Study On Antitumor Mechanism Of Trichinella Spiralis

Antitumor effect of Trichinella spiralis (T. spiralis) had been reported, nevertheless,alive T. spiralis was often employed in all studies. Alive T. spiralis was always involved into public health for coexistance of antitumor effect and pathogenicity.Currently,it was reported cytokines level of hosts was elevated during trichinization.The studies indicated that apoptosis play a role on antitumor of T. spiralis. Study showed that antitumor active ingredient of T. spiralis had still been a blank, however,with development of phage display technology. Stuty on its isolation, identification and clinical application will become new research focus. Using solid murine tumor models of MFC,H22 or S180 in ICR mice, antitumor effect of T. spiralis was evaluated in vivo. Infected mice by oral administration of 400 viable T. spiralis larvas per mouse previously grafted tumor for 7 days. The inhibit rate of infected mice was 72.35±18.82%,54.22±23.52%,55.62±28.05% with statistical significance compared with control group after 11 days. Other groups of tumor-bearing mice were given caudal vein injection of polypide proteins of adult and newborn larvae at 17.5, 35.0 or 70.0 mg kg- 1 . The inhibition rate was 68.99±7.46%, 74.49±13.38%, 86.88±11.09%(P<0.05). Those treatments to inhibit tumor growth was dose-dependent (P<0.05). Using MFC, H22, S180, human K562 and H7402 cells, antiproliferative activity of polypide protein was examined in vitro at 0.035, 0.070 or 0.140 mg ml- 1. Employing MTT stain, tumor cells proliferation in vitro was also inhibited in dose–dependent manner (45.65±6.23%, 40.39±7.28%, 38.21±9.06% , 50.63±4.03%,4 8.03±8.72%,P<0.05),Drugs of anti-adhesion,anti-motion and inactivation matrix protease could inhibit migration and invasion of tumor cell. Migration and invasion was a basic feature of tumor cell,a nd invasion for basal lamina was key phase during tumor metastasis. Inhibition migration and invasion of the polypide protein could block those procedure to step down migration and invasion of H7402 cells. Test of migration, invasion and colony formation ability further indicated that the polypide proteins has antitumor effect and will be exploited as a potential antitumor agent. At the same doses,polypide proteins induced apoptosis of K562 and H7402, measured by TUNEL, detected by DNA fragmentation,o bserved by TEM.In K562 and H7402 treated cells , microvilli abscission , mitochondria intumescence,c ell membrane Integrity,c ell nucleus schizolysis,high-density bolus chromatin of cytoplasm and apoptotic body was observed by TEM. Cell cycle was analysised after K562 and H7402 cells was treated with the polypide protein for 24h. Cell cycle analysis indicated that polypide proteins, at 0.140 mg ml- 1, arrested cell cycle of K562 and H7402 in G1 or S phase.Those results supports the hypothesis which the polypide protein impaired chk of K562 and H7402 cells. The hypothesis as follow: A. The polypide protein impaired chk function of the tumor cells in G1 phase and enhanced selectivity therapy effect for tumor cells in S phase B. The polypide protein induced apoptosis of the cells by incurable DNA damage C. The polypide protein increased sensitivity of tumor to DNA damage. It is concluded that Trichinella spiralis contains antitumor active agent. H7402 cells was treated with 0.140mg/ml polypide protein for 6h, its relative fluorescence intensity of mitochondrial membrane potential was 112.48±9.75, untreated cell was 189.93±14.16,(P<0.01). The results showed that the polypide protein exhausted intracellular Ca2 + of H7402 cells. It lead to alteration of mitochondrial membrane potential and apoptosis sensitivity of the cells. p53,Bcl-2,Survivin expression was analysised by immunohisochemistry, FCM and confocal microscopy respectively. The polypide protein upregulated expression of p53, downregulated expression of Bcl-2, survivin (P>0.05). Expression of IL-2, IL-10 and DR4 was effected by the polypide protein in RT-PCR assay. The results showed a possible antitumor mechanism of the polypide protein as follow A. As result of survivin down regulation, it remove inhibition of caspase-3 and caspase-7 from survivin so that caspase-3 hydrolyzed micro-tubular structure protein,destroied spindle integrity and terminated mitosis of tumor cells. The chain reaction also emancipated DNAase from inhibition of ICAD,activated CAD and lead to apoptosis of H7402 cells B.DR4 upregulated by the polypide protein raise sensitivity of TRAIL in DR4 signal path to avtivate caspase-8.It combined with Bid to avtivated Bax. Bax combined with Bcl-2 by dimeride form. Bcl-2 was downregulated by the polypide protein lead to Bax and dimeride increase and cytochrome C release. Apoptosis signal was amplified. It lead to apoptosis of H7402;C. Cellular damaged by the polypide protein remove inhibition of Ca2 + from Bcl-2. It lead to mitochondrial membrane potential drawdown,C ytc release,superoxide anion increase an activated AIF,finally,apoptosis of H7402 was accelerated. D.DNA damage from the polypide protein avtivated ATM, it boosted p53 upregulation. p53 upregulation lead to the results as follow a. The polypide protein impaired chk function of the tumor cells in G1 phase and enhanced selectivity therapy effect for tumor cells in S phase;b. The polypide protein induced apoptosis of the cells by incurable DNA damage;c . The polypide protein increased sensitivity of tumor toDNA damage. E. The polypide protein stimulated overexpression of IL-10. Overexpression of IL-10 upregulated activation of IКB and inhibite activity of NFКB combination with DNA, it inhibited expression of protooncogene regulated tumor cells proliferation and increase cytokine expression to depopulate tumor cells. Based on our datum,We speculate valid protein from T. spiralis regulate apoptosis-related genes that can lead to tumor or cancer cell apoptosis. We speculate this occurs by regulating apoptosis-related genes and triggering a mitochondrial pathway or death receptor pathway that can lead to tumor or cancer cell apoptosis. T. spiralis confers resistance to tumor cells and also produces resistance to Shope's fibroma virus and its neoplastic effect in rabbits. Eosinophils play important roles in immunity against T. spiraisl in human infection. Normal eosinophils are unexpectedly sensitive to apoptosis, whereas infected eosinophils become resistant to this form of cell death Consequently, further studies on the relationship of the proteins and apoptosis gene expression (P53,caspase or Bcl-2),may help to identify the antitumor protein of T. spiralis and permit further analysis of its antitumor action. Biopanning and rapid analysis of selective interactive proteins are likely to be a significant component of this work.To get valid protein from T. spiralis, T7 phage cDNAC display library was successful constructed. Organic phase multicell screen was employed to sift candidate protein combined with K562 and H7402 by the phage display. The protein was gained and analysised by protein analysis software. The result show that its molecular weight, molecular formula,hydrophobicity index was 16736.3,C₇₆₅H₁₁₆₃N₂₀₅O₂₀₅S₇ and 84.04 respectively. It was a stable protein. Those datum will be significant on study of its antitumor effect and target therapy of tumor.