| Colorectal cancer(CRC)is one of the malignant tumors that endanger human health.The incidence of CRC is increasing year by year,and it is ranked third in common malignant tumors around the world.Surgery is currently the preferred method of treatment for colorectal cancer,but chemical drugs are an important means of treating colorectal cancer.Natural-derived drugs or derivatives synthesized from natural products have achieved remarkable results in cancer treatment,which have become more and more hotspots in clinical anti-cancer drug research due to their low toxicity and multiple targets.Bursopentin and genistein are natural compounds.Bursopentin(Cys-Lys-Arg-Val-Tyr)is a new multifunctional active pentapeptide isolated from chicken bursa of Fabricius.It has been reported that BP5 can be involved in the regulation of multiple cellular signaling pathways related to anti-tumor activity.However,there is no relevant literature on the inhibition of tumor cell growth by BP5.Genistein(Gen)is widely distributed in Pueraria lobata,Sophora japonica,Broccoli,Sophora japonica and other leguminous plants.Genistein is one of the natural active ingredients of many Chinese herbal medicines.Studies have shown that Gen can induce apoptosis,inhibit proliferation and inhibit drug resistance.In this study,we first studied the effect and mechanism of BP5 on inhibiting the growth of colon cancer HCT116 cells.It was found that BP5 inhibited the growth of colon cancer HCT116 cells by inducing G1 phase cell cycle arrest,endoplasmic reticulum stress and mitochondrial caspase-dependent apoptotic pathway.On this basis,a preliminary study on the combined application of BP5 and Gen was carried out.The results showed that the two could synergistically inhibit the growth,induce apoptosis and autophagy of HCT116 cells.In addition,158 cases of colorectal cancer in Yangzhou Hospital of Traditional Chinese Medicine were collected.The clinical distribution,pathological characteristics,TCM syndromes and treatment methods of colorectal cancer were analyzed retrospectively.The results showed that there were significant differences in age,TCM syndromes and treatment methods between colon cancer and rectal cancer,but there were no significant differences in gender,pathological gross classification,histological classification,degree of tissue differentiation,clinical stage and metastasis.The research content of this topic is divided into three parts as follows.Part ? Bursopentin(BP5)induces G1 phase cell cycle arrest and endoplasmic reticulum stress/mitochondria-mediated caspase-dependent apoptosis in human colon cancer HCT116 cellsObjective:To investigate the anticancer effects of BP5 on HCT116 colon cancer cells and its possible underlying mechanisms.Methods:Cell viability was assessed using the Cell Counting Kit-8(CCK-8)assay.Cell cycle analyses and apoptosis ratios were assessed using propidium iodide(PI)and annexin V/PI staining,respectively.The underlying mechanisms were analysed by the detection of cell cycle-related and apoptosis-related proteins.Specific siRNAs targeting IRE1,ATF-6,and PERK were used for IRE1,ATF-6,and PERK knockdown,respectively.Intracellular reactive oxygen species(ROS)were determined by staining with an H2DCF-DA green fluorescence probe.Cytosolic free Ca2+ concentrations and mitochondrial membrane potentials(??m)were detected by flow cytometry by using Fluo-3 AM and JC-1 stains,respectively.Results:BP5 exerted a strong inhibitory effect on the proliferation of HCT116 cells in a dose-and time-dependent manner and induced a dose-dependent apoptosis.Mechanistically,BP5 arrested HCT116 cells at the G1 phase via the upregulation of p53 and p21 and the downregulation of cyclin E1 and CDK2.Meanwhile,BP5 treatment dramatically activated the endoplasmic reticulum(ER)stress-mediated apoptotic pathway,as revealed by the significantly enhanced expression of unfolded protein response(UPR)sensors(IRE1?,ATF6,PERK)as well as downstream signaling molecules(XBP-1s,eIF2?,ATF4 and CHOP).However,IRE1,ATF6,and PERK depletion with specific siRNAs in BP5-treated cells significantly impaired the BP5-induced expression of IRE1,ATF6,and PERK.Additionally,BP5-induced ER stress was accompanied by the accumulation of cytosolic free Ca2+and intracellular reactive oxygen species(ROS).Furthermore,BP5 treatment increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2 while reducing the mitochondrial membrane potential(??m),subsequently causing cytochrome c release from the mitochondria to cytoplasm and finally increasing the activities of caspase-9 and caspase-3.In addition,pretreatment with a pan-caspase inhibitor(z-VAD-fmk)markedly rescued the BP5-induced reduction in cell viability and reduced BP5-induced apoptosis in HCT116 cells,suggesting that BP5-induced apoptosis is associated primarily with the caspase-dependent pathway.Conclusions:Our present results suggest that BP5 has an anticancer ability to induce human colon cancer HCT116 cell growth inhibition.This ability is because BP5 can induce G1 phase cell cycle arrest and induce ER stress/mitochondria-mediated caspase-dependent apoptosis.Therefore,our findings provide insight into further investigations of the anticancer activities of BP5.Part ? Bursopentin and Genistein Act Synergistically Against Human Colon Cancer HCT116 Cells through Induction of Apoptosis and Autophagic Cell DeathObjective:To investigate the synergistic effects of a combination of BP5 and genistein against human colon cancer HCT116 cells and explore its possible mechanism.Methods:Cell survival was measured using the CCK-8 assay.Apoptosis was detected with flow cytometric analysis using Annexin V-APC/7AAD staining.Autophagy was evaluated by the Monodansylcadaverine(MDC)staining assay.Apoptosis-related proteins and autophagy-associated proteins were observed using western blot analysis.Results:Compared to BP5 or genistein treatments alone,the combination treatment of BP5(2 mM)and genistein(0.15 mM)synergistically inhibited the growth of HCT116 cells.The combination treatment induced apoptosis that was accompanied by increased expression levels of Bax,reduced expression levels of Bcl-2,activation of caspase-3 and the cleavage of PARP.Furthermore,the combination treatment synergistically induced autophagic cell death,which was accompanied by augmented-accumulation of LC3-? and reduction of p62.Conclusion:Combination of BP5 and genistein acts synergistically to suppress the growth of HCT116 cells by inducing apoptosis and autophagic cell death,illustrating the potential synergistic and combinatorial application of bioactive natural products.Part ? Retrospective analysis of 158 cases of colorectal cancerObjective:To explore the clinical distribution,pathological features,diagnosis and treatment of colorectal cancer.Methods:158 cases of colorectal cancer diagnosed by oncology department in Yangzhou Hospital of Traditional Chinese Medicine from October 2016 to October 2018 were retrospectively analyzed.The colorectal cancer was divided into two groups:colon cancer and rectal cancer.The patient's gender,age,pathological gross classification,histological classification,degree of tissue differentiation,clinical stage,TCM syndrome type,metastasis,and treatment methods were Integrated and analyzed.Results:1.There was no significant difference in gender,pathological gross classification,histological classification,histological differentiation,clinical stage and metastasis of colorectal cancer(P>0.05).The incidence of colorectal cancer was higher in males than in females,and the male proportion of colorectal cancer(62.37%)was slightly lower than that of rectal cancer(64.62%).More than 50%of the pathological types are ulcerative,80%of them are adenocarcinomas,a few are mucinous adenocarcinomas,and few are other types.More than half of them are moderately differentiated.The proportion of patients with advanced colorectal cancer is much higher than that of patients with early stage,and the proportion of simple lymph node metastasis and non-metastasis is more.2.There were significant differences in age,TCM syndromes and treatment methods of colorectal cancer(P<0.05).In terms of age,the incidence of colorectal cancer in elderly patients was higher than that in young and middle-aged patients.The middle-aged incidence of colorectal cancer(33.85%)was slightly higher than that of colon cancer(21.51%),while the incidence of colorectal cancer in young patients(1.54%)was lower than that of colon cancer(10.73%).The elderly patients with colon cancer(67.74%)were slightly higher than those with rectal cancer(64.61%).In TCM syndromes,the main type of colorectal cancer patients is phlegm-dampness stopping syndrome,followed by deficiency of temper-qi syndrome.The syndrome of Qi and blood injury in colon cancer is more than that in rectal cancer.The syndrome of liver and kidney yin deficiency,blood stasis and toxin internal knot and spleen and kidney yang deficiency are more common in rectal cancer than in rectal cancer.The treatment of colorectal cancer is mostly surgery+chemotherapy,followed by pure surgery,but the proportion of rectal cancer surgery+chemotherapy+radiotherapy is larger than that of colon cancer.Conclusions:There are significant differences in age,TCM syndromes and treatment methods between colon cancer and rectal cancer,but there are no significant differences in gender,pathological gross classification,histological classification,degree of tissue differentiation,clinical stage and metastasis. |
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