| Cisplatin(Cis-dischlorodiammine-platinm DDP),a kind of platinum metal complexes, has lethal effect on most of tumor cells, which was the common clinical chemical drugs, but had certain toxic effect when the concentration was too high. SIP was a kind of polysaccharide separated and purified from s.esculenta(Sepia esculenta Hoyle)Monei with high activity and novel structure. Pharmacological activity study showed that SIP had high immune enhancement, anti-oxidation and anti-tumor activity, which can exert its anti-tumor effect through regulating expression of apoptosis related protein to induce the apoptosis of tumor cells. In order to improve the anti-tumor effect without increasing toxicity and reduce the tumor drug resistance at the same time, multiple anti-cancer drugs were combined to play a synergistic effect between drugs. Carcinoma of the breast is the highest incidence among malignant tumor disease in the world. About 1.2 million women suffer from breast cancer and0.5million died yearly, in addition, the number is still rising. The significant cause of breast cancer to difficult to cure completely was cancer metastasis. Many patients did not died of primary cancer, but metastatic cancer.MDA-MB-231 cell was used as DDP chemotherapy model in vitro and SIP was as protective agent and sensitizer for DDP to observe and analyze the effect of the two drugs for single use and combined use on migration, apoptosis and autophagy of MDA-MB-231 cell.The median-effect principle was used to select out the appropriate drug dosage and contrast the relationship of drug dosage and effect. The results showed that, DDP had a certain inhibitory effect on MDA-MB-231 cells when applied alone, in which the median-effect concentration is 4.9 μg/mL and SIP had the same effect as DDP, in which the median-effect concentration is 1659.6 μg/mL. However, the median-effect concentration on MDA-MB-231 cells was much less than the total median-effect concentration of two drugs for single use. That is to say, the two drugs with low dosage had a higher inhibitory effect only when combined,which could reduce the drug resistance and the toxic damage on body caused by DDP for single use.Secondly, the influence of drug on cell migration was apparently observed according to the single cell cloning experiment and cell scar test, whose molecular mechanism was analyzed through western blot. The single cell cloning experiment and cell scar test showed that, whether DDP and SIP single use or combination had a certain inhibitory effect on the migration of cancer cells. The inhibitory effect of SIP was less than that of DDP, but asprotective agent and sensitizer for DDP, the inhibitory effect of migration reached to the best and expressed highly significant difference(P<0.01)compared with two drugs for single use when combined. Western blot experiment showed that, the group of SIP+DDP had better inhibitory effect on MMP-2 and MMP-9 than the two drugs for single use, which significantly reduced the expression of the two matrix metalloproteinases. Generally, the two drugs promote each other to improve the inhibitory effect on tumor cell metastasis in the course of drug action.In addition, the effect of drugs on apoptosis of MDA-MB-231 cells was observed and tested on the basis of Hochest33242/PI double-stained and the apoptosis-related protein expression was analyzed by western blot results. From flow cytometry results, both SIP and DDP could induce the apoptosis of MDA-MB-231 cells, but the level of DDP is less than that of SIP. When combined with two drugs, the pro-apoptotic level is significantly improved compared with two drugs for single use. Western blot results showed that, the apoptosis was mediated by SIP and DDP through regulating the ratio of Bcl-2/Bax and caspase protein expression.Finally, the effect on autophagy of MDA-MB-231 cells was measured through MDC staining. The autophagy-related proteins expression and the key regulatory factors of PI3K-Akt and p38 MAPK signaling pathway were analyzed according to western blot results.The interaction and molecular mechanism of apoptosis and autophagy in MDA-MB-231 cells in the action of drugs were initially discussed. MDC staining results showed that, DDP could induce autophagy in MDA-MB-231 cells, but the autophagy rate of SIP+DDP group was significantly lower than DDP group(P<0.05). Therefore, the autophagy induced by DDP in cancer cells could be alleviated by SIP. Western blot results showed that, compared with DDP group, among 3-MA+DDP group and SIP+DDP group, the transition level of LC3 I to LC3 II was reduced and the expression level of LC3 II protein was significantly decreased(P<0.01),and that expression level of Beclin-1 protein did(P<0.01). When combined apoptosis-related protein expression and p-Akt and p-p38 expression which is the key regulatory factors of PI3K-Akt and p38 MAPK signaling pathway, DDP could induce apoptosis and autophagy in tumor cells through PI3K-Akt signaling pathway for promoting autophagy and antagonistic apoptosis and p38 MAPK signaling pathway for promoting both autophagy and apoptosis. SIP could destroy tumor cells through inhibiting PI3K-Akt and p38 MAPK related signaling pathway to suppress autophagy and promote apoptosis for further development. |
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