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

Our previous work had demonstrated that myocardial cells culture medium（CMCM) can inhibit the growth of human nasopharyngeal carcinoma cells (CNE-2)in a time-dependent manner, and have no observable effect on normal cells ’proliferation. We speculated that cardiac muscles are capable of producing someactive factors with anti-cancer activity. Furthermore, we found that CMCMsuppressed the growth CNE-2cells involving both cell cycle (Go/GI and G2/M) arrestand apoptosis. In vivo, CMCM also showed significant inhibitory effect on the tumorgrowth of athymic nude mice and S180mice both with human nasopharyngealcarcinoma xenografts. But it did not affect the normal growth of the mice. Thisinhibitory effect is also related to apoptosis, and had no relationship with theinhibition of angiogenesis.Objective:Previous study showed that CMCM can inhibitthe growth of CNE-2cells through inducing cell apoptosis, However，the mechanismof CMCM-induced cell death is unclear. In the present study，we studied theantitumor effect of myocardial cells culture mediu（CMCM) on other tumor cells, andinvestigated the mechanism by which CMCM initiated apoptosis in order to providetheory support for the clinical practice of cardiac muscle low molecular tumorsuppressor.Methods: Collected myocardial cells culture medium（CMCM) throughprimary cultures of newborn rat cardiomyocytes; the abilities of CMCM(<10KD) toinhibit the proliferation of mice Lung cancer cells(Lewis), Cervical cancer cells(Hela) and Normal rat liver cells (BRL) were tested by MTT assay with cisplatin (DDP) aspositive control and DMEM as blank control; With the same method,the survivalrates of Hela cells treated with CMCM for24h,48h,72h were also determinedrespectively; human nasopharyngeal carcinoma cells (CNE-2) were cultured inRPMI-1640medium containing10%FCS and50%CMCM(<10KD) for48h.Cellstreated with RPMI-1640medium served as control. Then the activities ofCaspase-3,8,9were measured by ELISA reader.In addition， the differentiallyexpressed apoptosis genes in different groups were investigated by gene chip.Result:1. Compared to the blank group, CMCM suppressed the proliferation of Lewis cellsand Hela cells as well as DDP. The Lewis cell survival rates were64.69±15.86%and60.51±12.20%respectively，and42.97±6.45%，34.57±3.26%respectively for Helacells. And the difference between DDP group and CMCM group was not statisticallysignificant. However the normal cells such as BRL cells were not affected. What wasmore, CMCM inhibited the growth of Hela cells in a time-dependent manner. At24hand48h cell viability rates after been treated with CMCM were65.75±4.30%and42.97±6.45%respectively，which were lower than control group（P<0.05）. Themaximal inhibitory rate(23.95±4.56%) was at72h.There was also no statisticaldifference between DDP group and CMCM group at each time pointǜ2. After beentreated with CMCM (<10KD) for24h, activities of Caspase-3,8,9in CNE-2cells weremeasured by ELISA reader. We found that the activities of Caspase-3and Caspase-9increased compared with the control group. The OD values for Caspase-3andCaspase-9of CMCM group were0.271±0.016，0.215±0.018respectively.While the OD values of control group were only0.179±0.010，0.134±0.015respectively. Andthere was statistical difference (p <0.05) between the two group. However there wasno obvious difference about the activity of Caspase-8between CMCM group andcontrol group. The OD values were0.196±0.016，0.185±0.020respectively. Thedifference was not statistically significant (p>0.05).3. Compared with control group，there is3up—regulated and2down—regulated genes identified in CNE-2cellsafter been treated with CMCM (<10KD).Conclusion: CMCM showed significantinhibitory efforts on Lewis cells and Hela cells. However the normal cells such asBRL cells were not effected. CMCM-induced apoptosis0f CNE-2cells had positiverelationship with the activities of Caspase-3and Caspase-9.We speculated thatCMCM can mediated growth suppression, at least in part, employs regulation ofmitochondrial apoptotic pathways. The results also provided evidence usingmicroarray analysis for the first time that the expression of certain genes increased anddecreased to regulate the apoptosis pathway after been treated with CMCM，andwould assist in the further study of the mechanism of apoptosis meditated byCMCM.