| Part ?EFFECTS OF PEMETREXED COMBINED WITH ICOTINIB/ERLOTINIB AT DIFFERENT TIME ON LUNG ADENOCARCINOMA CELLS IN VITROBackgroundAt present,lung cancer is the world's highest morbidity and mortality of malignant tumors,of which more than 85%of non-small cell lung cancer,and adenocarcinoma accounted for a large proportion of clinical diagnosis of advanced patients,the loss of the timing of surgery,For in patients with advanced lung adenocarcinoma,chemotherapy is one of the essential treatments,of which pemetrexed is more tolerant and has been used in the first-line treatment of advanced lung adenocarcinoma.Pemetrexed as a novel chemotherapeutic agent that inhibits tumor growth by inhibiting thymidine synthase,dihydrofolate reductase and glycine amide nucleotide transcarboxylase antagonizing folate metabolism,blocking nucleic acid synthesis.However,pemetrexed did not improve the treatment of lung adenocarcinoma patients with the overall survival rate,the efficacy has entered the plateau,regardless of the recent or long-term,the effect is not very satisfactory.In recent years,with the development of molecular technology,molecular targeted drug therapy has become the focus of lung adenocarcinoma research,lung adenocarcinoma treatment has occupied a very important position.For patients with lung adenocarcinoma with activated epidermal growth factor receptor mutations,epidermal growth factor tyrosine kinase inhibitors have become the most effective first-line therapy,but many evidence suggests that EGFR-TKIs are not effective for all patients with mutations.EGFR-TKIs are recommended as first-line drugs for patients with advanced lung adenocarcinoma sensitive to EGFR mutations,but phase III clinical studies such as IPASS and OPTIMAL suggest that non-small cell lung cancer patients benefit from EGFR-TKIs alone without overall survival,progression-free survival was secondary resistance after 10-13 months or so,with objective and effective IPASS 43%;OPTIMAL 83%.To explore a better approach to comprehensive treatment is urgent,in order to further explore new treatment in order to extend the survival and improve the objective and effective,many scholars mentioned whether chemotherapy can be combined with EGFR-TKIs treatment of non-small cell lung cancer,so early,there are many studies about EGFR-TKIs treatment model,but the conclusion is different.Purpose1.To investigate the effects of wild-type A549 cells,EGFR exon 19 mutant HCC827 cells and T790M secondary drug resistant H1975 cells on the different combination of pemetrexed and icotinib/erlotinib.2.To further explore the mechanism of the difference in the efficacy of different combinations of drugs.Method1.Selection of cell linesEGFR-TKIs are not recommended for patients with lung squamous cell carcinoma.Therefore,we use three lung adenocarcinoma cell lines,A549 is wild type cell line,H1975 is T790M secondary resistant cell line,and HCC827 is EGFR exon 19 mutant cell line.2.drug selection(1)advanced lung adenocarcinoma patients with first-line chemotherapy drugs:pemetrexed(2)molecular targeted drugs:icotinib,erlotinib.3.The setting of the experiment sequence(1)application of icotinib/erlotinib for 48 h,then pemetrexed alone for 24 h;(2)pemetrexed application 24 hours,icotinib/erlotinib application 48h;(3)pemetrexed + icotinib/erlotinib 48h at the same time,then medium application 24h.4.The CCK-8 method was used to detect the cell viability,and the IC50 of pemetrexed and icotinib/erlotinib was established.The combined index Cl was calculated.5.Apoptosis and cell cycle were detected by flow cytometry.Result1.CCK-8 results(1)The IC50 values of pemetrexed in A549,HCC827 and H1975 cells were 1.9000 ± 0.2037umol/L,1.5780 ? 0.2504umol/L and 3.3723 ± 0.0824umol/L,respectively.(2)The IC50 values of icotinib in A549,HCC827 and H1975 cells were 7.7273 ±0.8093umol/L,0.0042 ± 0.0007umol/L and 5.7983 ? 0.998lumol/L,respectively.(3)The IC50 of erlotinib in A549,HCC827 and H1975 cells was 6.6643 ±0.3404umol/L,0.0034 ± 0.0005umol/L and 4.8120 ± 0.0576umol/L,respectively.(4)The combined index(CI)of pemetrexed followed by icotinib in A549,HCC827 and H1975 cells was 1.00 ± 0.06,0.67 ± 0.12 and 0.61 ± 0.07,respectively.(5)The combined index(CI)of pemetrexed followed by erlotinib in A549,HCC827 and H1975 cells was 1.00 ± 0.09,0.68 ± 0.12 and 0.72 ± 0.03,respectively.(6)The combined index(CI)of icotinib followed by pemetrexed in A549,HCC827 and H1975 cells was 1.00 ± 0.08,1.04 ± 0.18 and 1.03 ± 0.14,respectively.(7)The combined index(CI)of erlotinib followed by pemetrexed in A549,HCC827 and H1975 cells were 1.02 ± 0.06,1.00 ± 0.11,1.00 ± 0.05,respectively.(8)The combined index(CI)of icotinib combined with pemetrexed at the same time was e 1.46 ± 0.22,1.30 ± 0.20 and 1.20 ± 0.15,respectively.(9)The combined index(CI)of erlotinib combined with pemetrexed at the same time was 1.29 ± 0.05,1.37 ± 0.41,1.27 ± 0.08 in A549,HCC827 and H1975 cells,respectively.The results of CCK-8 showed that pemetrexed and icotinib/erlotinib could inhibit the proliferation of A549,HCC827 and H1975 cells in a concentration-dependent manner.In the HCC827 and H1975 cells,pemetrexed followed icotinib/erlotinib had a synergistic effect(CI<1),and icotinib/erlotinib followed pemetrexed had an additive effect(CI = 1),pemetrexed combined with icotinib/erlotinib at the same time had an antagonism effect(CI>1).In the A549 cells,pemetrexed combined with icotinib/erlotinib at the same tim also had antagonistic effects(CI>1),and pemetrexed followed icotinib/erlotinib had an additive effect(CI=1),icotinib/erlotinib followed pemetrexed had an additive effect(CI=1),so no synergistic effect was found.2.Apoptosis results(1)The apoptotic rates of pemetrexed followed icotinib were 6.36 ± 0.65%,30.22 ?7.97%and 18.12 ± 5.09%in A549,HCC827 and H1975 cells,respectively.(2)The apoptotic rates of pemetrexed followed erlotinib were 5.26 ± 0.3%,50.47±8.68%and 20.1 ± 4.07%in A549,HCC827 and H1975 cells,respectively.(3)The apoptotic rates of icotinib followed pemetrexed were 5.54 ± 0.77%,18.8 ±0.64%and 11.5±1.37%in A549,HCC827 and H1975cells,respectively.(4)The apoptotic rates of erlotinib followed pemetrexed were 5.83 ± 1.3%,17.91 ±1.35%and 11.05 ± 2.03%in A549,HCC827 and H1975 cells,respectively.(5)The apoptotic rates of pemetrexed combined with icotinib at the same time were 6.13 ? 1.89%,11.27 ± 2.26%and 11.76 ± 1.97%in A549,HCC827 and H1975 cells,respectively.(6)The apoptotic rates of pemetrexed combined with erlotinib at the same time were 5.71 ± 0.36%,14.35 ± 0.57%and 10.77 ± 0.26%in A549,HCC827 and H1975 cells,respectively.(7)The apoptotic rates of the control group were 1.39 in A549,HCC827 and H1975 cells,respectively 1.39 ±0.59%,1.47± 0.38%? 1.41 ± 0.43%,respectively.(8)The apoptotic rates of pemetrexed were 6.11 ± 1.81%,6.13 ± 0.96%and 7.55 ±0.56%in A549,HCC827 and H1975 cells,respectively.(9)The apoptotic rates of icotinib group were 4.59 ± 0.33%,6.82 ± 0.55%and 9.89± 1.64%in A549,HCC827 and H1975 cells,respectively.(10)The apoptotic rates of the erlotinib group were 3.55 ± 1.14%,7.41 ± 0.41%and 7.56 ± 0.79%in A549,HCC827 and H1975 cells,respectively.The apoptotic rates were significantly higher in the HCC827 and H1975 cell line than in the other groups,with significant efficacy(P<0.05).But there was no significant difference in A549 cells(P>0.05).3.Cell cycle results(1)The S phase of A549,HCC827,H1975 cell lines in the pemetrexed group was significantly increased,which were 34.11 ± 1.43%,34.46 ± 1.88%,34.48±1.26%,respectively,.(2)The G1 phase of icotinib of A549,HCC827 and H1975 cell lines were significantly increased,which were 65.97 ± 3.77%,63.78 ± 2.48%and 63.02 ± 1.30%,respectively.(3)The G1 phase of erlotinib of A549,HCC827 and H1975 cell lines were signjficantly,increased,which were 65.56 ± 2.55%,.67.33 ± 2.60%and 65.51±1.71%,respectively(P<0.05).(4)The S phase of A549,HCC827,H1975 cells were significantly increased in pemetrexed followed erlotinib group,which were 46.34 ± 4.64%,55.74 ± 7.38%and 46.82 ± 6.08%,respectively.(5)The S phase of A549,HCC827,H1975 cells was significantly increased in pemetrexed followed icotinib group,43.37±0.69%,49.26 ± 2.96%,44.05 ± 2.93%,respectively(6)The application of G1,G2 and S of A549,HCC827,H1975 cells in icotinib/erlotinib followed pemetrexed group and pemetrexed followed icotinib/erlotinib did not show obvious regularity.The results showed that A549,HCC827 and H1975 cells were arrested in the S phase in pemetrexed group,were arrested in G1 phase in icotinib/erlotinib group,were significantly higher in the S phase in pemetrexed followed icotinib/erlotinib group.The results suggested that pemetrexed followed icotinib/erlotinib strengthened the resistance to S phase.Conclusion1.Pemetrexed followed icotinib/erlotinib had a synergistic effect,and icotinib/erlotinib followed pemetrexed had an additive effect,pemetrexed combined with icotinib/erlotinib at the same time had an antagonism effect.In A549 cells,regardless of pemetrexed combined with icotinib/erlotinib at the same time,pemetrexed followed icotinib/erlotinib and icotinib/erlotinib followed pemetrexed no synergies were found.2.In HCC827 and H1975 cells,the apoptotic rate of pemetrexed followed icotinib/erlotinib was significantly higher,whereas in A549 cells,regardless of pemetrexed combined with icotinib/erlotinib at the same time,pemetrexed followed icotinib/erlotinib and icotinib/erlotinib followed pemetrexed apoptotic rates were not significantly affected.3.The cells of A549,HCC827 and H1975 were arrested in G1 phase in icotinib/erlotinib group,and the cells of A549,HCC827 and H1975 were blocked in S phase in pemetrexed group.The cells of A549,HCC827 and H197 were consistent significantly increased in the S phase in pemetrexed followed icotinib/erlotinib group.The results suggested the synergistic effect of the sequence of pemetrexed followed icotinib/erlotinib was considered to be related to the cell cycle.Part ?METFORMIN SYNERGISTIC PEMETREXED SUPPRESS LUNG ADENOCARCINOMA CELLS PROLIFERATION AND INVASION IN VITROBackgroundAt present,lung cancer is one of the malignant tumors with a high mortality rate.The prognosis is very poor.86%of patients die within 5 years after diagnosis.Progressive lung cancer patients accounted for about 85%,only 15%of patients had a limited lesion at the time of diagnosis and a 5-year survival rate of 50%.Most of the patients in the newly diagnosed in the ?B?? period,lost the timing of surgical treatment,it is difficult to cure by surgery,so the treatment is mainly dependent on chemotherapy or radiotherapy and other comprehensive treatment methods,which may be the main factors of higher lung cancer mortality,while adenocarcinoma is the most common type of histology.Because of its exact effect,and with other cytotoxic drugs compared to the smallest toxicity,pemetrexed is currently recommended the first-line treatment for advanced non-small cell lung cancer treatment.In recent years,pemetrexed has become a hotspot in lung adenocarcinoma.Pemetrexed as a multi-target anti-folic acid chemotherapeutic agent,can inhibit thymidine synthase,dihydrofolate reductase and glycine amide nucleotide transcarbylase,from a number of obstacles to the formation of thymine and purine,interfere with folic acid metabolism and nucleotide formation,and thus achieve the purpose of blocking the growth of cancer cells,and can reduce the resistance of tumor cells.Its main side effects are myelosuppression and mucositis,generally by taking folic acid intake and vitamin B12 way to reduce the incidence of its side effects.But a number of studies have shown that,compared with other chemotherapy drugs such as docetaxel pemetrexed is also difficult to improve the survival rate of patients.Therefore,the overall effect is not optimistic.Looking for new adjuvant drugs to enhance the efficacy of chemotherapy in order to improve the survival rate of patients with lung adenocarcinoma is the focus of the current study.Metformin is a well tolerated biguanide hypoglycemic drug and is used as a first-line drug to control blood sugar by more than 120 million patients with type 2 diabetes worldwide.Since the 1970s Dilman V M and other scholars found that biguanide anti-diabetic drugs had anti-aging ability and anti-cancer effect,metformin has attracted a large number of cancer researchers.Many studies have found that long-term use of metformin type 2 diabetes patients with colorectal cancer,lung cancer and other cancer risk was significantly reduced,and taking metformin in patients with type 2 diabetes mellitus can significantly reduce mortality.Many studies in vitro and animal experiments also found that metformin can inhibited the proliferation of cancer cells and promote the role of apoptosis.It was found that the inhibitory effect of metformin on tumor cells could activate JKK/p38MAPK signaling pathway and increase the expression of JNK and p38 in lung cancer cells.Induced induction of growth stagnation and DNA destruction gene GADD153 gene expression increased activation and then induced lung cancer cell apoptosis.At the same time the study also found that metformin combined with chemotherapy drugs cisplatin or etoposide and other chemotherapy drugs can inhibit the proliferation of lung cancer cells and improve lung cancer cells on cisplatin or etoposide and other chemotherapeutic drugs susceptibility,but the current study on metformin and pemetrexed in combination with lung adenocarcinoma cells has not been reported.Purpose1.To determine whether metformin combined with pemetrexed in vitro can inhibit the proliferation of lung adenocarcinoma cells,promote apoptosis,affect cell cycle and reduce the ability of migration.2.To further explore its related molecular mechanisms.Method1.Selection of cell linesWe used three lung adenocarcinoma cell lines,A549 was wild type cell line,H1975 was T790M secondary resistant cell line,HCC827 was EGFR exon 19 mutant cell line.2.Set the experimental method(1)metformin,pemetrexed monotherapy and combination of three lung adenocarcinoma cells 48 hours later,by CCK8 test method to detect IC50 value,and calculate the combination index(CI),and then clear metformin combined with pemetrexed whether anti-proliferative effect and therapeutic effect.(2)Cell apoptosis and cycle distribution were detected by flow cytometry.(3)Transwell method was used to detect the changes of migration ability of three lung adenocarcinoma cells after application of drug therapy.(4)The mechanism was further clarified by Western blotting.Result1.CCK-8 results(1)The IC50 values of A549,HCC827 and H1975 cells were 11.92 ± 0.11mmol/L,4.72 ± 0.14 mmol/L and 5.41 ± 0.55 mmol/L,respectively after 48 hours of metformin administration.(2)The IC50 values of A549,HCC827 and H1975 cells were 1.82 ± 0.17 umol/L,1.54 ± 0.30 umol/L and 3.37 ± 0.14umol/L,respectively after 48 hours of application of pemetrexed.(3)The combined index(CI)of A549,HCC827 and H1975 cells was 0.56,0.63 and 0.64,respectively after 48 hours combined with metformin and pemetrexed.The results suggest that metformin alone can significantly reduce the proliferation of three lung adenocarcinoma cell lines in a concentration-dependent manner.Metformin combined with pemetrexed at the same time play a synergistic effect on the antiproliferative effect of lung adenocarcinoma cells(CI<1).2.Apoptosis results(1)The apoptotic rates of A549,HCC827 and H1975 cells were 10.40 ± 0.57%,16.28 ± 1.21%and 12.68 ± 1.67%,respectively,which were significantly higher than those in the blank group(P<0.05).(2)The apoptotic rates of A549,HCC827 and H1975 cells were 14.26 ± 1.17%,14.65 ± 0.84%and 13.22 ± 1.60%,respectively,which were significantly higher than those in the blank group(P<0.05).(3)The apoptotic rates of metformin combined with pemetrexed were 48.34 ± 3.62%,35.55 ± 3.25%and 28.54 ± 4.07%,respectively,which were significantly higher than those in the pemetrexed group(P<0.05).(4)The apoptotic rates of A549,HCC827 and H1975 cells were 1.39 ± 0.59%,2.02± 0.68%and 1.47 ± 0.38%in the blank group,respectively.The results showed that in A549,HCC827 and H1975 three lung adenocarcinoma cells,metformin alone had significantly enhanced cell apoptosis in the blank group(P<0.05).The combination of metformin and pemetrexed significantly enhanced cell apoptosis compared with pemetrexed alone.(P<0.001).3.Cell cycle results(1)In A549,HCC827 and H1975 cells,the G1 phase of the blank group was 74.75 ±0.35%,62.48 ± 1.39%,53.25 ± 1.88%respectively;the S phase was 18.67 ± 1.27%,22.86 ± 0.52%,29.37 ± 2.11%;G2 phase was 6.59 ± 1.33%,14.66 ± 0.97%and 17.38 ± 0.69%respectively.(2)In A549,HCC827 and H1975 cells,the G1 phase of metformin alone treatment group was 48.09 ± 1.70%,41.36 ± 0.70%28.03 ± 1.54%,respectively.The S phase were 20.69 ? 4.64%,43.18 ± 7.07%and 28.03 ± 1.54%respectively.The G2 phase was 11.36 ± 4.54%,15.46 ± 7.7%and 25.79 ± 4.95%,respectively.In A549 cells,metformin alone treatment group had no significant effect on the cell cycle compared with the blank group(P>0.05).In HCC827 cells,the level of S-phase in the metformin treatment group was significantly higher than that in the blank group(P<0.01).In H1975 cells,the level of G2-phase in the metformin treatment group was significantly higher than that in the blank group(P<0.05).In conclusion,the effect of metformin on the cell cycle of A549,HCC827 and H1975 cells was not found to be common.(3)In A549,HCC827 and H1975 cells,the G1 phase of pemetrexed treatment group was 46.43 ± 0.38%,61.41±4.18%and 64.83 ± 1.06%,respectively.The S phase was 53.38 ± 3.34%,33.02 ± 3.32%,4.00 ± 3.64%.The G2 phase were 0.67 ± 0.65%,5.57± 1.03%,25.79 ± 4.95%respectively.(4)In A549,HCC827 and H1975 cells,the G1 phase of metformin combined with pemetrexed treatment group was 443.54±5.64%,36.43±1.28%,64.80±4.92%respectively.The S phase was 51.48 ± 10.99%,62.48 ± 0.97%,30.79 ±6.17%.The G2 phase were 7.20±5.91%,1.09±0.48%,4.41±1,35%respectively.In A549 cells,metformin combined with pemetrexed treatment group had no significant effect on cell cycle compared with pemetrexed group(P>0.05).In HCC827 cells,the S phase of metformin combined with pemetrexed-treated group were significantly higher compared with pemetrexed group(P<0.01).In H1975 cells,the S phase of metformin combined with pemetrexed-treated group were significantly higher compared with pemetrexed group(P<0.01).In summary,the effect of metformin combined with pemetrexed 48 hours on the cell cycle of A549,HCC827 and H1975 cells was not found to be common.In conclusion,the effect of metformin alone and metformin combined with pemetrexed on the cell cycle of A549,HCC827 and H1975 cells was not found in the common law.Analysis of metformin monotherapy and metformin combined with pemetrexed in lung adenocarcinoma cells may have little effect on cell cycle.4.Transwell experimental resultsThe treatment of metformin alone significantly inhibited the migration ability of three lung adenocarcinoma cells compared with the blank group(P<0.05).Metformin combined with pemetrexed-treated group significantly inhibited the migration of three lung adenocarcinoma cells(P<0.05).5.By Western blot analysis,compared with the blank group,the level of Bcl-2 in the metformin-treated group was lower and the level of Bax was increased(P<0.05).In the comparison of metformin combined with pemetrexed and pemetrexed in the treatment group,the level of Bcl-2 in the metformin combined with pemetrexed group was lower and the level of Bax was increased(P<0.05).These results suggest that the use of metformin alone and metformin combined with pemetrexed can significantly enhance the apoptosis of lung adenocarcinoma cells,so the analysis of metformin on lung adenocarcinoma cell proliferation and sensitization of chemotherapy drugs may be through the mechanism apoptosis occurs.ConclusionThe results of this study show that metformin monotherapy and metformin combined with pemetrexed at the same time can inhibit the growth of lung adenocarcinoma cell lines,enhance cell apoptosis,reduce the ability to migrate,and its mechanism may be induced by apoptosis to play a related role.Our data show that metformin monotherapy in vitro lung adenocarcinoma cells have significant anti-tumor effect,while metformin on the chemotherapy drug pemetrexed in the inhibition of tumor has significant sensitization,which the clinical type 2 diabetes patients regardless of whether it is combined with the tumor,in the choice of hypoglycemic drugs may have a certain guiding role,but our study is only in vitro,still need a lot of other related research,animal experiments and clinical studies to further confirm. |
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