Research Of MSN Induce Cancer Stem Cell Apoptosis

MSN is a member of TGF-βsuperfamily and plays a negative role in the control of skeletal muscle growth and the inactivation of MSN gene in mice leads to marked hypertrophy and hyperplasia of the skeletal muscle.Till now,most of studies regarding to MSN focus exclusively on muscle control.As a member of superfamily,MSN have the same recptor and signal.Therefor,there is no report about MSN and cancer.P6C cells are from patients of colorectal cancer,which have the character of stem cell .Despite the rapid development of medicine now, but still not very effective cancer treatments. The existence of cancer stem cells and metastasis of tumor key, so the treatment of cancer stem cells are the crux of the treatment of cancer, our experiments showed that MSN can lead to apoptosis of colon cancer stem cells. This anti-tumor study provides a new strategy for the treatment of cancer.AIMThe stem cell is a new appoint in these years,the stem cell also give some new strategies for the treatment of cancer.We use Flow cytometry,conforcal and western eatl,to detect MSN induce P6C cells apoptosis.METHODS1.P6C cells cultured in DMEM according to conventional methods in the culture medium, culture medium and the addition of 10% fetal bovine serum 1% penicillin and streptomycin each, placed in 37℃, 5% CO2 incubator (SANYO).2.Annexin V and PI detection of cell apoptosis. Flow cytometry, excitation wavelength used 488 nm, marked on the Annexin V FITC green fluorescence by excited-fat (530±30 nm), which binds to DNA in the PI of the emission wavelength is 630±22 nm, red fluorescence. This method can distinguish between living cells (Annexin V and PI double negative), early apoptotic cells (Annexin V positive, PI negative), late apoptotic and necrotic cells (Annexin V and PI double positive).3.westernblot. Cells were harvested, PBS washed 2 times by adding 106 cells per 100μL lysis buffer, mixed gently on ice cracking 30 - 45 minutes, centrifuge, Bradford protein, polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, and then transferred to nitrocellulose membrane protein; the first antibody for night at 4°C, PBST washing three times, and then with horseradish peroxidase (HRP) labeled secondary antibodies were incubated 2 hours at room temperature oscillation, and finally PBST washed three times, The ECL system (Pierce) color.4. Immunofluorescence. The cells were seeded into 6-well plate with small slice, according to the requirements of experiments were divided into experimental and control groups, with 3.7% paraformaldehyde fixed, and 37°C ,15-20 minutes, 0.2% Triton X-100 for 10 minutes for slotting, PBS diluted primary antibody containing 1%-5% FBS, first antibody incubated for 1 hour at room temperature. PBS washed cells, add the amount of markup or TRITC/cy3b FITC labeled secondary antibody, cells were incubated at room temperature for 1 hour in the cassette, PBS washed the cells, sealed tablet and observed the photographs.5. Mitochondria extraction. Hypotonic treated cells, Dounce homogenized cells moderate micro homogenizer .Homogenate was collected, differential centrifugation to collect mitochondria. Bradford method for quantitative protein concentration of each component, the SDS-PAGE and Western Blotting detection.6. Cell transfection. Plate until the cells grew to 70%-80% adherent, start transfection, choose different transfection methods according to cells.7.RNAI primer synthesis and determination.8. Quantitative cell proliferation was detected. Plant 1000-5000 cells in 96 poles plate,in 6 hours,12 hours,24 hours,36 hours,deal with MTT and DMSO and detected cell proliferation.RESULTS1. The proportion of apoptotic cells with the MSN of dose and time increases by detected flow cytomety. Caspase fluorescent substrate binding results of flow cytometry to detect Caspase activation after MSN, western results showed that the Caspase3, Caspase9 precursors decreased significantly.2. The assay of coloneffection show that MSN inhibit cell colony,assay of MTT show that MSN also can inhibit cell proliferation.3. We concentrated mitochondria,the result of western show that he cytochrome C releasing from mitochondria to the cytosol.By immunofluorescence we detected the red mitochondria and green bax can completely copy together in control,but not in MSN dealing cells.4. we can detected apoptosis-related proteins bax,bcl-xl,vdac upregulate by western, IP detected bax conformational change occurs ,and which translocate to mitochondria by confocal microscope5. We also detected the bcl-xl knockdown of P6C are less apoptosis than control.CONCLUSIONS1. MSN can cause cancer stem cells.2. MSN inhibit cell colony formation and cell proliferation significantly .3. MSN can cause cytochrome C releasing.4. Apoptotic-associate protein bax, bcl-xl ,VDAC increased.5. Bax conformational change occurs, and conformational bax translocate to mitochondria.6. bcl-xl knockdown can decrease the apoptosis and the bax expression.