The Growth Inhibition And The Apoptosis Of HeLa Cells With Trichosanthin

Trichosanthes kirilowii Maximowicz (Tian Hua Fen), used to induce abortion, is the Chinese traditional medicinal herb. In the 1980s, Trichosanthin (TCS) was isolated from the root tuber and proved to be the active component, a type I ribosome-inactivating protein (RIP) with 247 amino acids which inactivates eukaryotic ribosomes via its N-glycosidase activity. TCS has long been used to induce mid-term abortion and to treat ectopic pregnancies, expel retained placenta and trophoblastic moles in China. In recent years, TCS has also been found to posses various pharmacological properties including immunomudulatory, anti-tumor and anti-HIV activities. TCS has aroused extensive attention.In this study we focus on the anti-tumor activity of TCS and its mechanism. TCS has the activity of anti-tumor, such as choriocarcinoma, liver cancer and other cancers of digestive system. The forepart research reported that the specific toxicity of TCS to choriocarcinoma cells had already been regarded as a result of the inactivation of ribosomes. However, another study revealed that TCS can inhibit the growth of JAR cells by inducing apoptosis. So the researches of TCS on tumorswere mainly on gastrointestinal tumor and seldom on others. And the anti-tumour mechanism of TCS isn't clearly demonstrated yet. In the present study, we chose HeLa cells (human cervical cancer) to investigate the growth inhibitory effect of TCS, to demonstrate the anti-tumour mechanism of TCS though apoptosis and restrain of cell cycle and further to explore the underlying molecular mechanism of apoptosis induced by TCS.Proliferation, apoptosis, differentiation and senescence of cells all depend on cell cycle, so nowadays, exploiting new anti-tumor drugs from natural resource, especially from traditional Chinese medicines, which can selectively regulate cell cycle, induce apoptosis and arrest cell growth will be the most ideal path to conquer tumors. Therefore, in our study, we initially investigated the growth inhibition effect and the induction of cell cycle arrest and apoptosis of TCS on HeLa cells. Furthermore, we also study the pathway of apoptosis induced by TCS. The present study may provide experimental bases for the application of TCS in the therapy of cancer and give new insights on the mechanism of anti-tumour.Objective To investigate the growth inhibition effect of TCS on HeLa cells, to demonstrate the induction of cell cycle arrest and apoptosis of HeLa cells, and further to explore the underlying mechanism of anti-tumour and apoptosis induced by TCS.Methods The antiproliferative effect of TCS on HeLa cells was measured with CCK-8. The FCM analysis was carried out to examine the effect of TCS on the cycle distribution of HeLa cells. Electron microscopy and FCM analysis was employed to observe the apoptosis of HeLa cells. The expression of proCaspase-3 and Caspase-3 mRNA was determined by Western blot and RT-PCR. The activity of Caspase-3, 8,9 was determined by a Caspases colorimetric assay kit.Results (l)The proliferation of HeLa cells was significantly inhibited by TCS with a time and dose dependant manner. When the concentration of TCS wasincreased and the time was prolonged, the growth inhibitory rate increased gradually. After exposure to lOOug/ml TCS for 24h, the inhibitory rate is 31.613.92%. And after 48h, it obviously increased to 49.34±4.49%. Otherwise, when the time was prolonged, the median inhibitory rates of TCS for HeLa cells declined. The IC50 was 191.46u,g/ml after 36h and 101.34ug/ml after 48h, respectively. (2)After HeLa cells were treated by TCS (lOOug/ml) for 24, 36 and 48h respectively, flow cytometric analysis showed that the percent of S phase increased gradually with time and the G2/M phase reduced accordingly. And after HeLa cells were treated by different concentration of TCS for 48h, the percent of S phase increased and the G2/M phase reduced accordingly only by 50, lOOug/ml TCS. (3)After HeLa cells were exposed to TCS (100ng/ml) for 24h, the apoptotic cells were found with microvilli reduction, shrinkage in size, condensation of the nuclear chromatin to form dense masses that bordered the nuclear membrane. After 48h, typical apoptotic characteristics were showed, including microvilli disappearance, cell membrane bledding and apoptotic bodies;besides, the chromatin was hypercondensed, the nuclear envelope was largely disrupted and apoptotic cells showed an extensive vacuolation and a very smooth surface. (4)After HeLa cells were treated with TCS (100|xg/ml) for 24, 36 and 48h respectively, the apoptotic index increased gradually with time;After HeLa cells were treated by different concentration of TCS for 48h, the induction of apoptosis took on a dose-dependent manner. Apoptotic index of HeLa cells reached its peak 42.52% with 100ng/ml TCS for 48h. (5)Treatment of HeLa cells with 100|ig/ml TCS for 24, 36, 48h or with different concentration of TCS for 48h caused a time and dose-dependent decrease in the intensity of the proCaspase-3 band, and the ratio of optical density integral of proCaspase-3/p-actin decreased significantly in a time- and dose-dependent manner, these results suggested that the proCaspase-3 was activated. (6)HeLa cells were treated with 100ng/ml TCS for 12, 24, 36 and 48h respectively, the intensity of the Caspase-3mRNA band and the ratio of optical density integral of Caspase-3 mRNA/p-actin were increased with time-dependent. (7)It revealed that the activity of Caspase-3, 8, 9 increased with time-dependent though colorimetric assay of Caspases activity. As revealed by colorimetric enzyme assay, Caspase-8 activity was the highest at 24h following lOOug/ml TCS treatment. At 36h following 100ng/ml TCS treatment, Caspase-3 activity was obviously higher than the control and reached its peak at 48h. Caspase-9 activity was markedly higher than the control but lower than Caspase-3 activity at 36 and 48h following lOOug/ml TCS treatment. These results suggest that the Caspase activation has a temporal sequence and the activation of Caspase-8, 9 leads Caspase-3 activated.Conclusion The results of our study showed that TCS inhibit the growth of HeLa cell through cell cycle arrestted in S phase and the apoptosis of HeLa cells induced with TCS. And it demonstrated that the activation of Caspase-3 is a common pathway mediating the apoptosis of HeLa cells with TCS. At the same time, the activation of Caspase-8, 9 critically involve in the activation of Caspase-3.