| BackgroundAlpha-momorcharin(α-MMC), extracted from the seeds of momordica charantia, is a ribosome inactivating proteins(RIPs). α-MMC can specifically hydrolyze N–C glycosidic bond between the ribose and adenine base at position A4324 of the 28 S r RNA in eukaryotic ribosomes, and thus results in the release of an adenine base and the inhibition of binding of the elongation factor EF-2 to the ribosome, thereby inhibiting protein synthesis. α-MMC exerts anti-tumor, antiviral, anti-fungal, anti-fertility, and immunoregulation activities. It has been reported that α-MMC possesses obvious inhibitory effect on the choriocarcinoma cells, melanoma cells, human nasopharyngeal carcinoma cells, human breast cancer cells, liver cancer cells, and colon cancer cells, few in vivo pharmacodynamic and mechanism studies of anti-tumor have been performed, and little is known about the effect of α-MMC toxicity in vivo. Although α-MMC has a potent anti-tumor effect, it is after all a plant toxin that is heterologous and has a high toxicity in vivo. The toxicity of α-MMC can affect its clinical application. Previous pharmacodynamic studies on α-MMC and other RIPs were mostly carried out in vitro. Few in vivo pharmacodynamic studies have been performed, and thus little is known about the effect of α-MMC toxicity on the therapeutic efficacy. Thus, it is worth to study how the toxicity of α-MMC can affect the therapeutic efficacy of α-MMC. If α-MMC has no therapeutic window, that is, the minimum effective dose is equal to the minimum toxic dose, then it will not be useful for clinical use. ObjectiveThis research was aimed to grasp anti-tumor effect of α-MMC and explore the preliminary antitumor mechanism, find the lowest effective dose and the minimum toxic dose of α-MMC(therapeutic window) and to better evaluate the safety of α-MMC, we performed in vivo and in vitro experiments of α-MMC. MethodsMTT assay is used to know the cytotoxicity of α-MMC on human breast cancer cells(MDA-MB-231, MDA-MB-453 and MCF-7) in vitro. Human breast cancer cells were treated with α-MMC, its morphological changes were examined under a light microscope or fluorescence microscopy, the extent of apoptosis and cell cycle of was detected by flow cytometer, and the caspase3 activity was determined. We investigated the anti-tumor effect of α-MMC in Balb/c nude mice xenografted with human breast cancer MDA-MB-231 and MCF-7 cells, and obtained the minimal effective concentration of α-MMC(the lower limit of therapeutic window) by testing different concentrations of α-MMC(0.53 mg/kg、0.80 mg/kg、1.20 mg/kg). We also implemented TUNEL staining and HE staining in xenografted tumor, morphological changes and the condition of apoptosis of tumor cells were observed. Balb/C mice that no tumor were intraperitoneally injected with 1.80 mg/kg, 1.20 mg/kg, 0.80 mg/kg, 0.53 mg/kg, and 0.35 mg/kg α-MMC three times a week for 6 weeks, and observed its toxic manifestation, obtained the minimal toxic dose of α-MMC(the higher limit of therapeutic window). Results(1) Alpha-MMC significantly inhibited cell growth in MDA-MB-231, MDA-MB-453, and MCF-7 breast cancer cells. The inhibition was in a dose-dependent manner and in a time-dependent manner. The IC50 of α-MMC was 15.07 μg/m L for MDA-MB-231 cells, 33.66 μg/m L for MCF-7 cells, and 42.94 μg/m L for MDA-MB-453 cells.(2) Alpha-MMC can induce apoptosis of human breast cancer cells. Cells treated with α-MMC exhibited features of apoptotic changes, increased cytoplasmic density, increased nuclear chromatin condensation, and fragmentation of the nucleolus and nucleus.(3) Flow cytometer test exhibited that α-MMC can induce apoptosis of human breast cancer cells in a dose-dependent manner. Cells treated with 210 μg/m L α-MMC, the percentages of cells with early apoptosis were 22.7% for MDA-MB-231 cells, 23.2% for MCF-7 cells, and 16.0% for MDA-MB-453 cells, and the percentages of cells with late apoptosis were 19.2% for MDA-MB-231 cells, 38.0% for MCF-7 cells, and 9.4% for MDA-MB-453 cells.(4) Alpha-MMC increased caspase3 activities in those three breast cancer cell lines in a dose-dependent manner.(5) After in vitro treatment with α-MMC, the cell cycle was arrested at the G0/G1 phases in MDAMB-231 cells and at the G2/M phases in MDA-MB-453 and MCF-7 cells.(6) The minimal toxic dose of α-MMC in Balb/C mice was 1.20 mg/kg.(7) Alpha-MMC of 1.20 mg/kg and 0.80 mg/kg significantly inhibited human breast cancer xenografted tumors growth and played the therapeutic effect, and the minimal effective concentration of α-MMC was found to be 0.80 mg/kg.(8) TUNEL staining showed obvious apoptotic cells in tumors in mice transplanted with MDA-MB-231 and MCF-7 by α-MMC. The apoptosis level was in a dose-dependent manner. ConclusionAlpha-MMC exhibited anti-tumor effects in human breast cancer in vivo and in vitro. It inhibited breast cancer cells through the inhibition of tumor growth and induction of cell apoptosis. The therapeutic window of α-MMC for the inhibition of breast cancer growth in mice was 0.80 mg/kg~1.20 mg/kg, which may be considered as a quite narrow range for drug safety in vivo. |
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