TCS is a type I ribosome-inactivating protein with a molecular weight of 27 kilodaltons. It can be purified from the root of tubers of the Chinese medicinal herb Trichosanthes kirilowii Maxim. This protein possesses broad-spectrum of biolological and pharmaceutical properties, such as anti-tumor, immunosupression, mid-term abortion and antiviral activities. At the end of 1980’s, more researchers were attracted to study TCS after McGrath et al found that the plant protein could inhibit HIV-1 replication in the acutely infected T lymphocytes and chronically infected macrophage cells. But the anti-HIV-1 mechanism of TCS is still uncertain. The results of our previous research on anti-HIV-1 activity and the structure-functional relationship of TCS showed that TCS has strong cytotoxicity on HIV-1 infected cells but little direct effect on the HIV-1. This suggested that the antiviral action of TCS may be related to the interaction with host cells. So we investigated the cytopathic mechanism of TCS on the HIV-1 infected cells. Firstly, cytotoxicity of TCS on H9 T lymphocyte cell line and HIV-1 chronically infected H9 cell line (H9/HIV-1ⅢB) were tested by using MTT colorimetric assay. Apoptotic action of TCS on H9 and H9/HIV-1ⅢB cells were tested by flow cytometry assay and agarose gel electrophoresis. The results showed that TCS could induce more H9/HIV-1ⅢB cells apoptosis in a dose-dependant manner. At TCS concentration of 25μg/ml, 8.4% of normal H9 cells were found to be apoptotic whereas the same concentration induced 24.5% in HIV-1 chronically infected cells in flow cytometry study. DNA fragmentation study also confirmed more laddering in infected cells. Such difference was not found in the control experiments with D-sorbitol treatment. Then the selectively apoptotic action of TCS on H9/HIV-1ⅢB cells was further validated by establishing HIV-1 chronically infected Jurkat(Jurkat/HIV-1ⅢB) cell line. We found that TCS could induce H9/HIV-1ⅢB, Jurkat and Jurkat/HIV-1ⅢB cell lines apoptosis in the same measure. But the effects on H9 cells were unconspicuous.Such confirmed our suggestion that TCS just selectively induced H9/HIV-1ⅢB cells apoptsis and had little selective apoptotic action on other HIV-1 infected cells. Present research suggested that there were two apoptotic pathways according to different cell types. H9 cells were TypeⅠcells which had mitochondria-independent apoptotic pathway.Jurkat cells were TypeⅡcells which had mitochondria-dependent apoptotic pathway. Our results showed that H9/HIV-1ⅢB, Jurkat and Jurkat/HIV-1ⅢB cell lines had similar sensitivity to TCS induced apotosis. But the effects on H9 cells were very inapparent. The loss of mitochondrial membrane potential and activity of caspase-8 in apoptotic cells which measured by flow cytometry assay also proved such above results. So we infered that H9/HIV-1ⅢB cells were more similar to the TypeⅡcells other than TypeⅠcells. And TypeⅡcells have more apoptotic sensitivity to TCS. Such may be responsible for the selective apoptotic action to H9/HIV-1ⅢB cells of TCS.
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