| Recently MSC was applied for clinical use as its vital role in immune regulation, tissue repairing. At the same time, the effect of MSC on cancer is becoming a hot spot at the same time. While the effect of MSC on colon cancer and its mechanism remains unknown.Objectives:(1) Investigating the impact of MSC-CM on colon cancer cell lines.(2) Further study in the impact on the important pathways of colon cancer and its specific mechanisms.(3) Clearing the effect of MSC-CM on the tumorgensis of colon cancer cell.Methods:(1) α-MEM severs as control, stimulating colon cancer cell lines with 24 hours MSC-CM for 24 h, then measuring the change of proliferation of colon cancer cell lines in 6 days, detecting the percentage of cell in proliferation stage, S stage with Brd U, Ki67 fluorescent staining assay. The percentage of apoptosis of colon cancer cell were determined by the change of the membrane potential of mitochondria and the status of cell cycle in colon cancer cell were detected by PI staining. At the meantime, using Transwell assay to measuring the change of cell migration ability, mixing the MSC-CM with colon cancer cell and then seed in the semi-solid medium to measuring the change of ability of colon cancer cell lines.(2) After stimulation from MSC-CM for 24 h, using western blot to detect the change of cell cycle relative protein P53, P16, P21, employing QPCR to measure the change of RNA level, then using western blot, QPCR to detect the expression of apoptosis relative protein Bax, Bcl-2, stemness relative maker c-myc, oct-4, sox-2 in protein and RNA level. Using ELISA assasy to measure the expression of IL-6, IL-8 in MSC-CM and MSC-CM after concentration, at the same time, employing QPCR and western blot to confirm the change of AMPK/m TOR and NF-κB pathway, EMSA to confirm the change of binding activity of NF-κB pathway.(3) MSC-CM was concentrated 50 times and intraperitoneally injected in Balb/c mouse with HCT-116 cell, then monitoring the change of mouse’s weight, quantity of tumor in mouse’s colon and immunohistochemical of HE, Ki67 and E-Cadherin.Results:(1) Compared with control medium, the proliferation of HCT-116 and LOVO was significantly improved after the stimulation of MSC-CM, the percentage of proliferation stage and S stage were also improved. The percentage of migration of colon cancer cell after 12 h, quantity and size of clone of colon cancer were also improved after 14 days, the rate of apoptosis of colon cancer cell declined, in the cell cycle, percentage of G1 declined and S, G2 stage increased.(2) Compared with control medium, the expression of cell cycle, apoptosis relatively protein P53, Bax were inhibited both in protein and RNA level, the inhibitor of apoptosis protein Bcl-2 were induced, the expression of P21, P16 were inhibited at the same time. Stemness relatively genes c-myc, oct-4, sox-2 were induced in RNA level, c-myc, sox-2 were also induced in protein level. At the meantime, using ELISA to confirm the expression of IL-6, IL-8 in MSC-CM were improved from 240±52.91(pg/ml), 220±36.06(pg/ml) to 1083.33±144.34(pg/ml), 733.33±104.08(pg/ml). After stimulation, IL-6, IL-8 expression were promoted to 1406.67±143.64(pg/ml), 830±65.57(pg/ml), concentrated 50 times IL-6, IL-8 were increased to 41700±1808.31(pg/ml), 29600±1967.23(pg/ml). Detecting the change of AMPK/m TOR and NF-κB pathway, we find that the phosphorylation of AMPK was inhibited, the phosphorylation of m TOR was induced. Subunit of NF-κB pathway-P65 were induced in phosphorylation status, phosphorylation of inhibitor of NF-κB pathway- IKBα was decreased and promotion of degradation of IKBα was detected. At the same time, the ability of transduction of NF-κB pathway were also improved.(3) Further intraperitoneally injected with concentrated MSC-CM and HCT-116, we concluded that the decline of mouse’s weight and increase of tumors in mouse’s colon, the positive staining of HE, Ki67 were also improved, the positive staining of E-Cadherin decreased.Conclusions: With the stimulation of MSC-CM, proliferation, migration and the ability to form clone of colon cancer cell line were increased, apoptosis of colon cancer cells were inhibited, at the meantime, percentage of G1 stage of colon cancer cell lines was decreased, S and G2 stage were increased. Cell cycle related protein were promoted, and inhibitor of apoptosis were inhibited, stemness of colon cancer were increased. The mechanism of effect of MSC-CM were related to the high expression of IL-6, IL-8 and its subsequent stimulation of AMPK/m TOR、NF-κB pathways. The tumorgensis of colon cancer cell in Balb/c mouse were also improved by MSC-CM. |
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