| Objectives:Five human NSCLC cell lines,A549,H460,H226,H522 and LK2 cells were cultured to conduct the expression of VEGFR.Apatinib(APA),as a small molecule inhibitor of VEGFR-2,has been identified to promote tumor cell apoptosis,inhibiting cell proliferation.So far,we observed the role of APA targeted drugs in enhancing radiosensitivity in NSCLC cell lines and tumor bearing mice.we detected the expression of VEGFR2、PI3K、p-PI3K、AKT、p-AKT、PKC、MEK、p-MEK、ERK、p-ERK in Blank group,APA group,IR group,and APA+IR group.The present study aimed to explore the radiosensitivity effect of APA in NSCLC and its underlying mechanism as a radiosensitizer.Methods:The first part:APA was dissolved in 100%dimethyl sulfoxide(DMSO)as a 100mM stock solution,and diluted with RPMI-1640 medium to the desired concentration.The normal mammary epithelial cell line HBEC and five human NSCLC cell lines,A549,H460,H226,H522 and LK2 cells were cultured in RPMI-1640 medium and supplemented with 10%fetal bovine serum and 1%penicillin/streptomycin in a humidified incubator at 37℃ in 5%CO2.A 6-MeV X-ray medical linear accelerator was used to irradiate cells at a dose rate of 300 cGy/min(dose:0 to 8 Gy)at room temperature.RNA extraction,reverse transcription,and qRT-PCR was performed to conduct the relative expression of VEGFR2 in A549 and LK2 cells to verify the accuracy of sequencing results.Expression of VEGFR2 were used Western blot and RT-PCR respectively in NSCLC.The cell proliferation was measured by CCK-8 and colony formation assay.Cell Counting Kit-8 was used to determine cell viability after irradiation.The absorbance was measured with a microplate reader at a wavelength of 450 nm.IC50 value of each cell line was calculated using the graphpad Prism 7 software.To perform the colony formation assay,300 cells were seeded into 6-well plates and irradiated(0,2,4,6,and 8 Gy)the next day.Two weeks later,the cells were fixed in 4%paraformaldehyde and stained with 0.1%crystal violet,and the number of colonies per dish was counted.Apoptosis analysis were observed in radiosensitivity of NSCLC cells.Cell apoptosis was measured using FITC Annexin-V/PI apoptosis Detection Kit.Cell cycle analysis was performed using 50 μg/ml PI and 100 μg/ml DNase-free RNase A.Twenty-four hours post-irradiation,the cells were harvested with trypsin and washed with phosphate-buffered saline,and then fixed in 70%ice-cold ethanol at 4℃ for 12 hours.After washing,the cell pellet was resuspended in PI staining buffer and incubated at 37℃ for 30 min in the dark.Cell apoptosis and cell cycle status were analysed by flow cytometry.Clone formation assay were conducted to detect the effect of combining with irradiation on cell radiosensitivity.The plating efficiency and surviving fraction were calculated.The second part:CCK-8 was used to detect the cell proliferation activity.The previous study showed that results revealed that the ALK and ERK signaling pathway may be involved in the radioresistance of NSCLC.Overexpressiong of VEGFR2、PI3K、p-PI3K、AKT、p-AKT、PKC、MEK、p-MEK、ERK、p-ERK was found in radioresistant cell lines.Then we detected the expression of VEGFR2、PI3K、pPI3K、AKT、p-AKT、PKC、MEK、p-MEK、ERK、p-ERK in IR cells with Western blotting.Meanwhile,Western blotting was used to evaluate AKT and ERK phosphorylation level in A549 and Lk2 cell lines to observe the radiosensitization effect of APA in NSCLC in four groups to explore the underlying mechanism as a radiosensitizer.Immunofluorescence detection of γH2AX was expose cells that grow on glass coverslips to 0 or 8Gy irradiation.Then,the cells were fixed in 4%paraformaldehyde 4h post-irradiation or without irradiation,and incubated with the primary phospho-γH2AX antibody and a secondary antibody conjugated to Cy3 according to the manufacturer’s protocol.We analyzed the staining with a confocal laser scanning microscope the number of γH2AX focus nuclei of different cells postirradiation.The immunofluorescence assay was conducted to assess the effects of ability of APA on which DNA was damaged and repaired by irradiation.The third part:Two cell lines A549 and LK2 were selected for the experiment.Divided into blank group and IR group.The blank group cells were raised normally without treatment and were directly used for tumor formation experiments.The IR group cells were vertically irradiated with a conventional radiation dose of 8Gy,and then raised for 24 hours for subsequent tumor formation experiments.Randomly divide 48 nude mice into 8 groups,with A549 and LK2 tumor bearing mice each divided into 4 groups:Blank group,APA group,IR group,and APA+IR group,with 6 animals in each group.Blank and APA groups of nude mice were subcutaneously inoculated with blank A549 and LK2 cells in the axilla;The IR group and APA+IR group of nude mice were subcutaneously inoculated with IR group cells,After tumor formation,the blank group and IR group tumor bearing mice were routinely fed,while the APA group and APA+IR group tumor bearing mice were given APA suspension by oral gavage,2mg/20g body weight,once a day,for one week continuously.Two hours after the last administration,measure the length and diameter of the tumor body,according to the tumor volume calculation formula we can make out the following results and conduct statistical analysis.Immunohistochemical method was used to detect the expression level of VEGFR2 in tumor tissue.Western blot method was used to detect the expression levels of VEGFR2,PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,and p-ERK in tumor tissues of mice in each group.Results:The first part:1.APA inhibited cell proliferation in does and time dependent manner.The cells lines A549,H522,H460,LK2,and H226 were evaluated for VEGFR-2 expression by Western blotting analysis and qRT-PCR.VEGFR-2 protein expression differed among the five NSCLC cell lines.A549 and LK2 cells were selected for further study based on their expression of VEGFR-2.Human lung cancer cell lines A549 and LK2 were cultivated with a series of different concentrations of APA for 24h,48h,and 72h.APA remarkably inhibited cell proliferation of A549 and LK2 cells in vitro and significantly inhibited in a dose and time dependent mode.After calculation,the IC50 does of APA after treatment for 72h in Lk2 cell and A549 cell were 30.73μmol/L and 17.61μmol/L respectively.We chose the IC20 concentration for 72h in the following experiments;2.Compared to irradiation alone,cell growth was remarkably suppressed in APA pretreatment plus IR group at different time points,especially at 96h.In addition,the results demonstrated that APA had an radiosensitivity by increasing cell death combined with X-ray irradiation and improved efficacy compared with IR or apatinib alone;3.The flow cytometry detection indicated that APA significantly increased radiation-induced apoptosis;4.Compared with control group,the clone forming ability and survival fraction of APA combined IR are lower,colony formation assays revealed that APA enhanced the radiosensitivity of NSCLC cells;5.The flow cytometry indicated the APA strongly restored the radiosensitivity through increased IR-induced G2/M phase arrest by changing cell cycle redistribution.The second part:1.Immunofluorescence detection of yH2AX indicated that the number of yH2AX focus nuclei of A549 and LK2 cells post-irradiation was increased obviously,but after 4h the number of yH2AX focus nuclei of NSCLC cell was hardly decreased in combination group than IR group.Further experiments showed that the APA effectively inhibited repair of radiation-induced DNA double-strand breaks(DNA-DSBs);2.AKT and ERK pathways are known to be activated by IR,the higher phosphorylation of AKT and ERK might play a role in radioresistance in NSCLC cells.Our previous results found that downregulation of AKT and ERK signal pathways enhanced cell radiosensitivity.In order to determine the potential molecular mechanisms of APA to exert enhanced anti-tumor effects and regulate radiosensitivity,we examined the key signaling molecules of AKT and ERK signaling pathways by western blotting.The overexpression of AKT and ERK was found in different NSCLC cells after IR,The results showed that phosphorylated AKT(p-AKT)and ERK(p-ERK)reduced after APA treatment both in A549 and LK2 cells differentially.These data indicate that APA combined with ionizing radiation can more increasing radiosensitivity by downregulating AKT and ERK pathways in NSCLC cells,but APA can also significantly decreased the activities of AKT and ERK in A549 and LK2 cells;More western blotting experiment results showed:Compared with the blank group,The level of VEGFR2,PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,p-ERK,p-ERK significantly decrease in APA group(p<0.01),The level of VEGFR2 was significantly decreased in the IR group,meanwhile the expression levels of PI3K,p-PI3K,AKT,pAKT,PKC,MEK,p-MEK,ERK,p-ERK were significantly increased(p<0.01),and the difference was statistically significant;Compared with the APA group,the expression level of VEGFR2 in the APA+IR combination group cells was significantly reduced(p<0.01),while the expression levels of PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,pMEK,ERK,and p-ERK in the IR group cells were significantly increased(p<0.01),with statistical significance;Compared with the IR group,the expression levels of VEGFR2,PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,p-ERK in the APA+IR combination group cells were significantly reduced(p<0.01),and the difference was statistically significant.The third part:1.The statistical results of tumor volume in each group of mice showed that compared with the blank group,the tumor volume of A549 and LK2 cell bearing mice in the APA group,IR group,and APA+IR combination group was significantly reduced(p<0.01),and the difference was statistically significant;Compared with APA group and IR group,the tumor volume of A549 and LK2 cell bearing mice in the APA+IR combination group was significantly reduced(p<0.01),and the difference was statistically significant;2.The detection results of VEGFR2 expression level in tumor tissues of A549 and LK2 cell bearing mice in each group showed:Compared with the blank group,the expression levels of VEGFR2 in the tumor tissues of A549 and LK20 tumor bearing mice in the APA group,IR group,and combination group were significantly reduced(p<0.01),and the difference was statistically significant;Compared with the APA group and IR group,the expression level of VEGFR2 in the tumor tissues of A549 and LK20 tumor bearing mice in the combination group was significantly reduced(p<0.01),and the difference was statistically significant;3.The expression levels of VEGFR2,PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,and p-ERK in A549 and LK2 tumor tissues of nude mice in each group were detected.The results showed that:Compared with the blank group,the expression levels of VEGFR2,PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,and p-ERK in A549 and LK2 tumor tissues in the APA group were significantly reduced(p<0.01);The expression levels of VEGFR2 in A549 and LK2 tumor tissues in the IR group were significantly reduced,while the expression levels of PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,and p-ERK were significantly increased(p<0.01),with statistically significant differences;Compared with the APA group,the expression level of VEGFR2 in A549 and LK2 tumor tissues in the APA+IR combination group was significantly reduced(p<0.01),while the expression levels of PI3K,p-PI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,p-ERK in A549 and LK2 tumor tissues in the IR group and combination group were significantly increased(p<0.01),with statistical significance;Compared with the IR group,the expression levels of VEGFR2,PI3K,pPI3K,AKT,p-AKT,PKC,MEK,p-MEK,ERK,and p-ERK in A549 and LK2 tumor tissues in the APA+IR combination group were significantly reduced(p<0.01),and the difference was statistically significant.Conclusions:1.VEGFR2 is expressed at both mRNA and protein levels in NSCLC cells.The anti vascular drug APA targeting VEGFR can also directly act on NSCLC cells and produce various biological effects on NSCLC cells through the VEGF/VEGFR2 pathway;2.APA has a direct proliferative inhibitory effect on NSCLC cells,meanwhile APA can significantly enhance the inhibitory effect of IR on NSCLC cell proliferation and promotes radiation-induced NSCLC cell apoptosis;3.APA can also enhance to induce more NSCLC cells to enter the radiotherapy sensitive G2/M phase,increasing the efficacy of radiation therapy.APA significantly strengthens the damage effect of radiation induced DNA double strand breaks in NSCLC cells,while interfering with and delaying the process of DNA double strand break repair,and has a radiation sensitization effect;4.The NSCLC cells in the APA combined with IR group showed a significantly reduced ability to form clones,lower survival scores,and an increase in radiosensitivity,which was statistically significant.This further validated the role of APA in enhancing the radiosensitivity of NSCLC cells;5.APA may inhibit NSCLC cell proliferation and increase NSCLC radiosensitivity through the AKT and ERK pathways and downregulate p-AKT and p-ERK;6.The underlying mechanism of APA’s inhibitory effect on tumor growth may be related to its inhibition of the activation of the PI3K/AKT signaling pathway in tumor tissue;7.The underlying mechanism of APA’s inhibitory effect on tumor growth is related to its inhibition of the activation of MEK/ERK signaling pathways in tumor tissue;8.In summary,our study demonstrates that APA enhances radiosensitivity of NSCLC cells,our findings also provide a rationale drug for clinical setting of APA as a new agent in NSCLC,especially for the VEGFR-2 overexpression.Our results also indicate a novel role of APA as a the radiosensitizer in NSCLC and may provide clinical guidance to improve radiotherapy efficiency in NSCLC patients,but the optimal dose of APA requires further study. |
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