Effects Of Triptolide On CA46 Cell And Its Xenografts In Nude Mice

Triptolide(Molecular formula ,C20H24O6 ; molecular weight ,360.4) is a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii Hook f. Several reports have indicated TPL exerts antitumorigeniesis activity against several tumor cells. In the 1970s, Kupchan et al found that TPL had an anti-leukemia activity. Our previous investigates also indicated TPL can induce apoptosis of several hematological tumor cells, such as acute myelocytic leukemia cells HL-60, U937 and K562. So there was a wide application perspective for TPL in treatment of hematological tumors. Refering to the rcords of past years, there were no study in TPL to Burkitt lymphoma cells CA46 in vitro and in vivo. So we chose CA46 cells as objective and tried to reveal the effect of TPL on CA46 cells in vitro and its anti-tumor effects on xenografts of CA46 cells in nude mice.The subjects of the thesis were focused on the following aspects:The first part of this experiment was in vitro cell experiment. MTT and colony forming assay were used to examine the effects of TPL on the proliferation of CA46 cells; transmission electron microscope (TEM), Annexin -V flow cytometry analysis and TUNEL assay were used to examine apoptotic cells.The second part was to try to investigate the probable mechanism of its antitumor action. The expression of mRNA was determined by reverse transcript-polymerase chain reaction (RT-PCR) assay. The expression of protein was detected by ELISA assay and Western blot technique.The third part was to treat the xenografts of CA46 cells in nude mice with TPL. Mice bearing growing tumors were divided into six groups. There were 2% propylene glycolsaline (NS) negative control group, VP-16 positive control and three treatment groups in which the dose of TPL in every animal was 400μg/kg,200μg/kg,100μg/kg. Reagents were administered via injection into the vena caudalis every two days for seven times. The changes of tumor weights and histology were used to evaluate the effect of TPL on xenograft growth of CA46 cells. The apoptosis of tumor cell was tested by transmission electron microscope assay. The blood was gained to detect the the cell number, liver and renal function and the important organs were detected by histology assay to evaluate the toxic action of TPL to nude mice.Major results were obtained as follows:1. TPL could inhibit the proliferation of CA46 cells significantly in a dose- and time-dependent manner.2. TPL could induce apoptosis in CA46 cells by the following data: 1) Apoptotic cells were observed by TEM in CA46 cells treated with TPL. 2) Positive Annexin V -FITC on cell membrane showed that TPL induced apoptosis of CA46 cells in a dose-dependent manner. 3) TUNEL assay also showed that TPL induced apoptosis of CA46 cells in a dose-dependent manner.3. TPL could regulate the expression of apoptosis-related genes : RT-PCR analysis showed that the expression of c-myc,bcl-2 were down-regulated in CA46 cells treated with TPL , whereas the expression of TGF-β1,bax were up-regulated .4. TPL could regulate the expression of apoptosis-related proteins: 1) ELISA analysis showed that the level of autocrine TGF-β1 raised dramatically in CA46 cells treated with TPL. 2)Western blot analysis showed that the protein expression of Bcl-2,C-myc,Pro-caspase,NF-κb,I-κb were down-regulated in CA46 cells treated with TPL but the protein expression of Bax were advanced.5. In vivo TPL could inhibit xenograft growth of CA46 cells with the inhibition rate of 11.3%(p>0.05),24.6%(p<0.05)and51.3% (p<0.01)in the group where TPL was injected to each mouse with a dose of 100μg/kg,200μg/kg and 400μg/kg every two days. TPL can inhibit the proliferation of CA46 cells in vivo in a dose-dependent manner. There were more apoptotic cells in treatment groups than in control group by transmission electron microscopy (TEM) assay.From above results, we conclude that TPL can inhibit the proliferation of CA46 cells and induce CA46 cells apoptosis in vitro and in vivo.