Objective To investigate the role and mechanism of low-dose aspirin concurrent with IFN-a in inducing hepatocellular carcinoma apoptosis in BEL-7402 cells. Methods BEL-7402 cells were cultured and treated with IFN-α,or low dose aspirin or both.MTT and flow cytometry were used to measure the cell proliferation and apoptosis after treatment with a singular drug or the combined regiment.The expressions of the apoptosis-related proteins were detected by Western blot.Results MTT assay revealed after IFN α administration alone or combined with aspirin treatment for 48 h,the proliferation ratio of the IFN-α or aspirin group were 82.45％ ± 1.71％ and 83.22％ ±2.26 ％,compared with the control group.The group which received the combined therapy had a proliferation ratio of 69.84 ％ ±1.18 ％,which was significantly lower than the single groups (P＜0.05).The flow cytometry revealed that the apoptosis ratio in IFN-α group and aspirin group were 14.78 ％ ±1.93％ and 14.00％±0.61％,respectively,while the IFN-α + aspirin group was 21.68％±1.28％,which was also significantly higher than that of the single groups (P＜0.05).Western blot detected that IFN-α and aspirin (1 mmol/L) could promote caspase-3 and caspase-9 protein expressions,and when the two drugs were combined,caspase-3 and caspase-9 were also significantly activated.IFN-α alone or combined with aspirin can promote the expression of pro-apoptotic protein Bax (P＜0.05),while the anti-apoptotic proteins expression of Bcl-2 and Bcl-xl did not change significantly (P＞0.05).Conclusions Low-dose aspirin can cooperate with IFN-α in inhibiting the BEL-7402 cell growth and inducing the cell apoptosis by activating and increasing caspase-3 and caspase-9 levels,which may be related to the increased expression of pro-apoptotic protein Bax.

OBJECTIVE: To explore the therapeutic mechanism of Liushen Wan (LSW) against colitis-associated colorectal cancer (CAC) by network pharmacology. METHODS: TCMSP, BATMAN-TCM, CNKI, PubMed, Genecards, OMIM, and TTD databases were used to obtain the related targets of LSW and CAC. The common targets of LSW and CAC were obtained using Venny online website. The PPI network was constructed using Cytoscape 3.8.2 to screen the core targets of LSW in the treatment of CAC. GO and KEGG enrichment analysis were conducted using DAVID database. The therapeutic effect of LSW on CAC was evaluated in a C57BL/6J mouse model of AOM/DSS-induced CAC by observing the changes in body weight, disease activity index, colon length, and size and number of the tumor. HE staining and RT-qPCR were used to analyze the effect of LSW on inflammatory mediators. Immunohistochemistry and TUNEL staining were used to evaluate the effect of LSW on the proliferation and apoptosis of AOM/DSS-treated colon tumor cells. Immunohistochemistry and Western blotting were used to detect the effects of LSW on the expression of TLR4 proteins in CAC mice. RESULTS: Network pharmacology analysis identified 69 common targets of LSW and CAC, and 33 hub targets were screened in the PPI network. KEGG pathway enrichment analysis suggested that the effect of LSW on CAC was mediated by the Toll-like receptor signaling pathway. In the mouse model of AOM/DSS-induced CAC, LSW significantly inhibited colitis-associated tumorigenesis, reduced tumor number and tumor load (P < 0.05), obviously improved histopathological changes in the colon, downregulated the mRNA levels of proinflammatory cytokines, and inhibited the proliferation (P < 0.01) and promoted apoptosis of colon tumor cells (P < 0.001). LSW also significantly decreased TLR4 protein expression in the colon tissue (P < 0.05). CONCLUSION LSW can inhibit CAC in mice possibly by regulating the expression of TLR4 to reduce intestinal inflammation, inhibit colon tumor cell proliferation and promote their apoptosis.
Mice ; Animals ; Toll-Like Receptor 4 ; Colitis-Associated Neoplasms ; Network Pharmacology ; Mice, Inbred C57BL ; Colonic Neoplasms/pathology*

Objective : To study the effect of bufalin on the proliferation and apoptosis of human colorectal cancer cell line HCT116，and to explore the role of endoplasmic reticulum stress ( ERS) in this process． Methods : The effect of bufalin on the proliferation of HCT116 cells was determined by CCK-8 assay．After HCT116 cells were treated with different concentrations of bufalin for 48 hours，cell apoptosis was detected by Annexin V / PI assay， and the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot．At the same time，the expression of ERS-related proteins glucose regulated protein 78 ( GRP78) ，phosphorylated protein kinase R like endoplasmic reticulum kinase ( p-PERK) ，eukaryotic translation initiation factor 2 α ( eIF2 α) ，phosphorylated eukaryotic translation initiation factor 2 α (p-eIF2 α) and C / EBP homologous protein ( CHOP) was detected by Western blot．HCT116 cells were divided into control group，bufalin group and combination group (bufalin + 4-phenylbutyric acid) ，and the expression of apoptosis-related proteins Bax and Bcl-2 was observed by Western blot. Results: CCK-8 assay showed that bufalin could inhibit the proliferation of HCT116 cells．Apoptosis assay showed that bufalin could induce apoptosis of HCT116 cells．The results of Western blot showed that bufalin could up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2．It could also induce ERS and activate PERK / eIF2 α/ CHOP pathway．When bufalin combined with 4-phenylbutyric acid，the apoptosis-promoting effect of bufalin was inhibited． Conclusion Bufalin can effectively inhibit the prolif- erative activity and induce apoptosis of HCT116，which is achieved to some extent by activating ERS.