatin,the inhibition of cell cycle progression of SPC-A1 in lung adenocarcinoma cells was more obvious than that of cisplatin alone,and had synergistrc effect with them.The possible molecular mechanism may be downregulated the protein expression levels of Cyclin D1 and CDK4.3.When calycosin was combined with cisplatin,inducing apoptosis of SPC-A1 in lung adenocarcinoma cells was more obvious than that of cisplatin alone,the possible molecular mechanism may be achieved by regulating the protein expression levels Bcl-2?Bax?Cyt-c and Cleaved-caspase 9 in mitochondrial pathway of SPC-A1 cells and PTEN and p-AKT in PTEN/P13K/Akt signaling pathway4.When calycosin was combined with cisplatin,the inhibitory of migration and invasion of SPC-A1 cells in lung adenocarcinoma cells were more obvious than those of cisplatin alone,the possible molecular mechanism may be achieved by regulating the protein and mRNA expression levels of MMP2 and MMP9...