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Research article Effect of c-Met inhibitor SU11274 on human colon cancer cell growth Shuohui GAO1, Chao LIU2, Jun WEI1,Ye FENG1 1. The department of general surgery, the third hospital of Jilin University, 130033 China 2. The department of neurosurgery, the second hospital of Jilin University, 130021 China Correspondence to: XXXXX Keywords: Colon cancer, SU11274, cell growth, c-Met Abstract Background: Colon cancer is one of the major malignancies worldwide and still remains resistant to much of the currently available chemotherapy. Downregulation of HGF/c-Met signaling pathway is an emerging therapy for cancer treatment. Methods: In this study, the inhibitory effects of c-Met phosphorylation were observed with SU11274 on different colon cancer cell lines in vitro. Results: The results revealed the significant inhibitory effects of SU11274 on cell proliferation and cell survival, in a time and dose-dependent manner. Furthermore, the inhibitory effects of SU11274 on different subgroups of colon cancer cells via the signaling pathway were implicated in this study. Conclusions: The results suggested the possible selective therapeutic effects of c-Met inhibitor on colon cancer Colon cancer represents one of the most common malignancies worldwide, and despite advances in chemotherapy, this cancer still remains a major cause of death. The risk of severe adverse effects are also associated with currently available anti-cancer drugs [1]. Tumor invasion and lymph node metastasis are important factors determining the prognosis of the disease. Therefore, novel individualized treatment options are needed to improve patients’ survival, and the prevention of cancer invasion and metastasis. The receptor tyrosine kinase c-Met, a receptor to Hepatic Growth Factor (HGF) which activates the c-Met signaling pathway by phosphorylation, was first identified by chemical rearrangement in vitro [6]. The c-Met protein contains a tyrosine kinase domain that initiates a range of signals to regulate various cellular functions, including cell proliferation, motility, adhesion and tumor cell invasion [3]. In the past two decades, accumulating evidence suggests that overexpression of c-Met protein is strongly associated with poor patient survival or metastasis in numerous human malignancies [2,5]. Inappropriate HGF/c-Met signaling in cancers can resemble developmental transitions between epithelial and mesenchymal cell types. Thus, inactivation of c-Met phosphorylation has been considered a potential therapeutic target for influencing the events of tumor progression and some agents are currently in clinical trials [7]. Furthermore, c-Met signaling is complex, and how the regulation of its receptor subunits via HGF/c-Met signaling axis influences its oncogenic behavior is not fully understood. A small molecule inhibitor SU11274 has been shown to inhibit c-Met phosphorylation, and consequently decrease cell growth, motility, invasion and proliferation in lung cancer and ovarian carcinoma [4, 9]. These results support the therapeutic potential of targeting c-Met in cancers where c-Met plays a crucial role in tumor growth and metastasis. The overexpression of c-Met in colon cancer is an important prognostic marker for early stage invasion and regional metastasis, and the development of new targeted therapy inhibiting the c-Met signal pathway promises a new modality for treatment [8]. The aim of this present study was to study the inhibitory effect that SU11274 had on colon cancer cell growth via HGF/c-Met signal pathway. 1. Methods Cell lines and cell culture Four well-differentiated colon cancer cell lines (K-ras wild type HT-29, Hct-8, and K-ras mutant type Hct-116 and DLD-1) were purchased from the American Type Culture Collection (ATCC, Rockville,MD). Cells were maintained in RPMI 1640 medium (Cellgro), supplemented with 10% FCS, and 1% penicillin-streptomycin. After incubation at 37°C with 5% CO2, the cells were harvested during log phase growth. For analysis all the cultured cells were flash frozen in liquid nitrogen and stored at -80℃. Reagents and antibodies Rabbit anti-human Met, Akt, mTor, erk, ps6 and beta-actin monoclonal antibodies, as well as rabbit anti-human phospho-Met, phospho-Akt, phospho-mTor, phospho-erk, and phospho-ps6 monoclonal antibodies were all purchased from SIGMA, Inc. (Beverly, MA, USA), and were used as primary antibodies. Peroxidase-conjugated goat anti-rat monoclonal antibodies were used as secondary antibodies and were purchased from Jackson Immunnoresearch Inc. Hepatocyte growth factor (HGF) was purchased from Rocky Hill company. c-Met specific inhibitor SU11274 was purchased from SUGEN, Inc (South San Francisco, CA, USA). Cell viability assay Cell viability was measured by the acid phosphatase assay. Continuously cultured cells were harvested and seeded on 96-well plates at a density of 8x103 cells per 200 ul in medium containing 10% FBS and cultured for 24 h. Then, cells were treated with 30ng/ml HGF and with 0.01uM, 0.1uM, 0.5uM and 2.5uM of SU117274 separately. After incubation at 37˚C for 1.5 h, the reaction was stopped by adding 10 μl of 1 M NaOH and cell viablilty was evaluated using a microplate reader (Bio-Rad Laboratories, Hercules, CA) at 405 nm. Western blotting For stimulation studies with HGF, continuously cultured cells were deprived of growth factors by incubating the cells without FBS for 24 h and cells were homogenized in lysis buffer. Cells were treated with different doses of SU11274. The cells were tested for protein tyrosine phosphorylation in response to 30 ng/ml HGF for 15 minutes. Proteins were quantified by BioRad RC-DC assay. Proteins were resolved using SDS-PAGE gel electrophoresis and blots were incubated overnight at 4°C with specific primary antibodies, followed by 1 h incubation with appropriate peroxidase-coupled secondary antibodies and visualized. Western blotting band intensities were expressed in arbitrary units and normalized to fold increases compared with control. The same blot was also incubated with an anti--actin antibody (Sigma, St. Louis, MO) for the normalization of protein loading. The membranes were stripped between hybridizations. Cell cycle analysis by flow cytometry Cells were synchronized by serum deprivation, and the experimental cells were treated 24 h with 0.5 M SU11274 and untreated cells were used as controls. Cells were fixed with 75% ethanol in PBS, cells were treated with 30 ng/ml HGF in PBS at 37°C for 15 min and stained on ice with propidium iodide solution (20 mg/ml) (Invitrogen) containing RNase A (100 g/ml) (Promega, Madison, WI, USA) at 4°C for 30 min. The distribution of cells in the cell cycle was assessed by a FACSscan flow cytometer (Becton Dickinson, San Diego, CA, USA). The result is the mean of three independent experiments and was processed using Excel (Microsoft, Inc.). 2. Results Inhibition of cell proliferation Each cell line was treated with different concentrations of SU11274 (0, 0.1, 0.5, and 2.5 uM) for 24 h, 48h and 72h separately, and cell growth was determined by the acid phosphatase assay (Figure 1). Decreased proliferation was observed in all colon cancer cells in response to SU11274 in a dose and time dependent manner. The dose of 0.5 M SU11274 was sufficient to achieve the similar maxim inhibition after treatment for 72 h as the dose of 2.5 M, but in cell line DLD-1, the inhibition was similar after treatment with different doses of SU11274 for 48 h. These results suggest that the inhibitory effects of SU11274 correlate with the dose and the time of treatment. HT-29 HCT-8 120 120 Cell prolifiration (%) 100 80 60 40 20 0 HT-29 0 24 48 SU11274-0.1M SU11274-0.5M SU11274-2.5M 100 Cell prolifiration (%) SU11274-0.1M SU11274-0.5M SU11274-2.5M 80 60 40 20 0 HCT-8 72 0 Time effect (h) HCT-116 48 SU11274-0.1M SU11274-0.5M SU11274-2.5M 100 120 SU11274-0.1M SU11274-0.5M SU11274-2.5M Cell prolifiration (%) 100 80 60 40 20 0 HCT-116 0 72 DLD-1 120 Cell prolifiration (%) 24 Time effect (h) 24 48 72 80 60 40 20 Time effect (h) 0 DLD-1 0 24 48 72 Time effect (h) Figure 1: Graphs representing the effect of SU11274 on colon cancer cell viability. Different colon cancer cells were treated with increasing concentrations of SU11274. Cell viability was measured at different time points. The experiments were repeated 3 times and the results represent mean±SD. Expression of c-Met signaling in cell lines with or without SU11274 A monoclonal antibody against c-Met was used to detect the c-Met expression. Expression of Met proteins downstream c-Met signaling pathway in all the cells was strongly detected. In response to HGF (30 ng/ml, 15min; Figure 2), there was increased tyrosine phosphorylation of multiple proteins in the four cell lines used. SU11274 effectively decreased the amount of activated c-Met proteins to a detectable level within 30 min of treatment without significantly affecting the total c-Met protein levels. With the concentration of 0.1 uM SU11274 in all the cell lines, the tyrosine phosphorylation of Met was inhibited totally, and tyrosine phosphorylation in the rest of the downstream proteins like Akt, mTor, rpS6 and Erk were weakened. With the concentration of 0.5 uM SU11274, the tyrosine phosphorylation was totally inhibited for Akt in cell lines HCT-8 and HCT-116 and for rpS6 in cell line DLD-1. With the concentration of 2.5 uM SU11274, total inhibition of tyrosine phosphorylation was seen for Akt, rpS6 and Erk in cell line HT-29, and in other cell lines the activated c-Met signaling proteins were weakened significantly. HT-29 HCT-8 HCT-116 DLD-1 Figure 2: Western blot analysis showing the expression levels of phosphorylated proteins downstream of the c-Met signaling pathway. P42???? Modulation of cell cycle progression by SU11274 The effect of SU11274 on cell cycle progression in cells was tested by flow cytometry. The concentration of 0.5 M SU11274 was used to evaluate the effect of the compound on cell cycle progression. The data demonstrated that all the cells exhibited a dramatic reduction in DNA synthesis (Figure 3). The percentage of cells in S phase was reduced significantly (from 24% to 8% in HT-29, 25.3% to 6.3% in HCT-8, 25.4% to 13.9% in HCT-116, and from 27.9% to 16.7% in DLD-1 cells, respectively). This decrease in S-phase population was accompanied by a concomitant increase of the G0/G1 fraction (increasing from 58.2% to 80.5% in HT-29, 59.6% to 81.3% in HCT-8, 59.4% to 71.5% in HCT-116 and from 55.6% to 70.3% in DLD-1 cells, respectively) ,suggesting a strong G0-G1 cell cycle arrest. Furthermore, SU11274 treatment also led to a reduction in the G2-M phase (from 17.8% to 11.5% in HT-29, 15.1% to 12.4% in HCT-8, 15.2% to 14.6% in HCT-116 and from 16.5% to 13% in DLD-1 cells, respectively). Figure 3: Cell cycle analysis of different colon cancer cells treated with 0.5 M SU11274. 3. Discussion Normal epithelial cells express c-Met widely, including normal colorectal epithelium, and overexpression of c-Met has been shown to correlate with metastasis and poor patient prognosis in various human malignancies [7]. So it is suggested that c-Met expression plays an important role in the progression of the cancer. Targeting HGF/c-Met signaling pathway provides a potential therapy for cancer treatment, and the small molecule inhibitor SU11274 specific for c-Met has been used to test this hypothesis in vitro [9]. However, the effect of c-Met inhibition on colon cancer has not investigated widely. We endeavored to study the effects of SU11274 on the oncogenic behavior of c-Met in colon cancer. Four cell lines of colon cancers were used in this study, all of which were treated with HGF and displayed proliferation in the presence of HGF, indicating the initiation of HGF/c-Met signaling pathway, which is dependent on HGF. To our knowledge, HGF is the only ligand for c-Met and is used widely in targeting c-Met signaling pathway [10]. In the four cell lines used, the cell proliferation showed inhibition with increasing concentrations of SU11274 and with prolonged time periods as shown in Figure 1, indicating the blockage of HGF/c-Met signal pathway by SU11274 is both time-dependent and dose-dependent. The inhibition rate of cancer cells by chemotherapy differs significantly with different dosages and the administration conditions. A recent study on HCT-116 cell line reflected this efficacy of chemotherapy [19]. Similar results were also seen in our study. In this study, we did not test the inhibition of SU11274 in response to different doses of HGF, which might reflect the autocrine loops of HGF/c-Met which is responsible for increase in cell proliferation [11, 12]. Our study also demonstrates the differential effect of SU11274 on different colon cancer cell lines, so future studies are required to analyze the response of different colon cncer cell types toward chemotherapies [13]. HGF/c-MET signaling pathway regulates multiple cellular process, including cell proliferation, apoptosis and angiogenesis, and in recent years efforts have been made to investigate the functional effects of this pathway on the progression of human cancer [20]. Blockage of the downstream c-Met signaling pathways plays a potent role in inhibiting the c-Met oncogenic behavior [16,18]. Proteins downstream of this pathway include Erk, c-Met, Akt and mTor, which induce proto-oncogenic expression of c-Met. Studies targeting c-Met in vivo or in vitro hae demonstrated attenuated expression of these proteins leading to the inhibition of cellular proliferation and differentiation [21-22]. Western blotting in our study revealed the significant inhibition of phosphorylation of these proteins following treatment with SU11274, as shown in Figure 2. The tyrosine phosphorylation of c-Met was inhibited totally by 0.1 uM SU11274 in all the cell lines, while the phosphorylation of the remaining proteins were weakened with this dose and displayed decreasing expression levels in response to increasing amount of SU11274 with time. The differential effects of SU11274 on the inhibition of these proteins did not show any obvious significance in the different cell lines. Total inhibition of phosphorylation of mTor was not detected in this study, implicating that there might be a possible mechanism for regulation of c-Met signal in tumor microenvironments [17]. It is known that c-Met is expressed more strongly in primary colorectal cancers than in normal mucosa, and there is lack of evidence to show the correlation between the c-Met overexpression and cancer stages [14]. In our study, the percentage of cells in S phase arrested to G0/G1 phase was seen with SU11274 treatment, suggesting the inhibitory effect of this agent on the initiating events in the progression of cancer [15]. The significant reduction of DNA synthesis of cancer cells promises a therapeutic role of c-Met inhibitor in cancer treatment. However, modulation of cell cycle was not tested with different doses of SU11274 in our study. We analyzed the effects of c-Met inhibitor SU11274 on different colon cancer cell lines with proliferation, signal expression and motility in vitro. Our study strongly suggests that SU11274 suppresses the oncogenic behavior of c-Met via its signaling pathway, but different cancer subtypes have different responses to this inhibitor, and the possible mechanisms underlying the resistance of cancer to chemotherapies are yet to be elucidated. Disclosure The authors declare no conflicts of interest in this work. 1. Reference 1. Kelly H, Goldberg RM. Systemic therapy for metastatic colorectal cancer: current options, current evidence. J Clin Oncol. 2005; 23: 4553–4560. 2. Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat Rev Drug Discov. 2008; 7: 504–516. 3. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, et al. Identification of the hepatocyte growth factor as the c-met protooncogene product. Science .1991; 258: 802–804. 4. Ma PC, Jagadeeswaran R, Jagadeesh S, Tretiakova MS, Nallasura V, Fox EA, et al. Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res. 2005; 65:1479–88. 5. Raghav KP, Wang W, Liu S, Chavez-MacGregor M, Meng X, Hortobagyi GN, et al. cMET and phospho-cMET protein levels in breast cancers and survival outcomes. Clin Cancer Res. 2012, 15; 18(8):2269-77. 6. Cooper CS, Park M, Blair DG, Tainsky MA, Huebner K, Croce CM, et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature. 1984; 311:29-33. 7. Blumenschein GR Jr, Mills GB, Gonzalez-Angulo AM. Targeting the hepatocyte growth factor-cMET axis in cancer therapy. J Clin Oncol. 2012, 10; 30 (26):3287-96. 8. Takeuchi H, Bilchik A, Saha S, Turner R, Wiese D, Tanaka M, et al. c-Met expression level in primary colon cancer: a predictor of tumor invasion and lymph node metastasis. Clin Cancer Res. 2003; 9:1480-1488. 9. Koon EC, Ma PC, Salgia R, Welch WR, Christensen JG, Berkowitz RS, et al. Effect of a c-Met-specific, ATP-competitive small-molecule inhibitor SU11274 on human ovarian carcinoma cell growth, motility, and invasion. Int J Gynecol Cancer. 2008; 18:976-984. 10. Ma PC, Tretiakova MS, MacKinnon AC, Ramnath N, Johnson C, Dietrich S, et al. Expression and mutational analysis of MET in human solid cancers. Genes Chromosomes Cancer. 2008; 47:1025-1037. 11. Navab R, Liu J, Seiden-Long I, Shih W, Li M, Bandarchi B, et al. Co-overexpression of Met and hepatocyte growth factor promotes systemic metastasis in NCI-H460 non-small cell lung carcinoma cells. Neoplasia. 2009; 11:1292-1300. 12. Saigusa S, Toiyama Y, Tanaka K, Yokoe T, Fujikawa H, Matsushita K, et al. Inhibition of HGF/cMET expression prevents distant recurrence of rectal cancer after preoperative chemoradiotherapy. Int J Oncol. 2012; 40(2):583-91. 13. Yamashita N, Minamoto T, Ochiai A, Onda M, Esumi H. Frequent and characteristic K-ras cativation in aberrant crypt foci of colon. Is there preference among K-ras mutants for malignant progression? Cancer.1995; 75: 1527-33. 14. Fujita S, Sugano K. Expression of c-met proto-oncogene in primary colorectal cancer and liver metastases. Jpn J Clin Oncol. 1997; 27(6):378-383. 15. cagno SR, Li S, Colon M, Kreinest PA, Thompson EA, Fields AP, et al. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon. Int J Cancer. 2008; 122:2462-2470. 16. Lui VW, Wong EY, Ho K, Ng PK, Lau CP, Tsui SK, et al. Inhibition of c-Met downregulates TIGAR expression and reduces NADPH production leading to cell death. Oncogene. 2011, 3; 30(9):1127-34. 17. Xu C, Plattel W, van den Berg A, Rüther N, Huang X, Wang M, et al. Expression of the c-Met oncogene by tumor cells predicts a favorable outcome in classical Hodgkin's lymphoma. Haematologica. 2012, 97; 4: 572-8. 18. Sipos F, Galamb O. Epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions in the colon. World J Gastroenterol. 2012, 21; 18(7):601-8. 19. Wang ZX, Zhang B, Deng SM, Chen SJ. Early evaluation for treatment efficacy of 5-fluorouracil and hyperthermia on HCT-116 colon cancer cells by fluorine-18-fluorodeoxyglucose uptake.Chin Med J (Engl). 2012;125(4):657-61. 20. Sierra JR, Tsao MS. c-MET as a potential therapeutic target and biomarker in cancer.Ther Adv Med Oncol. 2011; 3(1 Suppl):S21-35. 21. Chen HT, Tsou HK, Chang CH, Tang CH. Hepatocyte growth factor increases osteopontin expression in human osteoblasts through PI3K, Akt, c-Src, and AP-1 signaling pathway. PLoS One. 2012;7(6):e38378. 22. Hung CM, Kuo DH, Chou CH, Su YC, Ho CT, Way TD.Osthole suppresses hepatocyte growth factor (HGF)-induced epithelial-mesenchymal transition via repression of the c-Met/Akt/mTOR pathway in human breast cancer cells.J Agric Food Chem. 2011 14;59(17):9683-90 .