| Objective:Both the human esophageal carcinoma and the gastric carcinoma have a higher morbidity in Jiangsu province and their main therapy methods are exairesis, chemotherapy and radiotherapy at present. These methods are not only higher-side effects, costly, drug fast, but also non-specificity (that is it will interrupt the metabolism of normal tissue, decrease the body’s immunity and down regulation the immune surveillance when treated the tumours by these means). In view of the theory that our traditional Chinese drugs have the supporting healthy energy to eliminate evils, so it can not only conduce to kill the tumour cells, but also adjust the immune balance from the global level if we apply our traditional Chinese drugs to assist the conventional therapies above-mentioned or decrement therapies to treat the tumours. So it’s important for us to development a kind of traditional Chinese drug with higher- anti-tumour therapeutic efficacy and lower- side effects. The bitter melons which liked by civilians are not only abundance resource, cheap, lower- toxicity, but also have many bioactivities such as heat-clearing and detoxicating, depressing blood sugar, anti-mutation, anti-virus, anti-HIV, anti-tumor, anti-bacteria, immunoregulation and so on. So it is very important for us to development the bitter melons into a kind of security and effective naturally occurring drugs. The most attractive bioactive substances isolated from the bitter melons are RIPs (ribosome inhibiting protein, RIP). RIPs have been classified intoⅠandⅡtypes. TypeⅠRIPs possesses many biologic activities such as anti-tumor and anti-virus activities. The most significant anti-tumor mechanism for typeⅠRIPs is inducing the tumor cells apoptosis. MAP30 (Momordica anti-HIV protein of 30ku) is a kind of RIP I and has no toxic effect on normal cells because MAP30 can’t ingress into these cells. Recently, interest in MAP30 has been stimulated by the reports that MAP30 has potent anti-tumor activity. MAP30 can induce many cells apoptosis, however, the precise apoptosis signaling pathway is still unknown. Here, we cloned MAP30 gene of the Momordica charantia (MC) protein, expressed and pured the MAP30 fusion protein. And then to investigate whether MAP30 could induce apoptosis in the human esophageal carcinoma cells EC-1.71 and the human gastric carcinoma cells BGC-823 in vitro and further to assess its apoptotic mechanism.Method:(1) Polymerase chain reaction (PCR) was used to amplify the MAP30 gene from bitter melon genome DNA. Then the target gene was inserted into the vector pGEM-T and identified by restriction digestion and DNA sequencing. Then the MAP30 gene fragment was inserted directionally into expression vector pET-28a. The pET-28a-MAP30 expression vector was transformed into E.coli BL21. The his-MAP30 fusion protein was expressed in E.coli BL21 with Isopropylβ-D-1-thiogalacto-pyranoside (IPTG), the target fusion protein was purified by Ni-column and identified by SDS-PAGE.(2) Recombinant MAP30-fusion protein were co-cultured with the human esophageal carcinoma cells EC-1.71 and the human gastric carcinoma cells BGC-823, respectively. The antiproliferative activities on the human esophageal carcinoma cells EC-1.71 were determined by MTT assay, Cytometry, colony formation and tumor xenograft model of nude mice and mice. The antiproliferative activities on the human gastric carcinoma cells BGC-823 were determined by MTT assay, Cytometry and tumor xenograft model of nude mice. Morphologic changes of the human esophageal carcinoma cells EC-1.71 and the human gastric carcinoma cells BGC-823 were detected by light microscope, electron microscope and fluorescence staining. The percentage of apoptosis and the proportion of the periodic cells of the human esophageal carcinoma cells EC-1.71 and the human gastric carcinoma cells BGC-823 were displayed by a flow cytometry (FCM) analysis. The MAP30-induced apoptosis of EC-1.71 cells was also determined by DNA ladder.(3) To further investigate the mechanism of MAP30-induced apoptosis of EC-1.71 cells and BGC-823 cells, the level of mitochondria membrane potential (ΔΨm) was displayed by JC-1 fluorescent probe assay. The effects of MAP30 on Cytc and TNF-αof EC-1.71 cells and the effects of MAP30 on Cytc of BGC-823 cells were investigated by ELISA. The expressions of ERK1/PERK1, AKT/PAKT, NF-κB and c-jun protein of EC-1.71 cells and the expression of NF-κB protein of BGC-823 cells were indicated by immunocytochemical stain. The activities of Caspase-12 of EC-1.71 cells and BGC-823 cells were showed by Caspase-12 Fluorometric Assay. The mRNA expression of caspase-9 and Apaf-1 of BGC-823 cells were investigated by RT-PCR. The activity of Caspase-3 of BGC-823 cells was showed by spectrophotometric method.Results:(1) The recombinant plasmid was constructed and the DNA sequence analysis showed that the length of the MAP30 sequence was 861bp. It has homology with DQ643967, DQ643968 and S79450. The prokaryotic expression vector pET-28a-MAP30 was constructed successfully and the recombinant MAP30 fusion protein which relative molecular weight was 30kDa was obtained.(2) Treated with MAP30, the proliferation of EC-1.71 cells was suppressed in a dose dependent manner and the IC50 was 82.86μg/mL After treated with MAP30 for 48 and 72h, some cells became round, blunt and shrinkage, smaller in size, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies and cells became detached and suspended in the medium under light microscopy. The ultrastructural and morphological changes in EC-1.71 cells were observed under electron microscope:nuclear fragmentation, chromosome condensation, abnormalities of mitochondrial cristae, cells shrinkage and endoplasmic reticulum (ER) expansion and broaden. The fluorescence staining indicated that MAP30 could induce the EC-1.71 cells apoptosis, but it wasn’t in a dose dependent manner. The proportion of EC-1.71 cells in S phase was decreased and the proportion of EC-1.71 cells in G0/G1 phases was increased. But there was no typical DNA ladder was observed.(3) Treateded with MAP30, the proliferation of BGC-823 cells was suppressed in a dose dependent manner and the IC50 was 51.67μg/mL When treated with MAP30, some cells became round, blunt and shrinkage, smaller in size, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies under light microscopy. The ultrastructural and morphological changes in BGC-823 cells were observed under electron microscope:the microvilli on the surface of the cells disappeared, cells shrinkage, nuclear condensation, chromosome agglutination, nuclear membrane fold, cytoplasm concentration. The fluorescence staining indicated that MAP30 could induce the BGC-823 cells apoptosis.The apoptosis peak of the hypodiploid was more evident indicated by FCM assay.(4) Treateded with MAP30, theΔΨm in EC-1.71 cells was decreased. The content of Cytc and TNF-a were increased in EC-1.71 cells supernatant. The expressions of ERK1 and AKT in EC-1.71 cells were increased in endochylema and the expressions of PERK1 and PAKT in EC-1.71 cells were decreased in nuclei. NF-κB protein translocation (from nuclei to endochylema) in EC-1.71 cells were indicated by immunocytochemical stain. The activity of Caspase-12 in EC-1.71 cells was increased, but the expression of c-jun protein in EC-1.71 cells was no marked change.(5) When cells were exposed to MAP30, theΔΨm in BGC-823 cells was decreased, the content of Cytc was increased in the cells’s supernatant and the mRNA expressions of caspase-9 and Apaf-1 were increased and the activity of caspase-3 was increased in BGC-823 cells. But the activity of caspase-12 was no significant change and MAP30 could activate the nuclear transcription factor NF-κB in BGC-823 cells.Conclusion:(1) The prokaryotic expression vector pET-28a-MAP30 was constructed successfully and the fusion protein MAP30 was obtained from E.coli BL21.(2) The recombinant MAP30-fusion protein could inhibite the proliferation of the EC-1.71 cells and the BGC-823 cells in a dose dependent manner.(3) The recombinant MAP30-fusion protein could induce the EC-1.71 cells and the BGC-823 cells apoptosis. (4) The recombinant MAP30-fusion protein could induce the EC-1.71 cells apoptosis in vitro via ER pathway and NF-κB pathway.But the specific mechanisms still need a further study.(5) The recombinant MAP30-fusion protein could activate the nuclear transcription factor NF-κB in the BGC-823 cells and could induce the BGC-823 cells apoptosis in vitro via the mitochondrial pathway. But the specific mechanisms still need a further study. |
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