| Malignant tumors in the peritoneal cavity, especially the poorly differentiated tumors originated from the gastrointestinal tract and the ovary, are of high recurrence rate after operation due to the exfoliation of tumor cells or peritoneal tumor dissemination through the lymphatic or blood vessels, resulting in decreased postoperative survival rate of patients. Intraperitoneal hyperthermic chemoperfusion (IHCP), an effective treatment of peritoneal tumor dissemination, can effectively kill the disseminating tumor cells, resulting in improved survival rate of patients. However, similar to chemo- and radiotherapies, IHCP can also produce brutal adverse effects on actively differentiating tissues or cells such as intestinal epithelial cells (IECs), lymphocytes, and even hematopoietic tissues, and hence the antitumor effect can be suppressed. It was reported that the adverse effects such as nausea, vomiting, and diarrhea were resulted from the IHCP-induced damages to IECs due to p53-mediated apoptosis of IECs. Therefore, how to decrease IHCP-induced apoptosis of IECs is the important measure in attenuating the adverse effects during the treatment of tumors. It was also reported that p-fifty three inhibitor-alpha (PFT-a), a p53 specific inhibitor, could survive the mice from the lethal dose of radiotherapy through the inhibition of p53, but did not induce additional tumors generation. It is important that PFT-a may be the useful drug for reducing the side effects due to cancer therapy through the suppression of p53-mediated apoptosis. Can PFT-a protect thermochemotherapy-induced apoptosis of IECs? What may be the mechanisms in this protective effect? Those questions are of great value in the investigation of the new therapeutic strategies in attenuating the side effects due to cancer therapy through the inhibition of p53.Objectives1. To investigate the effects of PFT-a on the proliferation of large intestinal epithelial cells and the efficacy of thermochemotherapy and the mechanisms in thermochemotherapy-induced apoptosis of IECs, cell cycles, and regulation of cell cycles.2. To investigate the effects of PFT-a on the subcellular localization and protein expression of p53 and the changes in the target gene expressions of p53, including the mRNA and protein expressions of Bax, MDM2, and p21WAF1/CIPI after thermochemotherapy in large intestinal epithelial cells, and to elucidate the mechanisms in this protective effect.3. To investigate the effects of PFT-a on the IHCP-induced apoptosis of small intestinal epithelial cells and to determine the mRNA and protein expressions of Bax, MDM2, and p21WAF1/clpl in small intestinal epithelial cells of BALB/c mice with large intestinal cancer cell line DLD1 (mutant p53) peritoneal carcinomatosis as well as the protein expression of cytochrome C in mitochondria and cytoplasm of small intestinal epithelial cells for investigating the mechanisms in the protective effect of PFT-a on IHCP-induced apoptosis of the small intestinal epithelial cells.MethodsLarge intestinal epithelial cells from normal human colon mucosa were primary cultured in vitro by using collagenase type I and thermolysin. The effects of PFT-a at different concentrations on proliferation of the large intestinal epithelial cells and the anti-tumor efficacy of thermochemotherapy were observed by methabenzthiazuron (MTT) assay. After the large intestinal epithelial cells were treated with thermochemotherapy in the presence or absence of PFT-a, the cell cycles were detected by flow cytometry (FCM) with propidium iodide (PI) labeling method and the apoptotic rate by FCM with Annexin V-FITC and PI double labeling method. Western blot and Reverse transcriptase polymerase chain reaction (RT-PCR) were performed to analysis the expression of protein and mRNA of CyclinBl of large intestinal epithelial cells. Simultaneously, the protein level of Cdc2 (Tyrl5) was observed by Western blot. The changes in subcellular localization and protein of p53 after treatment of the large intestinal epithelial cells with thermochemotherapy in the presence or absence of PFT-a were observed by immunocytochemistry and Western blot, respectively. Western blot and RT-PCR were also employed to measure the protein and mRNA levels of Bax, MDM2, and p21WAF1/clpl in large intestinal epithelial cells under thermochemotherapy with or without the serial dosages of PFT-a. Using BALB/c mice model of DLD1 peritoneal carcinomatosis in the present study, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) was applied to observe the apoptosis of smallintestinal epithelial cells of BALB/c mice. We detected the mRNA and protein expressions of Bax, Bcl-2, MDM2, and P21WAF1/CIPI in small intestinal epithelial cells of BALB/c mice by RT-PCR and Western blot, respectively. Meanwhile, cytochrome C in mitochondria and cytoplasm of small intestinal epithelial cells was measured by using Western blot. Results1. PFT-a at the concentrations lower than 50 uM had no effect on the proliferation of large intestinal epithelial cells, but PFT-a at the concentration of 60 uM could inhibit the growth of large intestinal epithelial cells and the inhibitory effect was significant at 70 uM (PO.01) and in a concentration-dependent manner. PFT-a, ranging from 5 uM to 40 uM, had no effect on the growth of DLD1 cells, but could inhibit the growth of DLD1 cells when the concentrations were 50 uM (PO.05) and 60 uM (/><0.01), respectively. PFT-a had cytotoxic effect at the concentration higher than 50 uM. After treatment of DLD1 cells with PFT-a lower than 40 uM and thermochemotherapy, PFT-a had no effect on the antitumor potency of hyperthermic chemotherapy. PFT-a at the concentrations of 50 and 60 uM could enhance the antitumor potency of hyperthermic chemotherapy in a concentration-dependent manner (7><0.01).2. After large intestinal epithelial cells were exposed to thermochemotherapy, the apoptotic rate was significant increased as compared with that in the control group (/><0.01). After treatment of large intestinal epithelial cells with PFT-a and thermochemotherapy, the apoptotic rate was decreased with the increasing concentrations. FCM analysis showed increased G2/M phase rate. In the meantime, Western blot measure displayed mRNA and protein expressions of CyclinBl and significantly higher protein level of Cdc2 (Tyrl5) than those in the hyperthermic chemotherapy group. Immunocytochemical studies of the subcellular localization of p53 and Western blot analysis of protein expression of p53 in large intestinal epithelial cells revealed that treatment with PFT-a was associated with the decreased nuclear-to-cytoplasmic p53 ratio and suppressed the nuclear translocation of p53 induced by thermochemotherapy. With the increasing concentrations of PFT-a, the transcription activities and protein expressions of the target genes of p53, including Bax, MDM2, and p2lWAF1/CIPI, were significantly lower than those in the hyperthermic chemotherapy group (P<0.0\). This inhibitory effect increased in a concentration-dependent manner.3. PFT-a could protect small intestinal epithelial cells of BALB/c mice against IHCP-induced apoptosis. PFT-a could down-regulate the levels of mRNA and protein expressions of Bax, MDM2, and p21WAFI/CIP1 in small intestinal epithelial cells of BALB/c mice in a dose-dependent manner, but could increase the mRNA and protein expressions of Bcl-2. PFT-a increased the ration of Bcl-2/Bax through increasing the Bcl-2 expression and attenuating the Bax expression. After treatment of BALB/c mice with PFT-a and IHCP, PFT-a could significantly suppress the release of cytochrome C from the mitochondria to the cytoplasm in small intestinal epithelial cells of BALB/c mice.Conclusions1. PFT-a at the concentration less than 50 uM had no effect on the proliferation of large intestinal epithelial cells, but could inhibit the proliferation when the concentration was higher than 60 uM. PFT-a at the concentration of less than 40 uM had no effect on the antitumor potency of hyperthermic chemotherapy, but could enhance the antitumor potency of hyperthermic chemotherapy and display synergistic anti-tumor effect with thermochemotherapy at the concentration higher than 50 uM.2. PFT-a could protect large intestinal epithelial cells against thermochemotherapy-induced damages. This protect effect enhanced in a concentration-dependent manner. The mechanisms may include: first, PFT-a decreased the apoptotic rate of large intestinal epithelial cells, but increased the G2/M phase rate and expression levels of mRNA and protein of CyclinBl.Meanwhile, significantly increased phosphorylation level of Cdc2 (Tyrl5) resulted in down-regulation of CyclinBl/Cdc2 complex activity. The inactivity of CyclinBl/Cdc2 was involved in the G2/M arrest. Second, PFT-a was associated with the decreased nuclear-to-cytoplasmic p53 ratio and suppressed the nuclear translocation of p53 triggered by thermochemotherapy. Third, with the increasing concentrations of PFT-a, the transcription activities and the protein expressions of the target genes of p53, including Bax, MDM2, and p21WAF1/CIP1, were significantly down-regulated.3. PFT-a could protect small intestinal epithelial cells of BALB/c mice from IHCP-induced apoptosis. PFT-a could down-regulate the mRNA and protein expression levels of Bax, MDM2 and p21WAFl/CIPI in the small intestinal epithelial cells of BALB/cmice, but PFT-a could increase the mRNA and protein expressions of Bcl-2. PFT-a could significantly suppress the release of cytochrome C from the mitochondria to the cytoplasm in small intestinal epithelial cells of BALB/c mice. PFT-a could regulate not only the target gene expressions of p53 but the expression of cytochrome C as well. PFT-a could protect small intestinal epithelial cells of BALB/c mice against IHCP-induced damages through the mitochondrial signal pathway.
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