| Objective: The inhibition effect of periplocin extracted from cortex periplocae (CPP) on proliferation of human colon carcinoma cell line SW480 in vitro and in vivo was studied, and the regulation effect of CPP on Wnt/β-catenin signaling pathway and its downstream target genes was investigated. The goal is to evaluate the anti-tumor mechanisms of CPP, and to provide some theoretical evidence for developing CPP as an anti-tumor drug.Methods: 1 Inhibition effect of CPP on cell growth of SW480 was measured by MTT assay in treated or untreated groups (0.125, 0.25, 0.5, 1.0, 2.0μg/ml CPP and control) for different treatment times (24h, 48h and 72h).2 The cell morphology and super-microstructural changes of apoptosis were observed under optical microscope and transmission electron microscope (TEM).3 Flow cytometry (FCM) was used to measure cell apoptosis rate and cell cycles of SW480 cells after treatment with CPP (0.5μg/ml) for different times (0h, 6h, 12h and 24h).4 Subcellular localization ofβ-catenin in SW480 cells after treated with CPP (0.5μg/ml) for 12h was observed by immunofluorescence and laser scanning microscope (LSM).5 Protein expression ofβ-catenin in total cell lysates, in cytosolic extracts, and in nuclear extracts of SW480 cells treated with CPP (0, 0.125, 0.5, 2.0μg/ml) for 24h were analyzed by Western blot.6 Binding activity of the TCF complex in nucleus to its specific DNA binding sites was measured by electrophoretic mobility shift assay (EMSA) after SW480 cells were treated with CPP (0, 0.125, 0.5, 2.0μg/ml) for 24h.7 Expression ofβ-catenin, survivin, c-myc and cyclin D1 mRNA in SW480 cells after treated with CPP (0, 0.125, 0.5, 2.0μg/ml) for 24h were detected by semi-quantitative RT-PCR.8 Construct human colon cancer SW480 cell xenografts in nude mice, 12 nude mice were randomly divided into CPP-treated group and control group. CPP (30 mg/kg) were injected around the tumor in CPP-treated group mice once a day, and control group mice were injected with normal saline (NS). The transplantation tumor volume was measured every 3 days. After given drug for 12 days, the mice were killed, and the tumor xenografts were removed and measured.9 Morphology of SW480 cells in transplanted tumors were observed under optical microscope after HE staining.10 Expression ofβ-catenin, survivin and c-myc were detected by immunohistochemistry.Results: 1 CPP (0.125~2.0μg/ml) could significantly inhibite the proliferation of SW480 cells in vitro (P<0.01), the growth inhibition was in a time- and dose-dependent manner. The greatest inhibition rate was 91.71% observed in the treatment group of SW480 cells with the concentration of 2.0μg/ml CPP for 72 hours.2 After treatment with CPP, SW480 cells showed some typical morphologic features and super-microstructural changes of apoptosis.3 The results of FCM showed that CPP could arrest cell cycle of SW480 cells at G0/G1 phase (the cell rate increased from 17.54% to 37.87%) and induce cell apoptosis. The apoptosis rate of cells treated with 0.5μg/ml CPP for 24h was as high as 36.52%.4 Immunofluorescence showed that the protein ofβ-catenin in SW480 cells were mainly in nucleus and cytoplasm, green fluorescence densely distributed in the nucleus and cytoplasm; After treated with CPP, the expression level ofβ-catenin in cytoplasm was significantly higher than it in nucleus.5 The results by Western blot reveals that after treated with CPP, the protein expression level ofβ-catenin in total cell lysates, cytosolic extracts, and in nuclear extracts were declined significantly (P<0.05).6 The results by EMSA reveals that binding activity of the TCF complex in nucleus to its specific DNA binding sites was suppressed after SW480 cells were treated with different concentrations of CPP for 24h.7 The results analized by semi-quantitative RT-PCR showed thatβ-catenin mRNA didn't have significant changes after SW480 cells were treated with different concentrations of CPP for 24h, there was no statistically significant differences compared to the control group (P>0.05). Butβ-catenin/TCF downstream target genes survivin, c-myc and cyclin D1 were down regulated (P<0.01).8 SW480 cell xenograft tumor model was successfully established. The average tumor volume of treated group was 0.515±0.184 cm3 and average weight was 1.367±0.398 g, the tumor inhibition rate was 61.41% (compared to the control group, P<0.01).9 There were inflamation cells and noticeable necrosis changes in transplanted tumor tissue of CPP treated mice by HE staining observed under optical microscope.10 The results of immunohistochemistry suggest that expression level ofβ-catenin, survivin and c-myc in transplantation tumor were significantly lower in CPP treated group than in control group (P<0.05).Conclusion: 1 CPP could significantly inhibit the proliferation of human colon carcinoma cell line SW480 in vitro and in a dose- and time-dependent manner; its inhibition effect on SW480 cells may be related to its inducing cell apoptosis and cell cycle arrest.2 CPP could inhibit Wnt/β-catenin signaling pathway. Its mechamisms may be through influencing subcellular localization ofβ-catenin, reducing protein expression level of β-catenin in total cell lysates, in cytosolic extracts, and in nuclear extracts, suppressing binding activity of the TCF complex in nucleus to its specific DNA binding sites, to inhibitβ-catenin/TCF transcription activity.3 CPP could downregulateβ-catenin/TCF downstream target genes survivin, c-myc and cyclin D1 mRNA expression, butβ-catenin mRNA didn't have significant change. This may illuminate that the regulation of CPP onβ-catenin was not on mRNA level, but most likely on protein degradation level.4 CPP could inhibit the xenograft growth of SW480 cells in vivo. There were inflamation cells and noticeable necrosis changes in transplanted tumor tissue of CPP treated group.5 Expression level ofβ-catenin, survivin and c-myc in transplantation tumor were significantly lower in CPP treated group than in control group.6 CPP has significant inhibition effect on colon carcinoma cell line SW480 in vitro and in vivo, the mechanisms may be associated with inhibiting Wnt/β-catenin signaling pathway.
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