The Effect Of Actinidia Arguta On Human Glioma Cell And Mechanism Study

BackgroundBrain glioma accounts for 50% of brain tumors and its risk factors are unclear. The median survival time was 5 to 8 years in low grade glioma (WHO grade Ⅰ/Ⅱ). However, in high grade glioma (WHO grade III/IV, also called malignant glioma), median survival time of astrocytic glioma (WHO grade III) was 3 years and merely 12 to 18 months in grade IV glioma. Survival time of glioma patients with an age of 60 years or older was even shorter[1，2]. Presently, two kinds of theories about origin of glioma occurred. The first theory held a view that glioma cell originated from normal glial cell which could become immature glial cells when its proliferation and differentiation were out of control. The other theory which attracting more and more attention since glial stem cells were separated from the striatum of mouse by Reynolds et al in 1992, supported that glioma cell originated from neural stem cell[3] After that, a few researches found some amount of glial stem cells with a state of dormancy existed in human mature or developing central nervous tissue. These glial stem cells in dormancy have a potential of infinite proliferation, self-renewal and multidirectional differentiation, which determines the characteristics of tumors including occurrence, development, invasion, metastasis, dissemination, recurrence and sensitivity to many kinds of treatments[4]. When central nerves damaged, these dormant glial stem cells would proliferate and differentiate finitely, which could not repair the damage completely. And the specific mechanism was unclear yet[5,6]. In the animal models of brain glioma, both mature astrocyte and glial progenitor cell (precursor cell of mature astrocyte) with a treatment of specific genes could develop to be glioma.Thus, the origin of glioma was unclear[7]. Glioma formatted to be with characteristics of high incidence, recurrence and death rate and low curability[8,9]. And invasion is the characteristic of almost all kinds of glioma. Glioma cells migrated to nearby or more distant tissues following vessels and myelinated fibers path. These biological characteristics of glioma made the survival time of most part of glioma patients extremely short. Although the prognosis of glioma patients was improved greatly by microscopic surgery technology, the present operation methods could not remove all glioma tissue completely. Postoperative radiation and chemotherapy were generally accepted. However, resistance to radiation of tumor cells usually made residual lesions recur[10]. In addition, due to the poor curative effect of conventional chemotherapy drugs and experimental stage of immunotherapy and gene therapy, there is still a lack of satisfactory treatment in clinical practice. At present, the key problem in the research of glioma was how to improve the therapeutic effect of glioma, reduce the recurrence rate and prolong survival time.Traditional Chinese medicines (TCMs) play roles in the occurrence and developing of tumor with multiple targets, links and effects. They played important roles in inhibiting and killing tumor cells, improving clinical symptoms and signs, reducing radiation and chemotherapy adverse events and prolonging patients’survival time. And TCMs could regulate the body’s immune system and have a characteristic of low adverse event and drug resistance. So, antitumor TCMs researches received more and more scholars’attention[11]. In recent years, exploring the antitumor mechanism of TCMs from tumor cells proliferation inhibition and apoptosis became the research hot spot. Radix actinidiae chinensis was commonly used TCM in the treatment of colorectal cancer with an exact anticancer effect and its antitumor activity was high-profiled[12-16]. Radix actinidiae chinensis could significantly inhibit liver cancer, esophageal cancer and gastric cancer. However, researches about its roles in glioma were very few. Thus, to carry out the radix actinidiae chinensis on human brain U251 glioblastoma cell and its mechanism research would provide basis for further development and utilization of radix actinidiae chinensis in the treatment of brain glioma. Due to widely distribution, easy to grow, rich resources, low price and drug production cost, radix actinidiae chinensis would bring good social and economic benefits. So, we researched the effect of radix actinidiae chinensis on human brain U251 glioblastoma cell and its mechanism research. Our research consisted of three parts:I. The effect of radix actinidiae chinensis on human brain U251 glioblastoma cell proliferation in vivo and in vitro;Ⅱ. Effect of radix actinidiae chinensis on cells cycle in U251 and U87 cells; III. Effect of radix actinidiae chinensis on STAT3 signal pathway in U251 and U87 cells.Part Ⅰ. Effect of radix actinidiae chinensis on human U251 and U87 glioblastoma cell proliferation inhibitionObjective:to explore the effect of radix actinidiae chinensis on human U251 glioblastoma cell proliferation inhibition.Methods:(1) Observe the effect of different concentrations of radix actinidiae chinensis on human brain U251 and U87 glioblastoma cells morphological structure in culture plate under inverted microscope. And then dye U251 and U87 glioblastoma cell with Hoechst33258 fluorescent staining to observe the change of chromatin in nucleus and the phenomenon of nucleus pycnosis, cell membrane shrivel apoptotic body. (2) Detect the effect of different concentrations of radix actinidiae chinensis on human brain U251 and U87 glioblastoma cell proliferation inhibition by MTT method. (3) Detect the effect of different concentrations of radix actinidiae chinensis on human brian U251 and U87 glioblastoma cell activity after treatment of 48 hours by CCK-8 method. (4) Detect the effect of radix actinidiae chinensis on glioblastoma growth in vivo.Results:(1) Radix Actinidiae extractive could inhibit glioblastoma cell proliferationBoth in U251 and U87 glioblastoma cells, the cell proliferation rate in group of radix actinidiae chinensis is significantly lower than that in group of control.(2) The inhibition role of Radix Actinidiae extractive on proliferation of glioma cells was of concentration dependenceAfter certain time treatment of radix actinidiae chinensis, the proliferation rate of human brain glioblastoma cell siginificantly reduced. And with the increase of concentration, the proliferation rate reduced (P<0.05).(3) The inhibition role of Radix Actinidiae extractive on proliferation of glioma cells was not time-dependentAfter 24h,48h and 72h,at the the same concentration of radix actinidiae chinensis,the cell proliferation rates have no obvious change(P>0.05)(4) radix actinidiae chinensis inhibited glioblastoma growth in vivoGlioblastoma can be seen in all the ctrl groups, however, there was only one glioblastoma can be seen in radix actinidiae chinensis group. There was significant difference between ctrl group and radix actinidiae chinensis group(P<0.05).Conclusion:Radix actinidiae chinensis could significantly inhibit U251 and U87 glioblastoma cell proliferation in vitro and in vivo.Part II. Radix actinidiae chinensis regulated glioblastoma cell proliferation via regulating its cell cycleObjective:to explore the effect of radix actinidiae chinensis on human glioblastoma cell cycle.Methods:Detect the effect of different concentrations of radix actinidiae chinensis on human brian U251 glioblastoma cell cycle after treatment of 24 hours by flow cytometry method. And detect the expression level of BCL-2 and BAX by western bloting.Results:(1) effect of different concentrations of radix actinidiae chinensis on brain U251 glioblastoma cell cycle after treatment of 24 hourThe human brain U251 glioblastoma cell cycle was changed after 24 hours1 treatment of radix actinidiae chinensis. Cell cycle was arrested in G1 phase. And with the increase of concentration, the cell cycle arresting enhanced (P<0.05).(2) expression level of BCL-2 and BAX in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensisThe BCL-2 expression level decreased and BAX increased after 24 hours’ treatment of radix actinidiae chinensis. And with the increase of concentration, differences became more significant (P<0.05).(3)Regulating BCL-2 and BAX can regulate glioblastoma proliferationThe cell number increased when Flag-BCL-2 plasmid was transfected into U251 cells, and there was significant difference compared to its control (P<0.05). The cell number decreased when siBCL-2 was transfected into U251 cells and there was significant difference compared to its control (P<0.05).The cell number increased when si-BAX plasmid was transfected into U251 cells, and there was significant difference compared to its control (P<0.05). The cell number decreased when Flag-BAX was transfected into U251 cells and there was significant difference compared to its control (P<0.05).(4) radix actinidiae chinensis regulated proliferation by negatively regulating Bcl-2 and positively regulating BAX.The cell number decreased when radix actinidiae chinensis treated U251 cells and there was significant difference between ctrl group and radix actinidiae chinensis group(P<0.001), however, when we transfected BCL-2 or siBAX to U251 cells, the cell number of radix actinidiae chinensis +Flag-BCL-2 and radix actinidiae chinensis +si-BAX group was increased and there is no significant difference compared to Ctrl group(P=0.291,0.319);Conclusion:Radix actinidiae chinensis arrested human brain U251 glioblastoma cell cycle at Gl phase by decreased BCL-2 expression level and increased BAX expression level.Part Ⅲ. Radix actinidiae chinensis regulated glioblastoma cell cycle via STAT3 signal pathwayObjective:To explore the mechanism of radix actinidiae chinensis regulating human brain U251 glioblastoma cell proliferation.Methods:Detect human brain U251 glioblastoma cell STAT3 and p-STAT3 protein expression level after treatment of different concentrations of radix actinidiae chinensis by western bloting. And detect STAT3 mRNA by q-PCR.Results:(1) STAT3 and p-STAT3 protein expression level in human brain U251 glioblastoma cell after 24 hours’treatment of different concentrations of radix actinidiae chinensisSTAT3 and p-STAT3 protein expression level decreased in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensis. And with the increase of concentration, STAT3 and p-STAT3 protein expression level decreased (P<0.05).(2) STAT3 and p-STAT3 mRNA expression level in human brain U251 glioblastoma cell after 24 hours’treatment of different concentrations of radix actinidiae chinensisSTAT3 and p-STAT3 mRNA expression level decreased in human brain U251 glioblastoma cell after 24 hours’ treatment of different concentrations of radix actinidiae chinensis. And with the increase of concentration, STAT3 and p-STAT3 mRNA expression level decreased (P<0.05).(3)Regulating STAT3 signaling can regulate glioblastoma proliferationThe cell number increased when STAT3 signaling was actived by Flag-STAT3, and there was significant difference compared to its control (P<0.05); Cell number decreased when STAT3 signaling was inhibited by siSTAT3 and there was significant difference compared to their control groups(P<0.05);(4)RND3 regulated proliferation by negatively regulating Notchl signalingThe cell number decreased when radix actinidiae chinensis treated U251 cells and there was significant difference between radix actinidiae chinensis group and Ctrl group(P< 0.001), however, when we transfected Flg-STAT3 to active STAT3 signaling, the cell number of radix actinidiae chinensis +Flag-STAT3 group was increased and there is no significant difference compared to Ctrl group(P=0.191);Conclusion:Radix actinidiae chinensis regulated glioblastoma cell cycle via regulating STAT3 phosphorylation and mRNA expression, and then regulated glioblastoma cell proliferation.