Study Of The Inhibitory Effect Of Src Kinase Inhibitor M475271 On NSCLC And The Mechanism

IntroductionNon-small cell lung cancer ( NSCLC) accounts for about 80% of all lung cancers. Although some progress has been made in the treatment of NSCLC, NSCLC remains the leading cause of cancer-related mortality in most countries. The majority of the diagnosed patients with NSCLC are in advanced stage because of its highly metastatic properties, and such patients are not candidates for surgical resection. Although systemic chemotherapy reduces the rate of death attributable to advanced NSCLC, disease progression is inevitable and dose-limiting toxicities restrict its use.A novel strategy for chemorefractory NSCLC in advanced stage is molecular-ly targeted therapy inhibiting specific pathways and key molecules implicated in tumor growth and progression. Among molecularly targeted therapies, inhibitors of epidermal growth factor receptor (EGFR) -tyrosine kinases have been successfully developed for advanced NSCLC. Although EGFR inhibitors are more effective for adenocarcinoma compared with squamous cell carcinoma, the clinical responses are observed only in some populations of the patients even in adenocarcinoma. Therefore, novel drugs targeting other critical molecules are required for patients with NSCLC.Src is the first reported oncogene and has been a prototype in identifying many characteristics of follow on oncogenes. C-Src and its retroviral form ( V-Src) are both non-receptor tyrosine kinases, encoding a Mr 60,000 membrane -associated protein tyrosine kinase ( PTK). Results from many different studies suggest a role of Src in various aspects of a cell's life, including proliferation,survival, motility, cell-cell and cell - matrix adhesion, and local invasion, all of which are deregulated during cancer progression. C-Src has been found to be over-expressed and highly activated in a variety of human cancers, including colon cancer, breast cancer, and pancreatic cancer. Recent studies showed that the proto-oncogene C-Src was also highly activated in NSCLC, especially in ade-nocarcinomas, suggesting the possibility of molecular targeted therapy for lung adenocarcinoma.M475271 is a novel, orally available selective inhibitor of Src kinases. It has potential to inhibit migration of the highly metastatic human pancreatic cancer cell line ( L3. 6pl) at submicromolar dose levels in vitro . In addition, oral treatment with M475271 suppressed the progression of orthotopically implanted L3.6pl cells in a nude mouse model. In the present study, we examined the effect of M475271 on in vitro properties of human lung cancer cell lines expressing various levels of Src protein. We further investigated the therapeutic effect of M475271 on the subcutaneous growth and experimental metastasis produced by human adenocarcinoma cell lines in SCID mice.Materials and methodsSrc expression and activation in NSCLC cell lines as well as M475271 mediated inhibition of phosphorylation of Src and MAPK were assayed with immu-noprecipitation and western blot.MTT assay was used to examine the effect of M475271 on NSCLC cells proliferation.We used collagen I coated transwell chambers to assess the effect of M475271 against cellular invasion in four NSCLC cell lines in vitro .We examined the level of VEGF, activated MMP-2 and MMP-9 in NSCLC cell lines as well as the effect of M475271 on VEGF expression, activated MMP-2 and MMP-9 by ELISA.For the subcutaneous tumor model, A549 cells or PC-9 cells were inoculated into SCID mice by subcutaneous injection. For the lung metastasis model, A549 cells were injected into NK cell-depleted SCID mice via the tail vein.Mice were orally given every day with or without M475271 to examine the effect of M475271 on the growth of subcutaneous tumor and experimental lung metastasis.Immunohistochemistry was used to show the effect of M475271 on PI and CD31 as well as VEGF expression in subcutaneous tumor and experimental lung metastasis.Results1. Expression of Src and Src phosphorylation in NSCLC cellspp60are was expressed in human bronchial epithelial BEAS2B cell line and all human cancer cell lines used in this study. All five NSCLC cell lines and BEAS2B cell line had autophosphorylated tyrosine-418. The level of phosphorylated Src was highest in PC-9 cells, which was 2-fold higher compared to normal level, followed by A549 cells, 1. 69-fold higher compared to normal level. While nearly no phosphorylated Src in small cell lung cancer SBC5 cell line was detected.2. Effect of M475271 on phosphorylation of SrcM475271 did not affect the level of Src protein expression in all cell lines, however, it reduced the autophosphorylation of Src in a dose-dependent manner in nearly all 5 NSCLC cell lines, especiall in PC-9 and A549 cell lines. 0. 3(xM M475271 nearly inhibited autophosphorylation of Src in PC-9 cells entirely.3. Effect of M475271 on phosphorylation of MAPKM475271 showed a dose-dependent inhibition on phosphorylation of ERK1/ 2. Submicromolar M475271 nearly inhibited phosphorylation of ERK1/2 in PC-9 and A549 entirely. While activated ERK1/2 in other 3 NSCLC cell lines was not sensitive to M475271.4. Effect of M475271 on cell proliferation in vitroThe effect of M475271 on cell proliferation varied significantly among the NSCLC cell lines. PC-9 and A549 cells showed a dose-dependent reduction by M475271. M475271 at 0. 03 ~ 3jjlM suppressed the cell proliferation of PC-9 cells by 11.7% ( P >0.05), 53.6% ( P <0.001), 72.22% ( P <0.001),82. 39% ( P <0.001) and 90. 56% ( P <0.001) , respectively. Same concentration of M475271 suppressed the cell proliferation of A549 cells by 24.03% ( P <0.05), 30.25% ( P <0.05), 38.54% ( P <0.001), 46.62%( P <0. 001) and 60. 78% ( P <0. 001) , respectively. M475271 at 3julM suppressed the cell proliferation of H226 cells by 20% ( P <0. 001). Same concentration of M475271 had no significant effect on proliferation of PC14PE6 and RERFL-COK ceUs.5. Effect of M475271 on cell invasiveness in vitroInvasiveness of NSCLC cells was suppressed by M475271 in a dose-dependent manner, though there was a little difference in degree of the inhibition among four cell lines. M475271 at 0. 03 ~ ljxM suppressed the cell invasion of PC-9ceUsbyl7.31%( P <0.05), 63.46% ( P <0.001), 82.69% ( P <0. 001) and 91. 69% ( P < 0. 001) , respectively. M475271 at 0. 3 ~ 3u,M suppressed the cell invasion of A549 cells by 36. 35% ( P <0.001) ,77. 9% ( P < 0.001) and 90. 96% ( P < 0. 001) , respectively. M475271 at 1 and 3uJVl could also suppress the cell invasion of PC14PE6 and H226 cells by 54. 73% ( P <0.001) and 79. 1% ( P <0.001) as weU as 36. 88% ( P <0. 001) and 68. 09% ( P < 0. 001 ) , respectively. The MTT assay demonstrated that M475271 did not reduce cells viability at concentrations and times we used for cell invasion assay.6. Effect of M475271 on activated MMP-2 and MMP-9 in NSCLC cells PC14PE6 and H226 cells expressed high MMP-2 and MMP-9 activity,whereas A549 cells expressed only MMP-9 activity. MMP-2 and MMP-9 activity in PC-9 cells was under detection limits. M475271 could obviously inhibited activated MMP-2 in PC14PE6 ceUs and MMP-9 in PC14PE6, H226 and A549 cells in a dose-dependent manner.7. Effect of M475271 on Production of VEGF by NSCLC Cells in vitro We found that M475271 could inhibit the production of VEGF in all threecell lines used in this experiment. In order to verify whether the reduction of VEGF was due to the reduction of cell proliferation, we also performed MTT assay. There was no significant reduction of cells in PC14PE6 and A549 cell lines. In PC-9 cell line, only the concentration of M475271 more than 300nMcould reduce cell proliferation. Standardized the level of VEGF by percentage of cell proliferation indicated that M475271 could significantly inhibit the production of VEGF in a dose-dependent manner, especially in PC-9 and A549 cells.8. Effect of M475271 on subcutaneous Tumor and metastatic lung tumors in SCID miceTreatment with M475271 ( both 10mg/( kg ? d) and 50mg/( kg ? d) ) markedly inhibited the growth of subcutaneous tumors produced by PC-9 cells. After oral feeding with 10mg/( kg ? d) and 50mg/( kg ? d) M475271 for 2 weeks, the volume of subcutaneous tumor was inhibited by 73.28% and 93.4% ( P < 0. 001) , respectively. While the same treatment was less effective to tumors produced by A549 cells, 50mg/( kg ? d) M475271 did not cause significant inhibition until treatment with M475271 3 weeks later, the inhibition rate was about 39.4% ( P <0.05-0.01).A549 cells developed numerous small lesions on the surface of the lung in all groups, but the nodules of the lung in 50mg/( kg ? d) group was smaller than that of control group , representing significantly less lung weight ( P < 0.05). Treatment with 10mg/( kg ? d) M475271 had no significant effect on the lung weight. Treatment with M475271 (both 10 and 50mg/(kg ? d) ) did not cause weight loss of mice significantly, However, inoculated A549 cells 28 days later, the weight in control group was significantly lost( P <0.01).9. Effect of M475271 on cell proliferation and vascularization in vivoIn PC-9 cells induced subcutaneous tumor, 50mg/(kg ? d) M475271 significantly reduced the number of Ki67-positive proliferating tumor cells. The proliferation index (PI) was 22 ±5 and 50 ±8 in treatment and control group, respectively ( P <0.001). In A549 cells induced subcutaneous tumor, 50mg/ (kg ? d) M475271 tended to reduce the number of Ki67-positive proliferating tumor cells. The proliferation index ( PI) was 34 ± Hand 42 ±7 in treatment and control group, respectively ( P >0. 05). Treatment with 50mg/( kg ? d) M475271 not only reduced the number and the size of metastatic lesions, but also inhibited the number of Ki67-positive proliferating tumor cells in lung metas-tases produced by A549 cells ( P <0. 001). PI in control group and treatment group was 59 ± 10 and 24 ±3.Treatment with 50mg/kg M475271 significantly reduced MVD in both PC-9 ( P < 0.001) and A549( P <0.01)- induced subcutaneous tumors.Moreover, M475271 could greatly reduce the production of VEGF in PC-9 and A549-induced subcutaneous tumors, as well as in lung metastases produced by A549 cells. 50mg/kg M475271 could substantially reduce the phosphorylation of Src at the Y418 site, especially in highly phosphorylated PC-9 induced subcutaneous tumors.DiscussionOver-expression of Src has been detected in a variety of epithelial and non-epithelial malignancy in humans. Mazurenko et al. reported that C-Src protein was found to be elevated in a half of lung cancer patients when determined by immunoblotting and immunohistochemistry, while no pp60 * rc protein was detected in epithelial cells of normal lung tissues. Regarding pp60 "Sre kinase activity, NSCLC, especially adenocarcinoma, had a higher level of pp60CSrc activity compared with colon cancer, which is most often associated with elevated pp60CSrc activity. These findings suggest possible implication of Src in progression of lung adenocarcinoma. Consistently, we found that NSCLC cell lines tested expressed Src protein (pp^SO"0) and have autophosphorylation of tyrosine-418, while there was no over-expression of Src compared with BEAS2B cells, the level of autophosphorylation of tyrosine-418 in NSCLC cells was higher than that in BEAS2B cells, suggesting that phosphorylation ( activation) , but not over-expression, of Src is crucial for tumor-cell invasion and proliferation.Src is a downstream molecule of various growth factor receptors, which regulate cell proliferation and invasion. Src interacts with EGFR that are recognized key molecules for progression of various solid tumors, including lung cancer. Furthermore, since several tumors are known to over-express both EGFR and Src, it is reasonable that they cooperate with each other in signal for cell growth and invasion. Ras/Raf/MEK/MAPK is one of critical cell signal transduction pathways of regulating cell proliferation by Src and EGFR.MAPK has been over activated in many human cancers. Our results showedthat inhibition of Src phosphorylation could suppress the phosphorylation of ERKl/2, which is parallel to the inhibitory effects on cell proliferation in NSCLC cell lines. Therefore, the inhibitory effect of M475271 on NSCLC cells proliferation maybe due to the suppression of ERKl/2, a downstream signal of Src.It is well known that MMPs are crucial for tumor invasion and metastasis. We found that M475271 at high concentration could inhibit MMP-2 and MMP-9 activity. MMP-2 and MMP-9 play a pivotal role in regulating cell mingation and invasion in some NSCLC cells, but other factors was also involved in regulating NSCLC cells invasion.Recent reports indicate that Src is one of critical molecules regulating an-giogenesis. Activation of Src stimulates VEGF protein production from various types of cell lines, and Src cooperates with VEGF receptors ( KDR/Flk-1) in endothelial cells, and hence stimulates endothelial proliferation. In fact, Ellis et al reported that transfection of antisense C-Src down-regulated VEGF production of colon cancer cells and inhibited tumor vascularization. In the present study, daily oral administration of M475271 suppressed the growth of subcutaneous tumors produced by PC-9 and A549 cells, accompanied with the inhibition of vascularization as well as tumor cell proliferation in vivo . VEGF production in the tumors was also inhibited by treatment with M475271, angiogenesis inhibition by this compound may be mediated by, at least in part, inhibition of the production of VEGF. Therefore, angiogenesis inhibition is regarded as one of the possible mechanisms of antitumor effects by M475271.The process of metastasis involves several sequentially linked steps. We e-quated experimental lung metastasis with the blood-borne spread of A549 cells. Although tumor cells had bypassed the initial steps of metastasis ( separation from the primary neoplasm, invasion, and release into blood vessels and lymphatics ) , all subsequent steps in the process for formation of metastases (arrest at a distant site, extravasation, invasion and proliferation with angiogenesis in the lung) must have occurred. M475271 treatment started 7 days after tumor-cell injection (after the establishment of micrometastasis) inhibited the production of experimental lung metastasis of A549 cells, representing reduced lung...