The Anticancer Mechanism Of Evodiamine And Berberine On HT29 Cell

Colorectal cancer is one of the most common malignant tumors in digestive system,threatening human life and health. Along with economic development and change of lifestyle, disease incidence of colorectal cancer is generally increasing. Although manymethods such as excision, radiotherapy and chemotherapy are adopted, therapeutic effectsare not satisfactory.Recent investigations demonstrated that the telomerase activity is significantly increased inhuman malignant tumors, but is not expressed in normal somatic cells, suggesting that thereis a close relation between telomerase activity and malignant tumors. So telomerase isregarded as tumor marker and a target of tumor therapy. With the development of molecularbiology and phaneogenetics, including methylation and acetylation, close attention is paidon the occurrence and development of cancer. At present methylation is studied deeply. Agreat deal of studies indicated that there is correlation between telomerase activity andhuman telomerase reverse transcriptase (hTERT). Methylation of hTERT play a importantrole in regulating telomerase activity.In recent years, the curative effect of Chinese traditional medicine and Chinese herbs in thetreatment of colorectal cancer gave promising results with more components, morefunctions and minimal side effects. More and more scientists focus on telomerase inhibitor.In our institution, Jiaweizuojin pellet for the prevention and treatment of gastrointestinal cancerhas been studied for several years. Results showed that Jiaweizuojin pellet candecrease the occurrence of gastrointestinal cancer and berberine restrain the proliferation ofcancer cells, induce apoptosis of tumor cells, promote differentiation of tumor cells anddepress metastasis of tumor. In order to study the mechanism of Jiaweizuojin pellet restraining colorectal cancer and the role of telomerase activity in the carcinogenesis andprogression of colorectal carcinoma as well as the correlation between telomerase activityand hTERT expression, we observed the effect of Evodiamine and Berberine on telomeraseactivity and hTERT expression in HT29 cell.1. Experimental Research1.1 Establishment of cell culture methodsIn this study, a HT29 cell line was cultured in vitro. We optimized the conditions of cellularpassage, cryopreservation and resuscitation, observed their shape and state by microscope,and used WST-8 assay to measure growth curve.Results showed that HT29 cells could pass generation at a ratio of 1/2 when they covered80% of the bottom of the flask, and optimal cryopreservation concentration is 5×10~6/ml.During resuscitation, cells were removed from liquid nitrogen to a waterbath at 38 degreecentigrade and defrosted within 1～2min. Cells were inoculated at a density of 1×10~5/ml,and adhered well after 24h. The growth curve showed that cells attained exponential phasegrowth after 2 or 3 days, and were available for experimental research.1.2 The effects of Evodiamine and Berberine on growth inhibition and apoptosis ofcolorectal cancer HT29 cells.The inhibitory effect of Evodiamine and Berberine on the growth of HT29 cell wasdetected by WST-8 assay, and the best time and effecture concentration were screened.Results showed that Evodiamine at the concentration of 7.5,15,30μM and Berberine at52.5,105,210μM were available and used in subsequent study. The time chosed is 72 hours.Evodiamine and Berberine could obviously inhibit proliferation of HT29 cells, whichshowed significant concentration-effect and time-effect relationship. Phase microscopyshowed that cells became shrunkened and rounded, the cytoplasm turbid and proliferationslowing, showing that the growth is inhibited.The induction of apoptosis was measured by Annexin-FITC/PI assay. Results indicated thatEvodiamine and Berberine could induce the apoptosis of HT29 cells. Along with theincrease of drug concentration, the apoptosis rate was gradually increasing, manifested asconcentration-effect dependent. Under scanning fluorescent electron microscope, themorphologic characteristic of apoptosis cells were observed, such as chromosomeconcentrating to the sides, nuclear condensation and fragmentation, apoptotic corpuscles,etc.In the mean time, we found that Evodiamine at concentration of 15μM and Berberine at13.13,26.25,52.5μM, are not synergetic in inhibiting the proliferation of HT29 cells. 1.3 The effects of Evodiamine and Berberine on telomerase activity.Telomerase activation play a important role in the carcinogenesis and progression oftumor., which is regarded as tumor marker and a target of tumor treatment. We investigatedEvodiamine and Berberine affected telomerase activity of HT29 cells by using in situtelomeric repeat amplification protocol-PCR) assay. Results indicated telomerase activity ofHT29 cells decreased remarkably after 72h. Along with the increase of drug concentration,telomerase activity was gradually decreased. Evodiamine and Berberine could decreasedtelomerase activity individually, but then combined effect was not much stronger thaneither of them alone.1.4 The interference of Evodiamine and Berberine on expression ofhTERThTERT, component of telomerase, has a close correlation with teomerase activity. Avariety of tumor controled factors have an effect on telomerase activity by regulatinghTERT expression. Also hTERT is regarded a target of restraining telomerase activity.We used RT-PCR to detect the changes of the expression of hTERT. Results indicated thatEvodiamine and Berberine could suppress the expression of hTERTmRNA in HT29 cells.The effects were concentration-dependent. So we deduced that Evodiamine and Berberinerepressed telomerase activity of HT29 cells by decreasing the expression of hTERT.2. ConelusionsWe built up the basis for subsequent study by optimizing the conditions of celluarpassage, cryopreservation and resuscitation. Evodiamine and Berberibine could restrain theproliferation of HT29 cells, which had a correlation with cellular apoptosis induction. Theycould low telomerase activity of HT29 cells and cut down the expression of hTERT. At thesame time we found that Evodiamine and Berberibine had no synergetic effect.3. LimitationsProblems in this study are major in two aspects: one is that the experiment of methylationstatus in HT29 cells is not finished as expected. The other is that we only studied tumorinhibition of evodiamine and berberine, two monomers of Jiaweizuojin pellet, which cannotrepresent other Chinese herbal recipes.4. ProspectiveOur experiments explored the tumor inhibitory mechanism of Evodiamine and Berberineon colorectal cancer from four aspects: inhibition of tumor cell, apoptosis induction,telomerase activity and expression of hTERT. In this study, Evodiamine and Berberine haveno synergic effect on proliferation of cells and telomerase activity, which is possiblly related to other mechanism of regulating telomerase activity. Based on this study, furtherstudy is required from more profound directions.