| Objective:Malignant neoplasm is one of the most serious diseases which damage human's health. It is of important significance to develop novel natural anti-cancer medicines with low toxicity and high efficacy to improve chemotherapy effect clinically. Elemene, extracted from the traditional Chinese medicinal herb Curcuma wenyujin, is a mixture ofβ-,γ-andδ-elemene withβ-elemene as the main component and the other two as the lesser amounts. It is a kind of anti-cancer natural medicine with low toxicity which has been developed by China independently. But mechanisms underlying the effectiveness of elemene on tumor growth and metastasis are not clear. Phospholipid is the dominant component of cell membrane and plays an important role in cell signaling and material transmitting. Therefore, lipid change in cell membrane may be one of mechanisms of anti-cancer medicine. Our study aimed at initially exploring the mechanism of anti-tumor effectiveness of elemene on cancer cells with lymphatic metastasis. Two murine hepatocarcinoma cell lines with high and low metastatic potential(HCa-P/L6 and HCa-P) were used to investigate and compare effects of elemene on some biological characteristics of these two cell lines. Live cell extracted and gas chromatography and mass spectrometry(GC/MS) were used to determine effective components in HCa-P/L6 and HCa-P treated with elemene. Differential centrifugation gradient centrifugation and high-performance liquid chromatography(HPLC) were applied to detect phospholipid in cell membrane. This study is not only of theoretical significance, but also of potentially applicable application. At the same time, this study can provide reliable theoretical proof in exploring natural anti-tumor medicines with cheaper price, lower toxicity and higher efficacy.Methods:The cytotoxicity of elemene on two murine ascites hepatocarcinoma cell lines(HCa-P/L6 and HCa-P) were studied by the use of MTT assay after these cells had been pretreated by elemene at concentration of 20-100μg/mL for 48h. Then, the maximum uncytotoxicity dose and the half maximal inhibitory concentration (IC50) were calculated. Pretreated by elemene at the maximum uncytotoxicity dose 10μg/mL, the effect of elemene against cell proliferation of HCa-P/L6 and HCa-P was observed by making cell growth curve and cell population doubling time; the effect of elemene on cell cycle of HCa-P/L6 and HCa-P were studied by flow cytometry(FCM). Live cell extraction and GC/MS were used to detect bioactive components in cells. Differential centrifugation gradient centrifugation and HPLC were used to determine phospholipid variation in cell membranes treated with elemene.Results:1. After HCa-P/L6 and HCa-P cells had been pretreated by elemene at concentration of 20-100μg/mL for 48 hours, elemene showed evidently growth inhibition on both cell lines in dose-dependent manner(Chi-square test P<0.01). Elemene had more inhibition on HCa-P/L6 with IC50 37.3μg/mL than on HCa-P with IC50 70μg/mL.2. The livability of HCa-P/L6 and HCa-P are 90% and 98.6%, respectively, which are higher than 90%, and thus 10μg/mL is the maximum uncytotoxicity dose.3. Cell growth curves and cell population doubling times are not different between HCa-P/L6 and HCa-P without elemene treatment. Pretreated by elemene at 10μg/mL for 7 days, the proliferation of HCa-P/L6 and HCa-P were inhibited in time-dependent manner(t test P<0.05) showing by cell growth curves and prolonging population doubling times (t test P＜0.05).4. Cell cycle progressions of HCa-P/L6 and HCa-P were different without elemene treatment. Pretreated by 10μg/mL elemene for 48h in HCa-P/L6 and HCa-P, cell cycle was redistributed with HCa-P/L6 and HCa-P being blocked in S phase and the blocking effectiveness in HCa-P/L6 was stronger than that in HCa-P.5. Elemene could induce cells apoptosis. The induced apoptosis potential in HCa-P/L6 was stronger than that in HCa-P.6. Pretreated with elemene at 10μg/mL with 48 h, eight bioactive components were detected includingβ-,γ-andδ-elemene by live cell extraction and GC/MS.β-elemene was validated the main bioactive component whose content level in HCa-P/L6 is lower than that in HCa-P.7. Pretreated with elemene at concentration of 10μg/mL for 48 h, the quatity of phospholipid in cell membrane was determined by use of differential centrifugation, gradient centrifugation and HPLC. Contents of phosphatidylinositol(PI) and phosphatidylethanolamine(PE) were found decreased in both HCa-P/L6 and HCa-P cells treated with elemene, and the decreased content level in HCa-P/L6 was higher than that in HCa-P(t test P<0.05).Conclusions:1. Elemene could inhibit the proliferation of HCa-P/L6 and HCa-P in dose-dependent manner and the IC50 of HCa-P/L6 was lower than that of HCa-P.10μg/mL Elemene for 48 h could prolonged population doubling times of HCa-P/L6 and HCa-P, redistributed cell cycle progressions of HCa-P/L6 and HCa-P with being blocked in S phase and the blocking effectiveness in HCa-P/L6 stronger than that in HCa-P. The induced apoptosis potential of elemene in HCa-P/L6 was stronger than that in HCa-P. HCa-P/L6 was validated more sensitive to elemene than HCa-P was. The cell cycle progressions redistribution and apoptosis induced by elemene might be potential mechanisms underlying the effectiveness of elemene on tumor growth and metastasis.2. The bioactive components of elemene interacting with HCa-P/L6 and HCa-P were same, which includedβ-,γ-,δ-elemene andβ-elememe was the main component. But the relative amount level ofβ-elememe in HCa-P/L6 was lower than that in HCa-P. It may be related with the inhibition effectiveness of elemene on tumor growth and metastasis by the amount variation of cell membrane phosphatides.3. Elemene could lead to PE and PI decrease, and the decreased amount level in HCa-P/L6 was higher than that in HCa-P. The content variation of cell membrane phosphatides induced by elemene might be associated with the mechanism underlying the effectiveness of elemene on tumor growth and metastasis.
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