Apatinib preferentially inhibits PC9 gefitinib-resistant cancer cells by inducing cell cycle arrest and inhibiting VEGFR signaling pathway.

BACKGROUND: Lung cancer is one of the most common and deadly tumors around the world. Targeted therapy for patients with certain mutations, especially by use of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), has provided significant benefit to patients. However, gradually developed resistance to the therapy becomes a major challenge in clinical practice and an alternative to treat such patients is needed. Herein, we report that apatinib, a novel anti-angiogenic drug, effectively inhibits obtained gefitinib-resistant cancer cells but has no much effect on their parental sensitive cells. METHODS: Gefitinib-resistant lung cancer cell line (PC9GR) was established from its parental sensitive line (PC9) with a traditional EGFR mutation after long time exposure to gefitinib. Different concentrations of apatinib were used to treat PC9, PC9GR, and other two lung cancer cell lines for its anti-growth effects. RNA sequencing was performed on PC9, PC9GR, and both after apatinib treatment to detect differentially expressed genes and involved pathways. Protein expression of key cycle regulators p57, p27, CDK2, cyclin E2, and pRb was detected using Western blot. Xenograft mouse model was used to assess the anti-tumor activity of apatinib in vivo. RESULTS: The established PC9GR cells had over 250-fold increased resistance to gefitinib than its sensitive parental PC9 cells (IC50 5.311 ± 0.455 µM vs. 0.020 ± 0.003 µM). The PC9GR resistance cells obtained the well-known T790M mutation. Apatinib demonstrated much stronger ( ~ fivefold) growth inhibition on PC9GR cells than on PC9 and other two lung cancer cell lines, A549 and H460. This inhibition was mostly achieved through cell cycle arrest of PC9GR cells in G1 phase. RNA-seq revealed multiple changed pathways in PC9GR cells compared to the PC9 cells and after apatinib treatment the most changed pathways were cell cycle and DNA replication where most of gene activities were repressed. Consistently, protein expression of p57, CDK2, cyclin E2, and pRb was significantly impacted by apatinib in PC9GR cells. Oral intake of apatinib in mouse model significantly inhibited establishment and growth of PC9GR implanted tumors compared to PC9 established tumors. VEGFR2 phosphorylation in PC9GR tumors after apatinib treatment was significantly reduced along with micro-vessel formation. CONCLUSIONS: Apatinib demonstrated strong anti-proliferation and anti-growth effects on gefitinib resistant lung cancer cells but not its parental sensitive cells. The anti-tumor effect was mostly due to apatinib induced cell cycle arrest and VEGFR signaling pathway inhibition. These data suggested that apatinib may provide a benefit to patients with acquired resistance to EGFR-TKI treatment.

Neighbors' death is required for surviving human adenocarcinoma PC-9 cells in an early stage of gefitinib treatment.

Acquired drug resistance is a major problem in chemotherapy, and understanding of the mechanism, by which naïve cells defend themselves from drugs when the cells exposed to the drugs for the first time, may provide a solution of the problem. Gefitinib is an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, and used as an anticancer drug; however, gefitinib treatment may sometimes lead cancer cells gradually into a gefitinib-tolerance. Here we describe that human adenocarcinoma PC-9 cells even under the presence of gefitinib were able to survive by activating another signaling pathway involving fibroblast growth factor receptor (FGFR) and its signaling molecule, FGF2; and further suggest that the FGF2 for initiating the pathway might be supplied from neighboring cells which were killed by gefitinib, i.e., the survival might be founded on neighbors' sacrifice in an early stage of gefitinib treatment. Our findings suggested that whether cells had a chance to encounter to survival factors such as FGF2 soon after gefitinib treatment might be an important crossroads for the cells for survival and for gaining a gefitinib tolerance.

Pemetrexed Disodium Heptahydrate Induces Apoptosis and Cell-cycle Arrest in Non-small-cell Lung Cancer Carrying an EGFR Exon 19 Deletion.

BACKGROUND/AIM: Non-small-cell lung cancer (NSCLC) remains a significant cause of death despite the recent introduction of several improved therapeutics. Pemetrexed disodium heptahydrate (pemetrexed) is currently available in combination with a platinum derivative for patients with advanced non-squamous NSCLC for first-line treatment, and as a single agent for second-line treatment. However, the mechanisms underlying its anticancer activities are still not well understood. In this study, we evaluated the growth inhibitory effects of pemetrexed on PC9 (EGFR exon 19 deletion) cells and elucidated the underlying molecular mechanisms. MATERIALS AND METHODS: PC9 cells were treated with pemetrexed and then assessed for the cell viability, morphological and nuclear changes, antigenic alterations, SA-ß-gal staining, and changes in protein expression. RESULTS: Pemetrexed reduced the cell viability of PC9 cells and initiated cell morphological changes in a concentration-dependent manner. Pemetrexed significantly induced G1 phase arrest in a dose-dependent manner. The results demonstrated that pemetrexed induced apoptosis in PC9 cells, a change coupled with an increase in reactive oxygen species and a decrease in mitochondrial membrane potential. Pemetrexed decreased Bcl-2 expression, while Bax expression was increased, and cytochrome c was released. Furthermore, the expression of extrinsic pathway proteins, e.g. Fas/FasL, DR4/TRAIL, and Fas-associated protein with death domain, was increased by pemetrexed, which then activated caspase-8, caspase-9, and caspase-3 and induced poly (ADP-ribose) polymerase proteolysis. CONCLUSION: This study revealed the mechanisms by which pemetrexed works an anticancer drug in the treatment of NSCLC.

Enhancive effect of FGL1 gene silencing on sensitivity of human lung adenocarcinoma PC-9 cells to docetaxel / 中国药理学通报

Aim To investigate the effect of fibrinogenlike protein 1 (FGL1)) silencing on docetaxel sensitivity in human lung adenocarcinoma PC-9 cells. Methods Western blot was used to detect the expression of FGL1 protein in human normal bronchial epithelial cell line BEAS-2B and human lung adenocarcinoma cell line PC-9. The FGL1 gene in PC-9 cell line was silenced by siRNA. CCK-8 assay was used to detect the inhibitory effect of silencing FGL1 on PC-9 cell proliferation and its effect on docetaxel sensitivity. Results Compared with BEAS-2B cell line, FGL1 was highly expressed in PC-9 cell line, and the relative expression of FGL1 protein was 6. 5 times that of BEAS-2B cell line with statistically significant difference (P <0. 01). Silencing FGL1 by transfection with FGLlsiRNA could enhance the inhibitory effect of docetaxel on PC-9 cells. Compared with FGLlsiNC group, the IC50value of PC-9 cells in FGL1siRNA group was significantly reduced with statistically significant difference (P < 0. 01). Conclusions Specific silencing of FGL1 gene could inhibit the expression of FGL1 in human lung adenocarcinoma cell PC-9, inhibit the proliferation of PC-9 cells and increase the sensitivity to docetaxel.

Effect of axitinib on the proliferation and apoptosis of human lung adenocarcinoma PC9 cells and its mechanism / 肿瘤研究与临床

Objective To study the effect of axitinib on the proliferation and apoptosis of human lung adenocarcinoma PC9 cells and its mechanism. Methods PC90 cells were treated with different concentrations (0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, and 64 μmol/L) of axitinib for 72 h, and half-inhibitory concentration (IC50) was calculated. The cell proliferation ability was detected by CCK-8 method. Plate cloning experiments were performed to observe the effect of axitinib on the formation of PC9 cell clones. The mitochondrial membrane potential and apoptosis of PC9 cells were detected by flow cytometry. The expression of cleaved-Caspase-3 protein in PC9 cells was detected by Western blot. Results Asitinib inhibited the proliferation of PC9 cells in a concentration-dependent manner. The IC50 at 72 h was 10.18μmol/L. The clone formation rates of PC9 cells were (100.0±3.2)％, (58.6±2.7)％, (29.3±3.3)％, and (10.9±3.0)％10 d after treatment with 0, 1, 2 and 4 μmol/L axitinib, and the difference was statistically significant (F= 316.922, P< 0.01). The apoptotic rate of PC9 cells at early and late stages increased after treatment with different concentrations of axitinib for 48 h, and the differences were statistically significant (both P< 0.01). After treatment with 0, 4, 8 and 16 μmol/L axitinib for 24 h, the percentage of PC9 cells with low mitochondrial membrane potential was (11.9±1.9)％, (38.5±2.3)％, (56.3±2.7)％, and (76.9±3.1)％, and the difference was statistically significant (F=234.320, P<0.01). The expression level of cleaved-Caspase-3 protein in PC9 cells increased, and the relative expression levels were 1.00±0.04, 1.26±0.09, 1.78±0.12, and 2.10±0.11, respectively, and the difference was statistically significant (F=55.670, P<0.01). Conclusions Axitinib could inhibit the proliferation of human lung adenocarcinoma PC9 cells. Axitinib induces the apoptosis of PC9 cells possibly through decreasing the mitochondrial membrane potential of PC9 cells.