| Colon cancer is one of the most common malignant tumors in the gastrointestinal system and is ranked third among cancer incidence for males and females.In recent years,there have been clear upward trends in both the incidence and mortality of colon cancer in China,the incidence of male is higher than that of female.The incidence and mortality of colon cancer in rural areas are higher than that in urban areas.The increasing number of persons with colon cancer poses a serious threat to public health.Therefore,it is of great practical significance to prevent and cure colon cancer.Toddalia asiatica Lain belongs to Toddalia genus of Rutaceae family that is mainly distributed in the region south of the Qinling Mountain ranges in China.It has good medicinal value.Various parts of this plant have been used in the treatment of different diseases,with the root often being taken as a cure for a wide variety of pains,bruises,rheumatoid arthritis and cough and so on.The main chemical compositions from Toddalia asiatica are coumarins and alkaloids.Modern pharmacologic researches on both the crude extracts and some isolated compounds of Toddalia asiatica,substantiated its multiple biological activities,including anti-oxidant,anti-inflammatory,anti-bacterial,anti-tumor and so on.So it can be speculated that Toddalia asiatica has a good development and application value.Reports on the active extracts and compounds from this plant with anti-colon cancer effects and their mechanism of action are rare.The aims of this work are to first screen the extracts from the root of Toddalia asiatica with inhibition of the colon cancer cell growth,and to further elucidate the underlying molecular mechanisms.1.Four fractions from the root of Toddalia asiatica:n-hexane extract,dichloromethane extract,ethyl acetate extract,and water extract,were obtained by systematic solvent extraction methods.The SRB colorimetric assay was used to determine the growth inhibition effect of different extracts on cancer cell lines derived from different tissues.These results found that dichloromethane extract showed highest inhibitory activity against human colon cancer HT-29 cells.Therefore,we can think that the dichloromethane extract has the value of further research.2.We further investigated the effects of the dichloromethane extract on cell proliferation and cell cycle progression of HT-29 cells.Firstly,inhibition of HT-29cells proliferation in time-and dose-dependent manner by the dichloromethane extract was observed.Then,Flow cytometry was used to detect the cell cycle distribution after the HT-29 cells treatment with the dichloromethane extract.The results indicated that the dichloromethane extract arrested cell cycle at the G2/M phase in HT-29 cells.The inhibition of cell proliferation may be possibly attributed to cell cycle arrest by dichloromethane extract treatment.3.The p53 protein plays an important role in the control of cell cycle progression,to determine whether the cell cycle arrest induced by dichloromethane extract was dependent on p53 status,we chose to use colon cancer cell lines with wild-type p53?wt-p53?and those with mutant-type p53?mt-p53?to evaluate the effects of dichloromethane extract on cell proliferation and cell cycle distribution.The similar trends in cell growth inhibition and induction cell cycle arrest caused by dichloromethane extract were observed in cell lines with different p53 status.These results indicated that p53 status was not directly linked to the response observed in cell cycle following treatment with dichloromethane extract.4.In order to investigate whether inhibition of proliferation of HT-29 cells by dichloromethane extract was due to apoptosis,Annexin-V/PI double staining was performed to analyze cell apoptosis ratio,the results showed that the rate of apoptosis in HT-29 cells was significantly increased in dose dependent manner after 24 h treatment with the dichloromethane extract,indicating that the dichloromethane extract has apoptosis-promoting effect.At the same time,westem-blotting was used to determine whether the the active/cleaved form of caspase-3,caspase-8 and caspase-9appeared predominantly in the cells treated with dichloromethane extract.The results showed that the dichloromethane extract can activate caspase-3,caspase-8 and caspase-9.This indicated that the dichloromethane extract could induce cell apoptosis through both intrinsic and extrinsic pathways.5.The intrinsic apoptosis pathway is regulated by the Bcl-2 family proteins Bax,in order to elucidate the role of Bax in the dichloromethane extract-induced cell apoptosis,we used colon cancer cell lines with different Bax status to evaluate the effects of dichloromethane extract on cell apoptosis.It was found that the dichloromethane extract induced apoptosis of wild-type Bax and mutant-type Bax colon cancer cell lines to a similar extent,proving that the dichloromethane extract-induced apoptosis did not correlate with Bax status.6.In this study,H2DCF-DA fluorescent probe was also used to investigate the effects the dichloromethane extract on reactive oxygen species?ROS?production in HT-29 cells.The results showed the capability of the dichloromethane extract to significantly induce ROS production.Studies have shown that ROS can cause cell cycle arrest and induce apoptosis.In order to investigate whether induction of cell apoptosis and cell cycle arrest by dichloromethane extract was due to excess ROS production,pretreatment with ROS scavenger NAC could reverse dichloromethane extract-induced cell cycle arrest and did not prevent dichloromethane extract-induced cell apoptosis Our results indicated that ROS generation is at least partially required for cell cycle progression,and the induction of apoptosis did not directly associate with ROS.In this paper we found that the dichloromethane extract of root of Toddalia asiatica?L.?Lam.has a significant inhibitory effect on the proliferation of human colon cancer HT-29 cells.Inhibition of HT-29 cells proliferation in vitro by the dichloromethane extract may be though cell cycle arrest and induction of apoptosis.At the same time,it was found that the cell cycle arrest induced by the dichloromethane extract was not related to p53,and the induction of apoptosis by the dichloromethane extract was not related to Bax.In addition,it was also found that the dichloromethane extract induced the production of reactive oxygen species?ROS?in HT-29 cells.The dichloromethane extract-induced cell cycle arrest was related to an ROS-dependent cell cycle progression,however,the induction of apoptosis did not involve ROS.Altogether,this works provide a theoretical basis for the development and utilization of Toddalia asiatica?L.?Lam.,and open a new avenue for further exploration of Toddalia asiatica?L.?Lam.as a new natural medicine for battling colon cancer. |
PDF Full Text Request