天龙合剂及蛇莓总酚抗肿瘤作用研究

【作者】 彭博；

【导师】 刘新民； 王立为； 常琪；

【作者基本信息】 中国协和医科大学 ， 生药学， 2008， 博士

【摘要】 恶性肿瘤是严重威胁人类健康和致死率最高的疾病之一。本文选择临床治疗肿瘤有效的天龙合剂为研究对象,应用子宫颈癌HeLa、卵巢癌SKOV-3细胞模型和子宫颈癌U14移植瘤动物模型,在筛选出天龙合剂有效组分—一蛇莓总酚（DPF）的基础上,从诱导细胞凋亡、抑制细胞增殖、阻滞细胞周期和调节免疫功能等方面探讨DPF抗肿瘤作用机制。连续灌胃给予接种子宫颈癌U14细胞的荷瘤小鼠天龙合剂水提物（相当于生药90g/kg）、白英水提物（相当于生药22.5g/kg）、蛇莓水提物（相当于生药22.5g/kg）和龙葵水提物（相当于生药22.5g/kg）10天,结果表明,天龙合剂和蛇莓有明显的抑制肿瘤生长的作用,抑瘤率分别达到34.64%和40.9%,其他成分为未见显著的抗肿瘤作用。以HeLa和SKOV-3细胞为体外模型,对天龙合剂、龙葵、白英、蛇莓、郁金分离提取获得的15个提取物进行细胞毒筛选,发现天龙合剂和蛇莓提取物抑制肿瘤细胞生长的作用最明显。体内外的结果均显示蛇莓与天龙合剂的抗肿瘤作用相当,提示蛇莓可能是天龙合剂抗肿瘤作用的主要有效组分。通过体外MTT筛选,发现蛇莓抑制肿瘤增殖的有效组分是蛇莓总酚,对HeLa和SKOV-3细胞48h的IC₅₀分别达到57.53μg/mL,86.12μg/mL。选用多种组织来源的肿瘤细胞模型,应用MTT法,对DPF体外抗肿瘤作用进行研究,发现20-160μg/mL DPF能显著的抑制肿瘤细胞增殖,且呈现明显的剂量和时间依赖依赖关系。同其它组织来源的细胞相比,DPF对子宫颈癌和卵巢癌显示出较高的敏感性(72h的IC₅₀:HEC-1B＞NCI-H460＞BGC-823＞SKOV-3＞CNE＞HepG2＞C33A＞HeLa);并且DPF对正常细胞的细胞毒性较弱,其抗肿瘤作用具有相对选择性。对U14子宫颈癌移植瘤小鼠灌胃给予0.25g/kg、0.5g/kg、1g/kg DPF后,能明显延长腹水瘤动物的生存时间、增加动物的生存率;同时能显著抑制实体肿瘤的生长,不同剂量的蛇莓总酚抑瘤率分别达到35%、45%、70%。AO/EB荧光染色、DNA Ladder、流式细胞法检测发现DPF处理细胞后出现明显的凋亡形态学改变;DNA琼脂糖电泳观察到典型的梯度条带;流式细胞法DNA含量测定证实20-320μg/mL DPF作用后可以检测到明显的亚二倍体峰,且此作用具有剂量和时间效应关系。采用Western blot法、RT-PCR和分光光度法测定凋亡相关基因的表达,结果发现DPF能上调Bax表达,下调Bcl-2表达,增加Bax/Bcl-2比例,促进Bax蛋白转位至线粒体,细胞色素c释放至胞浆,从而进一步激活caspase-3,通过线粒体途径诱导子宫颈癌和卵巢癌细胞凋亡。PI染色法测定肿瘤细胞周期发现,20-320μg/mL DPF可使HeLa、C33A和SKOV-3细胞阻滞在S期,同时使细胞周期相关蛋白cyclin E、cyclin D1、cyclin A和CDK2表达降低。U14荷瘤小鼠灌胃给予0.25-1g/k DPF 20天,HE染色和TUNEL染色实验显示DPF能明显诱导小鼠子宫颈癌肿瘤组织细胞凋亡。采用Western blot法、免疫组化法和分光光度法对荷瘤动物肿瘤组织中相关蛋白进行测定发现,DPF增加Bax表达,降低Bcl-2表达,增加Bax/Bcl-2比例,激活caspase-3:并且DPF能剂量依赖性降低肿瘤组织中PCNA和ki67表达。通过MTT法、ELISA法和RIA法检测发现DPF能够提高溶血素水平;促进脾淋巴细胞增殖;降低血清TNF-α、IL-4、IL-10水平;升高血清中IL-2、IFN-γ的水平。从而提高机体细胞免疫和体液免疫功能,刺激机体Th1型细胞因子分泌,产生明显的Th1型免疫应答,逆转荷瘤动物Th1/Th2偏移。综上所述,我们通过细胞水平的药物敏感性分析和动物水平的在体实验,确定天龙合剂抗肿瘤作用的主要成分是蛇莓总酚;并从整体、细胞和分子水平对DPF的抗肿瘤作用机制进行研究,证明DPF的抗肿瘤作用是通过诱导肿瘤细胞凋亡、抑制细胞增殖、阻滞细胞周期和调节机体免疫功能等方面实现。本文的研究为天龙合剂的临床应用提供了实验依据;并且对DPF的进一步研究将有可能提供一种有效的抗肿瘤新药。更多还原

【Abstract】 Cancer is a major public health problem and one of the most lethal diseases in the world, and the incidence and mortality rates are increasing in recent years.Tianlong formula is a folk recipe with traditional reputed benefits for the treatment of gynecological cancer and hepatoma.The aim of the present research is to seek the active consituents of Tianlong and investigate the underlying mechanisms of the anticancer action of the active consituents.In in vivo study,mice were implanted cervical cancer U14 cells and administrated with the aqueous extracts of Tianlong（90 g crude drug/kg）,Duchesnea indica（22.5 g crude drug/kg）,Solanum lytatum Thumb（22.5 g crude drug/kg） and Solanum nigrum L（22.5 g crude drug/kg）.The results demonstrated that the extract of D.indica and Tianlong had similar inhibitory effect with tumor inhibition rate of 40.9%and 34.64%,respectively, other extracts did not show significantly inhibitory effect on tumor growth.And the cytotoxic activity of 15 different fractionsfrom Tianlong and its four main herbs on HeLa and SKOV-3 cells was determined using MTT assay,the fractions of Tianlong and D.indica demonstrated the most sensitive effect on cell proliferation,and the anticancer effects of Tianlong and D.indica were comparable.Based on the in vivo and in vitro findings,Duchesnea phenolic fraction（DPF） was found to be the most potent fraction in Tianlong formula,and the IC₅₀ values of DPF on HeLa and SKOV-3 cells were 57.53μg/mL and 86.12μg/mL,respectively.Cytotoxic activity of DPF on different cancer cells was determined by MTT assay.DPF at concentrations of 20-160μg/mL significantly inhibited cancer cell proliferation in a dose and time dependent manner,and showed the most sensitivity to the ovarian cancer and cervical cancer cells(IC₅₀ at 72 h were HEC-1B＞NCI-H460＞BGC-823＞SKOV-3＞CNE＞HepG2＞C33A＞HeLa).And the cytotoxic activity of DPF was characterized by tumor-selective manner,as reflected by the comparatively high IC₅₀ values on normal cells for 72 h.In mice bearing intraperitoneal U14 tumors,treatment of 0.25-1 g/kg doses of DPF significantly prolonged the survival time compared with control.In mice implanted subcutaneously cervical cancer U14 cells,administration of 0.25-1 g/kg doses of DPF markedly inhibited tumor growth in dose-dependent manner,the tumor inhibition rates of DPF were 35%,45%and 70%.Importantly,DPF showed no significant toxicity to tumor transplanted mice under this condition. AO/EB staining,DNA ladder and flow cytometry analyses revealed that DPF induced apoptosis in SKOV-3,HeLa and C33A cells evidenced by characteristic apoptotic morphological changes,nuclear DNA fragmentation and sub-G1 peak.Then the role of the molecules in apoptosis was analyzed by Western blot,RT-PCR and coloremetric assay. DPF could suppressed Bcl-2 levels,enhanced Bax levels and Bax/Bcl-2 ratio,and simultaneously translocated Bax to mitochondria followed by mitochondrial release of cyctochrome c into the cytosol and activation of effector caspase-3.Furthermore,DPF at concentrations of 20-160μg/mL arrested the cells at the S phase,with down-regulation of cyclin A,cyclin E,cyclin D1 and CDK2.Then,we use cervical cancer U14 transplanted mice to translate the in vitro findings into an in vivo animal model.The increased TUNEL-positive cells in U14 tumors of DPF-treated mice demonstrated that the anticancer effect of DPF in vivo was mediated through activation of apoptosis.Western blot and immunohistochemistry analysis showed that DPF could decrease Bcl-2 expression,increase Bax expression and Bax/Bcl-2 ratio, and accompanied by caspase-3 activation in vivo.In addition to induction of apoptosis, tumor tissues derived from DPF-treated mice showed that DPF inhibited tumor proliferation evidenced by downregulation of PCNA and ki67.Additionally,our results indicated that DPF improved function of T lymphocyte proliferation and released antibodies of B lymphocytes;decreased serum TNF-α、IL4 and IL-10;increased serum IL-2 and IFN-γ.Collectively,DPF could exert antitumor efficiency not only through reinforcing cell-mediated and humoral immune response,but also enhancing Th1-type immune response and reversing Th2 to Th1.In conclusion,we found that DPF was the active constituents of Tianlong formula.Our pronounced in vitro and in vivo studies suggested that DPF,a mixture of plant polyphenols, had potent anticancer effect and the mechanism were associated with cell cycle arrest, inhibition of cell proliferation,induction of apoptosis via mitochondrial pathway and enhancing immunological reaction.All these results provide experimental evidence to the use of Tianlong and D.indica in clinic and sustain our contention that DPF has anticancer property and merits further investigation as a potential therapeutic agent.更多还原