Purification Of An Anticancer Protein From Trichosanthes Kirilowii And Its Mechanism Of Inducing Apoptosis In Human Colorectal Adenocarcinoma Cells

Colorectal cancer(CRC),a common malignant cancer of the digestive tract,is one of the leading causes of cancer mortality worldwide.At present,chemotherapy is one of the most common methods for CRC treatment.However,many chemo-therapeutic drugs have great side effects on the human body and hence restrict their clinical application.Many studies reported that the plant-derived protein has anticancer activity and has the advantage of low toxicity and low side effects.It has great protential in anticancer.Trichosanthes kirilowiiis is a member of cucurbitaceae family.In our country,it is a long history of common Chinese herbal medicine.Studies had shown that decoction of Trichosanthes kirilowii could inhibit growth of ascites cancer cell and cervical cancer cell in dose-dependent manner.At present,reports of anticancer active components from Trichosanthes kirilowii fruits is less.In this research,a kind of anticancer protein was obtained from Trichosanthes kirilowii fruits and the mechanism of its inducing apoptosis was studied.The research contents include the following aspects:Part 1:An anticancer protein,named as TKP,was purified from Trichosanthes kirilowii fruit by phosphate buffer extraction,employing acetone precipitation,ammonium sulfate fractionation precipitation,Q-Sepharose anion-exchange column and Sephacryl S-200 gel-exclusion column.MTT assay was used to assess the anticancer activity of the purified protein.Mass spectrometry revealed that TKP has high homologous with cucumisin of Siraitia grosvenorii.Moreover,it also showed that the native molecular weight of TKP was about 46.621 kD.Then the enzyme activity of TKP was identified.PMSF,an inhibitor of serine proteases,exerted a significant inhibition.The result suggested that TKP is a serine protease.Part 2:The effects of TKP on human colorectal adenocarcinoma cells(DLD1,HCT116,SW620)and normal colon epithelial cell(NCM460)were detected.MTT assay results showed TKP significantly inhibited viabilities of human colorectal adenocarcinoma cells,however,it had no such effect on normal colon epithelial cell.Clonogenic survival assay results suggested TKP inhibited the proliferation of human colorectal cancer cells.Results of DAPI staining and flow cytometric analysis showed TKP significantly induced apoptosis of human colorectal adenocarcinoma cells.JC-1 staining indicated TKP decreased the mitochondrial membrane potential in human olorectal adenocarcinoma cells.Western blotting results indicated that Cytochrome c,Bax,Activated-caspase-9 and Activated-caspase-3 levels were increased.All these results suggested TKP induced apoptosis via mitochondria pathway in human colorectal adenocarcinoma cells.Part 3:The effects of TKP on MAPK and PI3K/AKT signalings in human colorectal adenocarcinoma cells were detected.Western blotting showed TKP induced the increasing of p-ERKl/2 significantly but had no such effect on p-JNK and p-p38.Meanwhile,it decreased the protein level of p-AKT and had no obvious effects on AKT.These results indicated TKP activated MAPK/ERK1/2 signaling and inhibited PI3K/AKT signaling.In order to study the effect of MAPK/ERK1/2 signaling on TKP-induced apoptosis,cells were treated with inhibitor of MAPK/ERK signaling(PD98059),results showed PD98059 could inhibit phosphorylation of ERK1/2 induced by TKP,but further strengthened the apoptosis induced by TKP.These results showed that MAPK/ERK1/2 signaling was not involved in TKP-induced apoptosis.Moreover,cells were treated with inhibitor of PI3K/AKT signaling(LY294002)and activator of PI3K/AKT signaling(IGF-1).Results indicated LY294002 could further inhibited the decreasing of p-AKT and elevated the increasing of Cytochrome c,Bax,caspase-9 and caspase-3 along with the decreasing of Bcl-2 induced by TKP.Meanwhile,LY294002 could exacerbate TKP-induced reduction in cell viability and??m as well as apoptosis.The reduction of cell viability and apoptosis were also inhibited by IGF-1.All these results suggested TKP induced apoptosis via PI3K/AKT signaling in human colorectal adenocarcinoma cells.In summary,TKP,which was purified from Trichosanthes kirilowii fruit,could significantly inhibited proliferation and induced apoptosis in in human colorectal adenocarcinoma cells.The study founded TKP induced apoptosis via PI3K/AKT-mediated mitochondrial pathway in human colorectal adenocarcinoma cells.