Study On Inhibitory Effect Of Myocardial Cells Culture Medium (CMCM) And The Related Molecular Mechanism

Objective：To investigate the antitumor effects of cardiac muscle cells conditionedmedium（CMCM) and its possible molecular mechanism in vitro. Analyze theeffects of different temperatures and protease on CMCM to explore the physicaland chemical stability of CMCM in order to provide theoretical support for theclinical practice of cardiac muscle low molecular tumor suppressor. Methods:Collected fibroblast cells conditioned medium(FCM), smooth muscle cellsconditioned medium(SMCM), skeletal muscle cells conditioned medium(MMCM), myocardial cells culture medium(CMCM), the cell viability rates ofhuman nasopharyngeal carcinoma cell(CNE2) treated with cells culture mediumabove for48h were tested by CCK8assay. With the same method, we investigatedthe inhibitory effect of different diluted CMCM on CNE-2cells with fibroblastsconditioned medium as negative control.In addition, the effect of CMCM on theproliferation of mice lung cancer cells (Lewis), high-metastatic large cell lungcancer cell (L9981), cervical cancer cells (Hela), human nasopharyngealcarcinoma cell(CNE2) and normal rat liver cells (BRL), rat mesangial cells(MCs)were also tested. The morphological characteristics of CNE-2cells or BRL cellscultured alone or co-cultured with myocardial cells were observed by invertedphase contrast microscope. Then, the cell viability rates were also determined by CCK8after the CNE-2cells were treated with CMCM at different temperaturesand proteolytic enzymes (trypsin or pronase) for48h. Morphology of cellapoptosis was observed by TUNEL detection kits and DAPI stain. The mRNAexpression of eight apoptosis-related genes--Caspase3, BCL2L11, PERP, ENDOG,PPP3R1THAP1, CCAR1, AATF, was detected by Real-time PCR. The expressionof Caspase3、BCL2L11（Bim）、PERP、ENDOG、P53at the protein level weredetected by Western blot. Results:1. Compared to the cell viability rate of thecontrol group, the cell survival rates (X±S) were105.56±7.53%,116.00±7.00%,35.90±9.20%,35.00±3.42%respectively for fibroblasts conditioned medium,smooth muscle cells conditioned medium, skeletal muscle cells conditionedmedium and cardiac muscle cells conditioned medium.2. CMCM inhibited thegrowth of CNE-2cells in a time-and dose-dependent manner. At12h, higherconcentration of CMCM group showed higher inhibition rate on CNE-2cellproliferation compared with that of the group with lower concentration of1:2dilution group, but no significant difference (P=0.18) could be found between thetwo groups. At24h、36h and48h, cell viability rates after been treated withCMCM and1:2dilution group were decreased (P<0.05), between its internaldifferences were statistically significant (P<0.05), the inhibition with decreasingdrug concentration decreased, showing a dose-dependent manner. With theextension of treatment time, cell growth inhibition of CMCM group and1:2dilution group gradually increased, showing a time-dependent manner.3.Compared to the control group, CMCM suppressed the proliferation of Lewis cells，L9981，and Hela cells as well as CNE-2cells. The cell survival rates (X±S)were64.69±15.86%,45.90±5.50%,42.97±6.45%, and39.10±4.0%respectively.However, the normal cells such as BRL cells and MCS cells were not affected.4.Co-culturing of cardiomyocytes with CNE-2cells showed marked tendency toshrinkage in contrast to the same cells when incubated without cardiomyocytes.5.No decrease in inhibitory potency at different temperatures ranging from4to100°C.6. After different proteolytic enzymes (trypsin or pronase) treatment, thegrowth inhibition effect of myocardial cell conditioned medium (CMCM) has nostatistical change compared to the control group.7. CMCM caused apoptosissignificantly in a time-dependent manner. The cell apoptosis rates after beentreated with CMCM in12h,24h,48h were10.17±3.04%、19.60±5.84%and33.80±4.23%respectively.8. CMCM up-regulated the mRNA expression ofCaspase3, BCL2L11, PERP, ENDOG, PPP3R1, THAP1and down-regulate themRNA expression of AATF at the same time.9. CMCM could up-regulate theexpression of Caspase3、BCL2L11（Bim）、PERP、ENDOG、P53at the proteinlevel. Conclusion: CMCM may become a safe and effective drug for cancertherapy. And the active component was found to be stable under differenttemperatures and proteolytic enzymes (trypsin or pronase). Addtionally, Wespeculated that CMCM may up-regulate the expression of bim、Caspase3、P53、PERP and ENDOG, which induced apoptosis.