Function Analysis Of Autophagy In The Antitumor Effects Of Triptolide To Colorectal Cancer Cells

The incidence and mortality rates of colorectal cancer(CRC)are high,which rank the third and fourth of tumor spectrum these years.CRC occurs in the rectum and sigmoid colon junction,which is one of the common gastrointestinal malignancies because of its increasing incidence.Therefore,the researching of CRC’s prevention and treatment is also ongoing.Triptolide(TPL),also known as triptolide or triptolide alcohol,derived from the roots of Chinese medicine Tripterygium,which is a natural product with a variety of biological activities.Modern medicine research showed that TPL has the activity of anti-oxidation,anti-rheumatism,and anti-cancer,both in breast cancer and pancreatic cancer.However,the role of TPL on CRC remains unclear.Since autophagy and apoptosis has become hotspots in the researching of treatments of anticancer diseases nowadays.Many researches has showed traditional chemotherapeutic drugs can induce positive autophagy during the course of chemotherapy,thus decreasing the sensitive of chemotherapeutic drugs’ inhibits tumor cells’ proliferation.In this study,we try to research on the role of autophagy in TPL against colorectal cancer cell.(1)TPL induced the cell proliferation inhibition and apoptosis in CRC cellsThe CRC cell lines SW480 and HCT116 were treated with different concentrations of TPL for 48 h,and the cell growth activity was analyzed by CCK8.The results showed that TPL could significantly inhibit the proliferation of two kinds of CRC cell lines.The results showed that the apoptosis rate of TPL treatment group was significantly higher than that of the control group.Western blot analysis showed that the apoptosis factor Cleaved-Caspase3 and Cleaved-PARP1 was gradually activated with TPL increased concentration.The data showed that TPL can induce CRC cell apoptosis,thereby inhibiting its proliferation.(2)TPL inhibits the initiation of autophagy and autophagy flux in CRC cellsTo investigate whether TPL could regulate autophagy in colorectal cells,the processing of LC3-I to its PE-conjugated LC3-II,which is correlated with the extent of autophagosome formation was measured by immune blotting.The results showed the conversion of LC3-I to LC3-II was significantly decreased after TPL treatment in a dose dependent manner in CRC cells suggesting that TPL can inhibit the basic level of autophagy in CRC cells.Then we used the m TOR inhibitor Rapamycin or Serum starvation to induce the autophagy.The number of Acidic autophagic vacuoles(AVOS),another major feature of autophagy,decreased significantly with the TPL increased treatment concentration.Under the same conditions,the results also showed that TPL treatment induced a significant decrease of LC3 punctuates in the cells.The distribution of endogenous LC3 in CRC cells was also analyzed by indirect immunofluorescence staining,and less specific punctuate LC3 proteins dots were found in the TPL-treated cells.Western Blot data showed that autophagy-related proteins were inhibited when the CRC cells were treated with TPL under the treatment of Rapamycin or Serum starvation conditions.These results suggest that TPL inhibits autophagy in human CRC cell lines.To determine TPL can also inhibit autophagy flux in CRC cells,the co-localization of LC3 and LAMP1 was firstly analyzed by indirect immunofluorescence assay.The results showed that the co-localization of LC3 and LAMP1 was significantly decreased in TPL-treated HCT116 cells,suggesting TPL can also inhibit autophagy flux in CRC cells.(3)TPL inhibits autophagy by reducing the transcription of autophagy related genesTo detemine the molecular mechanism of TPL on inhibiting autophagy in CRC cells,the expression of autophagy related genes are examined by q RT-PCR assay.The results showed that TPL can significantly inhibit ATG5 ATG7 and ATG12 transcription in a dose-dependent manner.Then we use Western-blot method to further verify the obtained results.These data suggest that TPL induced inhibition of autophagy may bemediated bythe reduction of autophagy related gene transcription(4)Inhibitory effect of TPL on autophagy can enhance the anticancer effect of some chemotherapeutic drugs5-Fluorouracil(5-FU)can induce protective autophagy,Therefore we asked whether TPL can mediate chemosensitization in response to conventional genotoxic drugs such as 5-FU.The results showed that combination effects of 5-FU and TPL kill more tumor cells compared with5-FU or TPL treatment alone.It is suggest that TPL plays an role of autophagy inhibition,which enhance the effect of the drugs as well as induce protective autophagy in CRC cells.In summary,this study shows that TPL inhibits the proliferation of human CRC cells by inducing apoptosis and inhibiting autophagy.Moreover,TPL significantly enhanced the sensitivity of CRC cells to protective autophagy induced chemotherapy drugs such as 5-FU,suggesting might serve as a novel desirable adjuvant drug by inhibiting chemotherapy induced protective autophagy.