| Lung cancer is a malignant tumor with the highest incidence and the leading cause of cancer-related death worldwide. Non-small cell lung cancer(NSCLC) accounts for 85% of all lung cancer cases. The vast majority of lung cancer patients are in the advanced stage at diagnosis, and the 5-year survival rate in those metastases patients is only 4%. Even for the patients who are diagnosed at early stage and receive standard treatment, the 5-year survival rate is as low as 17%. Therefore, how to cure for patients with lung cancer and prolong their survival time is very important.Personalized therapy is the most effective method for the treatment of lung cancer, which is based on the genetic characteristics of patients. The ALK inhibitor crizotinib in treating NSCLC is a successfully, which can greatly improve the prognosis of lung cancer patients with ALK gene mutations. Crizotinib is a tyrosine kinase inhibitor with activity against MET, ALK and ROS1. As molecular targeted drug, crizotinib had been used to treat ALK-positive advanced NSCLC. The objective response rate(ORR) reached nearly 60% in one clinical trial and approximately 50% in another. Based on these promising results, FDA has approved the use of crizotinib in treating ALK-positive lung cancer patients.Recently, a phase III clinical trial(PROFILE 1014) evaluated the crizotinib versus standard first-line chemotherapy(pemetrexed-plus-cisplatin) in patients ith advanced ALK-positive non-small cell lung cancer(NSCLC).The results suggested that the patients treated with crizotinib showed significantly longer progression-free survival(PFS) and higher ORR, and had much milder adverse effects. Therefore, targeted therapy with crizotinib has been recommended as the first-line therapy for ALK-positivelung cancer patients.Unfortunately, during the process of crizotinib therapy, drug resistance will occur on all of the patients in one year, and the resistance mechanism is extremely complex. It will be a new approach for solving drug resistance to looking for a method to increase the sensitivity of conventional treatment methods, such as chemotherapy, radiation therapy, molecular targeted therapy, and delay the occurrence of resistance.mi R-200 c is a important member of mi R-200 family. It is expressed at low levels in many tumor tissues, and can regulate EMT, tumor invasion and metastasis. Overexpression of mi R-200 c in mesenchymal cells promotes their transition to epithelial cells, and enhances the sensitivity of lung cancer cells to various chemotherapeutic agents, EGFR-TKI drugs and radiotherapy. However, whether it has similar effect in ALK-positive lung cancer cells is unknown. The present study aims to investigate the role of mi R-200 c in regulating the sensitivity of ALK-positive lung cancer cells to crizotinib, and tries to explore its molecular mechanism.Part 1 The effect on the proliferation and migration of crizotinib in H2228, A549 and H460 lung cancer cellsObjective: To investigate the effects of crizotinib on the proliferation and migration of ALK positive lung cancer cells and other lung cancer cells.Methods: H2228 cells, EML4-ALK fusion gene positive lung adenocarcinoma cells, were purchased from American Type Culture Collection(ATCC). A549(lung adenocarcinoma cells) and H460(human metastatic non-small cell lung carcinoma cells) were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were cultured in RPMI1640/Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum out of antibiotics and maintained in a humidified 5% CO2 atmosphere at 37?C. Cells were treated with crizotinib(Sigma-Aldrich, USA).The cells of H2228, A549 and H460 were treated with different concentrations of crizotinib(0, 25, 50, 100, 200 nmol/L) for 48 h, and then cellproliferation activity was determined by MTT and cell count assays. As the same, these three cells proliferation activity was determined by MTT and cell count assays when treatment with 100 nmol/L crizotinib for different time(0, 24, 48 and 72 h).Transwell assay was used to measure the rate of cell migration. The cells of H2228, A549 and H460 migration ability were detected by transwell migration assay, after the cells were treated with different concentrations crizotinib(0, 25, 50, 100 nmol/L) for 48 h.Results:1 the inhibitory effect on the proliferation of crizotinib in H2228, A549 and H460 lung cancer cellsAfter cells of H2228, A549 and H460 were treated with different concentrations of crizotinib(0, 25, 50, 100, 200 nmol/L) for 48 h, the cells of H2228, A549 and H460 proliferation activity was determined by MTT and cell count assays. The results showed that The inhibitory effect of crizotinib in ALK positive lung cancer cell line H2228 was remarkable, and with the concentration of crizotinib increased, the inhibitory effect on the proliferation of the cells was increased(P<0.05, P<0.01). The IC50 was calculated as 121.91nmol/L. Crizotinib showed much lower inhibition in ALK mutation-negative lung adenocarcinoma cell A549 and metastatic non-small cell lung carcinoma cell H460, only at a large dose 200nmol/L, obvious inhibitory effect was observed(22% and 19.7%, P<0.05).The H2228 cell proliferation activity was determined by MTT and cell count assays when treatment with 100 nmol/L crizotinib for different time(0, 24, 48 and 72 h). The results showed that with the extension of the action time of crizotinib, the inhibitory effect on the proliferation of the cells was enhanced(P<0.05). In summary, the results showed that crizotinib had a dramatic inhibitory effect on proliferation of H2228 cells in a dose- and time-dependent manner2 the inhibitory effect on the migration of crizotinib in H2228, A549 and H460 lung cancer cellsAfter lung cancer cells of H2228, A549 and H460 were treated with different concentrations of crizotinib(0, 25, 50, 100, 200 nmol/L) for 48 h,It can be shown from the results of Transwell trial about cell migration ability assays that inhibitory effect of crizotinib in cell line H2228 was obvious, and with the concentration of crizotinib increased, the inhibitory effect on the migration of the cells was increased(P<0.05, P<0.01)Crizotinib showed no inhibition in the cell of A549 and H460, In summary, the results showed that crizotinib had a dramatic inhibitory effect on migration of H2228 cells with a dose-dependent manner.Conclusions: ALK inhibitor crizotinib had a dramatic inhibitory effect on proliferation viability and migration of H2228 cells with a dose- and time-dependent manner.Part 2 the regulation mechanism of mi R-200 c on H2228 cell proliferation and migration ability to crizotinibObjective: To observe the effect of mi R-200 c on H2228 cell proliferation and migration when treated crizotinib, and to explore its regulation mechanism.Methods: H2228 cells were transient transfected with mi R-200 c mimicis, mi R-200 c inhibitor, mi R-200 c mimicis negative control(NC) and mi R-200 c inhibitor NC. MTT assay was performed to measure the proliferation of H2228 cells which transfected with mi R-200 c mimicis, mi R-200 c inhibitor, mi R-200 c mimicis negative control and mi R-200 c inhibitor negative control. Transwell assay was used to measure the H2228 cell migration ability after transfected with mi R-200 c mimicis, mi R-200 c inhibitor, mi R-200 c mimicis negative control and mi R-200 c inhibitor negative control. The candidate targets of mi R-200 c were predicted by bioinformation and the ZEB1 as predicted target gene was determined by Luciferase report. The m RNA levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228 transfected with mi R-200 c mimicis or mi R-200 c mimicis negative control was measured by q RT-PCR. Western blot was used to measure the protein levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228 transfected with mi R-200 c mimicisor mi R-200 c mimicis negative control.Results:1 Transfected with mi R-200 c mimicis, mi R-200 c inhibitor, mi R-200 c mimicis NC or mi R-200 c inhibitor NC, the expression of mi R-200 c in H2228 cells were measured.To elucidate the regulation mechanism of mi R-200 c expression in the NSCLC cell H2228. H2228 cells were treated with mi R-200 c mimicis, mi R-200 c inhibitor, mi R-200 c mimicis NC or mi R-200 c inhibitor NC, respectively, and expression of mi R-200 c was analyzed by q RT-PCR. Compared with the mimicis NC, mi R-200 c expression levels increased 310-fold after transfected with mi R-200 c mimicis(P <0.01). While compared with the inhibitor NC, mi R-200 c expression level decreased 3.5-fold after transfected with mi R-200 c inhibitor(P <0.05).2 mi R-200 c increased the sensitivity of H2228 cells to crizotinibH2228 cells transfected with mi R-200 c mimicis and mi R-200 c mimicis NC, were treated with different concentrations( 50, 100, 200 nmol/L) of crizotinib, we found that the sensitivity of the cell to crizotinib was increased when mi R-200 c was overexpressed(P<0.05). While sensitivity of H2228 cell to crizotinib in various concentrations was decreased when transfected with mi R-200 c inhibitor and the expression of mi R-200 c was inhibited(P<0.05, P<0.01).3 Overexpression of mi R-200 c suppressed migration ability of H2228 cellTranswell assay was used to measure the migration ability of H2228 cells which transfected with mi R-200 c mimicis, mi R-200 c mimicis NC, mi R-200 c inhibitor or mi R-200 c inhibitor NC. We found that migration ability was inhibited when mi R-200 c was overexpressed(P<0.01)while, the migration ability of H2228 cell was promoted when transfected with mi R-200 c inhibitor and the expression of mi R-200 c was reduced(P<0.05).4 mi R-200 c directly target ZEB1 in H2228 by luciferase reporter assayThe coaction of mi R-200 c and mi R-200 c targets ZEB1 were analyzed bluciferase reporter assay. The mi R-200 c was transfected with the ZEB1 3’-UTR luciferase reporter gene into H2228 cells. The result showed that transfection of mi R-200 c mimics exhibited a striking reduction of luciferase activity(P<0.05).5 Effect of mi R-200 c on expression of EMT markers in H2228 cellsq RT-PCR was used to measure the m RNA expression levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228 transfected with mi R-200 c mimicis and mi R-200 c mimicis NC. Results from q RT-PCR indicated that the expression of N-cadherin, Vimentin and CD24 was decreased(P<0.05,P<0.01), while the expression of E-cadherin was increased when mi R-200 c was overexpressed in H2228 cells(P<0.01).Western blot was used to measure the protein levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228 transfected with mi R-200 c mimicis and mi R-200 c mimicis NC. We found that the expression of N-cadherin, Vimentin and CD24 was decreased( P<0.05), while the expression of E-cadherin was increased when mi R-200 c was overexpressed in H2228 cells(P<0.01)It suggested that forced expression of mi R-200 c induced mesenchymal-epithelial transition(MET) and enhanced the sensitivity of cells to crizotinib.Conclusions: Overexpression of mi R-200 c suppressed migration ability and increased sensitivity of ALK-positive lung cancer cell H2228 to crizotinib by reversing epithelial to mesenchymal transition through targeting ZEB1Part 3 the crizotinib-resistant ALK-positive cell line H2228/CR was establishedObjective: To observe the changes of crizotinib-resistant cell line H2228/CR and reserch the mechanism of drug resistanceMethods:Crizotinib-resistant H2228/CR cells were established by continuous stepwise exposure to crizotinib combined with pulse therapy in vitro. Morphology changes of ALK-positive lung cancer cell H2228 andcrizotinib-resistant cell H2228/CR were examined. MTT assay was used to measure proliferation of crizotinib-resistant H2228/CR cells in different concentrations of crizotinib, and the results were used to determine the IC50 of crizotinib in H2228 CR cells. Transwell assay was used to measure the migration rate of H2228 and H2228/CR cells. q RT-PCR was used to measure the m RNA expression levels of E-cadherin, N-cadherin, Vimentin, ZEB1 and ZEB2 in H2228 and crizotinib-resistant H2228/CR. Western blot was used to measure the protein levels of E-cadherin, N-cadherin, Vimentin, ZEB1 and ZEB2.Results:1 The cell of H2228/CR was resistant to crizotinibAfter culturing for more than 6 months, the resistant cells could grow in media containing 800 nmol/L crizotinib, Cell morphology of H2228 and H2228 CR cells was observed under a low magnification microscope. The drug-resistant H2228/CR cells changed from oval to spindle-shaped and arranged more dispersed. MTT assay was performed to measure the proliferation cells the IC50 of crizotinib in H2228 cells increased from the initial121.9 n M to 2618.45 n M, and thus the resistance increased 21.5 fold.2 Crizotinib-resistant H2228 CR cells had more migration abilityTranswell assay was used to measure the migration ability of H2228 and H2228/CR cells. It can be shown from the results of cell viability and migration assays that Crizotinib-resistant H2228/CR cells had more invasive ability than H2228 cell(P<0.05).3 EMT occurs in H2228/CR cellsThe drug-resistant H2228 CR cells changed from oval to spindle-shaped and arranged more dispersed, suggesting the presence of mesenchymal change. To elucidate the resistance mechanism of H2228/CR cells to crizotinib, the expression of markers in epithelial and mesenchymal cells were analyzed by q RT-PCR and western blot analysis. The results showed that N-cadherin and Vimentin were increased, while E-cadherin was decreased in H2228/CR cells. It suggested that the EMT occurred in H2228/CR cells.Conclusions:1 The crizotinib-resistant ALK-positive lung cancer cell line of H2228/CR was successfully established, the resistance increased 21.5 fold, and reached further experimental requirement.2 Crizotinib-resistant H2228/CR cells had more invasive ability and exhibited characteristics of EMTPart 4 mi R-200 c increased sensitivity of crizotinib-resistant H2228/CR to crizotinib by reversing epithelial to mesenchymal transitionObjective: To research the changes of proliferation and migration ability on crizotinib-resistant H2228/CR cell transfected with mi R-200 c and to explore its regulation mechanism.Methods:q RT-PCR was used to measure the expression of mi R-200 c in H2228 and crizotinib-resistant H2228/CR cells. H2228/CR cells were transient transfected with mi R-200 c mimicis, mi R-200 c mimicis NC. MTT assay was performed to measure the proliferation of H2228/CR cells which transfected with mi R-200 c mimicis, mi R-200 c mimicis NC. Transwell assay was used to measure the H2228/CR cell migration ability after transfected with mi R-200 c mimicis, mi R-200 c mimicis NC. The m RNA levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228/CR transfected with mi R-200 c mimicis or mi R-200 c mimicis NC and H2228 cell was measured by q RT-PCR. Western blot was used to measure the protein levels of N-cadherin, Vimentin, E-cadherin and CD24 in H2228/CR transfected with mi R-200 c mimicis or mi R-200 c mimicis NC and H2228 cell.Results:1 Crizotinib-resistant H2228 CR cells had reduced mi R-200 c expressionq RT-PCR was used to measure the expression of mi R-200 c in H2228 and crizotinib-resistant H2228/CR cells. The result showed that the expression of mi R-200 c in H2228/CR cells was 24.93 times lower than that in H2228 cells.2 mi R-200 c partially restored sensitivity of H2228/CR cell to crizotinibq RT-PCR was used to measure the expression of mi R-200 c in H2228 andcrizotinib-resistant H2228/CR transfected with mi R-200 c mimicis or mi R-200 c mimicis NC. Expression of mi R-200 c in H2228/CR cells was restored by transfection of mi R-200 c mimics. Results from MTT assay showed that these cells had increased proliferation compared to untransfected H2228/CR cells treated with different concentrations of crizotinib. It indicated that forced expression of mi R-200 c restored the sensitivity of crizotinib-resistant H2228/CR cells to crizotinib but unreached the level of H2228 cell.3 Forced expression of mi R-200 c inhibited the migration ability of crizotinib-resistant H2228/CRTranswell assay was used to measure the migration ability of H2228/CR cells transfected with mi R-200 c mimicis or mi R-200 c mimicis NC and H2228 cell. It can be shown from the results of cell viability and migration assays that Crizotinib-resistant H2228/CR cells transfected with mi R-200 c mimicis had less invasive ability than H2228/CR cells transfected with mi R-200 c mimicis NC but more than H2228 cell(P<0.05)4. mi R-200 c reversed EMT of H2228 CR cellsq RT-PCR was performed again to measure the m RNA expression of EMT markers, such as E-cadherin, N-cadherin, Vimentin and CD24 in the H2228/CR cells transfected with mi R-200 c mimicis or mi R-200 c mimicis NC and H2228 cell. Results showed that E-cadherin expression was higher in H2228/CR cells transfected with mi R-200 c mimicis than transfected with mi R-200 c mimicis NC(P<0.05), but more lower than H2228 cell(P<0.01). While the expression of N-cadherin, Vimentin and CD24 in H2228/CR cells transfected with mi R-200 c mimicis was lower than transfected with mi R-200 c mimicis NC(P<0.05),but higher than H2228 cell(P<0.05).Western blot was used to measure the protein levels of EMT markers, E-cadherin, N-cadherin, Vimentin and CD24 in the H2228/CR cells transfected with mi R-200 c mimicis or mi R-200 c mimicis NC and H2228 cell. Results showed that E-cadherin expression was higher in H2228/CR cells transfected with mi R-200 c mimicis than transfected with mi R-200 c mimicisNC(P<0.05), but more lower than H2228 cell(P<0.01). While the expression of N-cadherin, Vimentin and CD24 in H2228/CR cells transfected with mi R-200 c mimicis was lower than transfected with mi R-200 c mimicis NC(P<0.05),but higher than H2228 cell(P<0.05).Conclusions:1 Crizotinib-resistant H2228 CR cells had reduced mi R-200 c expression,Forced expression of mi R-200 c partially restored sensitivity of H2228/CR cell to crizotinib and inhibited the migration ability of crizotinib-resistant H2228/CR.2 Through analyzed expression of markers in epithelial and mesenchymal cells by q RT-PCR and western blot trail, we found that mi R-200 c regulates sensitivity of crizotinib-resistant H2228/CR to crizotinib by reversing epithelial to mesenchymal transition(EMT). |
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