小檗碱和甲基莲心碱对人成骨肉瘤细胞的生长抑制作用及其机制研究
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摘要
成骨肉瘤是恶性骨肿瘤中最常见的一种,发病年龄多在10—25岁,严重威胁儿童和青少年的健康。成骨肉瘤对放射治疗不敏感,主要采取手术联合化疗的手段进行治疗,但是目前使用的肿瘤化疗药物存在副作用大和肿瘤细胞易产生耐药性等缺陷,因而从天然化合物中寻找低毒、高效的抗癌药物对于成骨肉瘤的治疗有重要意义。
小檗碱也叫黄连素,是一种异喹啉类生物碱,是中药黄连的主要成分。研究表明:小檗碱在体外对肝癌、肺癌、食道癌、胃癌、前列腺癌等肿瘤细胞具有明显的抑制效果。小檗碱可以导致肿瘤细胞周期停滞、诱导肿瘤细胞凋亡;并且可以抑制肿瘤的侵袭和转移,阻止肿瘤新生血管形成。小檗碱在体内也有明显的抗肿瘤作用。然而小檗碱是如何导致肿瘤细胞周期停滞并引起细胞凋亡的,有哪些上游分子和信号通路参与该过程的启动,小檗碱作用肿瘤细胞的直接靶点是什么现在还不清楚。
甲基莲心碱是从睡莲科植物莲成熟种子的绿色胚芽即莲子心中提取的一种双苄基异喹啉类生物碱。具有降压、抗心律失常、抗血小板聚集、抗氧化等药理作用。近年来研究发现甲基莲心碱对肿瘤细胞有明显的化疗增敏作用,并能克服肿瘤细胞的多耐药性。但甲基莲心碱自身对肿瘤细胞是否有抑制作用目前未见报道。
为深入研究小檗碱及甲基莲心碱的抗肿瘤作用及其分子机制,本课题进行了以下四个方面的工作:
第一部分小檗碱和甲基莲心碱对成骨肉瘤细胞和正常成骨细胞生长的抑制作用
为了检测小檗碱及甲基莲心碱对成骨肉瘤细胞和正常成骨细胞的抑制作用,我们选用了人成骨肉瘤细胞U2OS、Saos-2、HOS和正常成骨细胞HCO进行实验。通过形态学观察、四唑盐比色法(MTT)、BrdU掺入法分析小檗碱及甲基莲心碱对成骨肉瘤细胞及正常成骨细胞的抑制作用。
1、形态学观察和MTT法检测发现:
(1)小檗碱及甲基莲心碱对人成骨肉瘤细胞U2OS具有明显的抑制作用并呈剂量依赖性和时间依赖性。
(2)对比分析小檗碱和甲基莲心碱对U2OS的抑制作用发现,甲基莲心碱具有更显著的抑制作用,如药物处理24h后分析,4μmol/L甲基莲心碱处理的细胞存活率为50.6%,而135μmol/L小檗碱处理的细胞存活率为75.7%。
2、采用BrdU掺入法检测小檗碱处理后U2OS细胞的增殖情况发现,U2OS细胞中处于增殖期的细胞数目随药物浓度的升高而逐渐减少,与对照细胞相比有显著差异。该结果进一步证实了小檗碱对U2OS细胞的抑制作用。
3、MTT法检测小檗碱及甲基莲心碱对人成骨肉瘤细胞Saos-2、HOS和正常成骨细胞HCO生长的抑制作用时发现:
(1)小檗碱及甲基莲心碱对成骨肉瘤细胞均有抑制作用并呈明显的剂量依赖性和时间依赖性。
(2)小檗碱及甲基莲心碱对正常成骨细胞HCO的毒性明显低于肿瘤细胞,低浓度的甲基莲心碱(1,2,3,4μmol/L)对正常成骨细胞不仅没有抑制作用,而且能促进正常成骨细胞的存活。
以上结果表明小檗碱及甲基莲心碱对成骨肉瘤细胞的生长具有明显的抑制作用;而且对多种成骨肉瘤细胞均有抑制作用,具有普遍性;更重要的是两种生物碱对肿瘤细胞具有较好的杀伤作用,而对正常细胞毒性较小。因此深入研究两种药物的抗肿瘤机制具有重要意义。
第二部分小檗碱和甲基莲心碱对成骨肉瘤细胞和正常成骨细胞细胞周期的影响及其作用机制
导致细胞周期停滞是抗肿瘤药物抑制肿瘤细胞生长的重要机制之一。因此,我们采用流式细胞仪、Real-time PCR及Western Blotting等方法研究小檗碱和甲基莲心碱对成骨肉瘤细胞细胞周期的影响及其作用机制。
1、PI染色流式细胞仪检测细胞周期发现:
(1)U2OS(p53野生型)和正常成骨细胞HCO经小檗碱处理后G1期细胞随药物浓度升高而逐渐增多,S期细胞随药物浓度升高而逐渐减少,小檗碱使U2OS和HCO细胞停滞于G1期,研究结果还显示50μg/ml(135μmol/L)的小檗碱使U2OS细胞出现G2/M期的累积;Saos-2(p53缺陷型)细胞经小檗碱处理后G1期和S期细胞没有规律性变化,G2/M期细胞随药物浓度升高而逐渐增多,小檗碱使Saos-2停滞于G2/M期。
(2)U2OS(p53野生型)和Saos-2(p53缺陷型)细胞经甲基莲心碱处理后G1期细胞随药物浓度升高而逐渐增多,S期细胞随药物浓度升高而逐渐减少,甲基莲心碱处理后使U2OS和Saos-2细胞停滞于G1期。
2、Western Blotting和Real-time PCR方法检测两种药物处理后p53基因及与G1期停滞相关蛋白的变化发现:
(1)小檗碱处理下调U2OS细胞cyclinD1、cyclin E的表达,而上调p53、p21和p27的表达;小檗碱可以从mRNA和蛋白质两个水平来激活p53。
(2)甲基莲心碱引起U2OS细胞G1期停滞是通过上调p21的表达而下调Cyclin E的表达实现的,cyclin D1和p53的表达没有规律性改变。
3、降低U2OS细胞p53蛋白活性对小檗碱引起U2OS细胞G1期停滞的影响。
稳定转染p53突变载体降低U2OS细胞内源性p53蛋白的活性,并用流式细胞仪检测细胞周期变化发现:转染空载体的细胞随药物浓度升高G1期细胞逐渐增多,S期细胞逐渐减少;转染mtp53载体的细胞,G1期细胞逐渐增多、S期细胞逐渐减少的趋势被明显削弱。
以上结果显示,小檗碱诱导U2OS细胞G1期停滞并下调cyclin D1、cyclinE的表达,而上调p53、p21和p27的表达:小檗碱可以从mRNA和蛋白质两个水平来激活p53。p53参与了小檗碱引起的成骨肉瘤细胞的G1期停滞,G2/M期的停滞与p53无关。甲基莲心碱引起成骨肉瘤细胞G1期停滞是通过上调p21的表达而下调CyclinE的表达来实现的,与p53无关。
第三部分小檗碱和甲基莲心碱对成骨肉瘤细胞凋亡的影响
化疗药物抗肿瘤的另一重要机制是诱导肿瘤细胞凋亡。因此,本部分采用Annexin V-PI双染法、TUNEL法检测了小檗碱及甲基莲心碱处理后成骨肉瘤细胞的凋亡情况;并用Real-time PCR法检测了U2OS-neo和U2OS-mtp53细胞经小檗碱处理后p53下游与凋亡相关基因的表达情况。
1、形态学观察及TUNEL法检测小檗碱对成骨肉瘤细胞及正常成骨细胞凋亡的影响。结果显示小檗碱能够诱导成骨肉瘤细胞和正常成骨细胞发生凋亡;凋亡率随小檗碱浓度的升高而增加;相同浓度的小檗碱处理后正常细胞的凋亡率低于肿瘤细胞,p53缺陷细胞Saos-2在低浓度时凋亡率与正常细胞没有显著差异。
2、TUNEL法检测小檗碱处理U2OS-mtp53和U2OS-neo细胞的凋亡。小檗碱可以诱导U2OS-mtp53细胞及U2OS-neo细胞凋亡,U2OS-mtp53细胞的凋亡率明显低于同样浓度小檗碱处理的U2OS-neo细胞。该结果初步说明p53参与小檗碱引起的U2OS细胞凋亡。
3、Real-time PCR检测p53下游凋亡相关基因的表达。
为了进一步证明p53参与了小檗碱引起的U2OS细胞凋亡,我们用小檗碱处理U2OS-mtp53和U2OS-neo细胞,检测p53下游与凋亡相关基因mRNA的表达水平。U2OS-neo细胞经小檗碱处理后,p53下游与凋亡相关的基因BAX、PUMA和FAS的mRNA表达显著上升,并呈剂量依赖性;U2OS-mtp53细胞经小檗碱处理后,BAX、PUMA和FAS的mRNA表达水平没有明显变化。该结果进一步证明p53参与小檗碱引起的U2OS细胞凋亡。
4、Annexin V-PI双染法检测甲基莲心碱对U2OS细胞凋亡的影响。结果显示甲基莲心碱处理后细胞的凋亡率与对照组细胞相比没有明显差别,说明甲基莲心碱对U2OS细胞凋亡没有明显诱导作用。
以上结果表明小檗碱诱导成骨肉瘤细胞和正常成骨细胞凋亡,相同浓度的小檗碱引起正常细胞凋亡率低于肿瘤细胞。p53通过激活下游与凋亡相关基因BAX、PUMA和FAS的表达而参与小檗碱诱导的U2OS细胞凋亡。诱导凋亡不是甲基莲心碱抑制成骨肉瘤细胞生长的机制。
第四部分初步探讨小檗碱引起肿瘤细胞周期停滞和细胞凋亡的上游因素
前期实验结果证实,p53在小檗碱抑制成骨肉瘤细胞生长过程中发挥了重要作用,参与了小檗碱引起的细胞周期停滞和细胞凋亡。小檗碱升高p53的磷酸化水平并上调p53 mRNA表达水平。那么p53又是怎样被激活的?我们推测DNA损伤可能是小檗碱激活p53并引起细胞周期停滞和细胞凋亡的上游因素。本部分采用免疫荧光结合Western Blotting方法检测小檗碱处理U2OS细胞后γ-H2AX的累积;并用微核率检测U2OS细胞基因组的不稳定性。结果如下:
1、免疫荧光和Western Blotting检测小檗碱处理U2OS细胞后γ-H2AX的累积。免疫荧光结果显示小檗碱可以导致γ-H2AX在细胞内累积,随药物浓度的升高γ-H2AX阳性细胞增多,焦点更多更亮。Western Blotting结果显示,随小檗碱浓度的升高,磷酸化H2AX的表达水平逐渐增强。
2、相同浓度的小檗碱引起正常成骨细胞HCO中γ-H2AX累积要低于U2OS细胞。相同浓度的小檗碱处理后在U2OS细胞中的荧光累积要比HCO细胞强,说明与HCO细胞相比小檗碱更容易进入U2OS细胞。
3、微核计数发现,小檗碱处理后随药物浓度的升高,U2OS细胞微核率与对照细胞相比有显著上升趋势。
4、免疫荧光检测甲基莲心碱处理U2OS细胞γ-H2AX的累积。甲基莲心碱处理U2OS细胞未发现典型的γ-H2AX焦点出现,说明甲基莲心碱不能导致γ-H2AX在细胞内的累积。
以上结果表明,小檗碱处理U2OS细胞引起γ-H2AX的累积和微核率的上升,说明导致DNA损伤是小檗碱激活成骨肉细胞p53并引起细胞周期停滞和细胞凋亡的始动因素。甲基莲心碱不能导致U2OS细胞γ-H2AX的累积。甲基莲心碱抑制成骨肉瘤细胞生长的机制与小檗碱是不同的。
Osteosarcoma is the most common type of bone cancer in children and adolescents,although cancer in young people is rare in general.Most osteosarcomas develop between the ages of 10 and 25 when the bones are rapidly growing.Since osteosarcomas are usually resistant to ionizing radiation,its treatment primarily relies on the combination of chemotherapy and surgical removal of the tumor. Chemotherapy plays a very important role in cancer treatment.However,multidrug resistance and toxicity greatly limit its application.Great efforts have been devoted to the search for new cancer chemopreventive agents from natural products.
Berberine,an isoquinoline alkaloid component in several Chinese herbs including Huanglian,has been shown to have antimicrobial,anti-inflammatory,anti-diabetic and anti-angiogenesis and cholesterol-lowering effects.A large number of studies also showed that berberine possesses anti-tumor activity,against cancer cells established from cervical,esophageal,oral,colonic,prostate cancers,leukemia,melanoma and glioblastoma.Many studies showed that berberine inhibits tumor cell growth by inducing cell cycle arrest and/or apoptosis.Some in vitro studies showed that berberine could inhibit tumor cell adhesion,migration and invasion.Recent studies also showed the anti-tumor activity of berberine in vivo.However,there are many unanswered questions such as follows.How does berberine initiate the cascade that eventually leads to cell cycle arrest and/or apoptosis? Why does berberine induce some cancer cells arrest at G1 phase and other cancer cells at G2/M phase? What is the direct target of berberine in tumor cells?
Neferine(Nef) is a bis-benzylisoquinoline alkaloid isolated from the green seed embryo of Nelumbo nucifera Gaertn.Neferine has been shown to have anti-inflammation,anti-oxidant,anti-hypertensive,anti-arrhythmic anti-platelet aggregation effects.Recently,neferine has been found to be a potent agent for reversing Adriamycin resistance of tumor cells in vitro.Neferine can also enhance the anti-tumor effect of adriamycin on osteosarcoma cells in vitro.However,it is not known whether neferine has direct anti-tumor effect.
We evaluated the anti-tumor effects of berberine and neferine on osteosarcoma cells and characterized the mechanisms underlying the anti-tumor effect of these two natural products.
PART ONE The inhibitory effects of berberine and neferine on the proliferation of human osteosarcoma cells and normal osteoblast cells.
We analyzed the inhibitory effects of berberine and neferine on osteosarcoma cell lines U2OS,Saos-2,HOS and normal osteoblast HCO by morphological observation, MTT and BrdU assays.
1.Analysis of the growth-inhibitory effects of berberine and neferine on U2OS cells by MTT assay.
(1) Berberine and neferine significantly inhibited growth of U2OS cells in a time- and dose-dependent manner.
(2) Neferine showed a more potent inhibitory effect than berberine.Such as the viability of U2OS cells after 24 hours was 50.6%with 4μmol/L neferine and 75.7%with 135μmol/L berberine.
2.Berberine reduced the proportions of cells in S phase in a dose-dependent manner,as measured by BrdU incorporation assay..
3.Inhibitory effects of berberine and neferine on additional human osteosarcoma cells and normal osteoblast cells by MTT assay.
(1) Both berberine and neferine exhibited significant inhibitory effects in a time- and dose- dependent manner on osteosarcoma cell lines.
(2) Normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells to berberine and neferine.Neferine promoted HCO cell survival atlowdose(1,2,3,4μmol/L).
The results in this part suggested that berberine and neferine exhibited a dose-dependent growth-inhibitory on osteosarcoma cells.However,normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells.Neferine showed a more potent inhibitory effect than berberine.
PART TWO Cell cycle analysis of human osteosarcoma cells in response to berberine and neferine
Induction of cell cycle arrest is one of the most important mechanisms of antitumor drugs.In this part,we studied the effect ofberberine and neferine on cell cycle progression and the underlying mechanism by flow cytometry,Real-time PCR and Western Blottinging.
1.U2OS,Saos-2 and HCO cells were analyzed for their cell cycle distribution by flow eytometry treated with berberine and neferine.
(1) In U2OS and HCO cells(p53 wild type) treated with berberine,the percentages of cells in G1 phase were increased at the expense of cells in S phase.While there was no change in the percentage of cells in G2/M at lower doses,the percentage of cells at G2/M was markedly increased at the highest dose tested,50μg/ml.The p53-deficient Saos-2 cells preceded into S phase in the presence of berberine.However,a great proportion of cells were arrested in G2/M.These results suggest that berberine arrests cells at G1 and/or at G2/M,depending on the concentration of berberine and on the cells it is applied to.
(2) In U2OS(p53 wild type) and Saos-2(p53-deficient) cells treated with neferine,the percentages of both two cell lines in G1 phase were increased in dose dependent manner at the expense of cells in S phase.
2.Responses of p53 and cell cycle effectors/regulators to berberine and neferine treatment.
(1) Berberine treatment resulted in a marked decrease in the expression of cyclin E in a dose-dependent manner.The expression level of cyclin D1 decreased only at the high concentration(50μg/ml).Berberine treatment resulted in a dose-dependent enhancement of p53 in protein and mRNA level.The level of phosphorylated p53 at serine 15 was also elevated in a dose-dependent manner.Berberine treatment resulted in a dose-dependent enhancement of p21WAF1/CIP1 and p27KIP1 protein level compared with control cells.
(2) Neferine caused G1 cycle arrest accompanied by an up regulation of p21WAF1/CIP1 and a down regulation of Cyclin E.There were no obvious changes of cyclin D1 and p53.Importantly,we observed that the up regulation of p21WAF1/CIP1 by neferine is independent on functional p53.
3.Cell cycle arrest at G1,but not G2/M,depends on functional p53 treated with berberine.Berberine treatment of U2OS-neo cells showed an accumulation at the G1 phase.U2OS-mtp53 cells,however,experienced no obvious changes. In contrast to the berberine-induced reduction of S phase cells in U2OS-neo, the percentage of U2OS-mtp53 cells at S phase was not significantly decreased upon berberine treatment.
The results above suggested that berberine induced U2OS cells arrest at G1 phase through down regulation of Cyclin E,cyclin D1 and up regulation of p21WAF1/CIP1 and p27KIP1.Cell cycle arrest at G1 in U2OS cells,but not G2/M,depends on functional p53.Neferine induced G1 arrest both in U2OS and Saos-2 cells.Neferine caused p53 independent G1 cycle arrest which was accompanied by up regulation of p21WAF 1/CIP1 and down regulation of Cyclin E in U2OS cells.
PART THREE Induction of apoptosis in osteosarcoma cells by berberine and neferine
In addition to cell cycle arrest,apoptosis has also been reported to be responsible for inhibition of tumor cell proliferation.In this part,we studied the effect of berberine and neferine on human osteosarcoma and normal osteoblast cells by Annexin V-PI and TUNEL assay.And we also analyzed the mRNA levels of p53 target genes involved in apoptosis in U2OS-neo and U2OS-mtp53 cells.
1.U2OS,HOS,Saos-2 and HCO cells were analyzed by TUNEL assay.After berberine treatment for 48h,the percentage of TUNEL-positive cells was increased in a dose-dependent manner in all the cell lines tested.Interestingly, the induction of apoptosis was less pronounced in the non-neoplastic cells HCO.There was no obvious difference of apoptotic cells between HCO and Saos-2 at low dose.
2.The induction of apoptosis by berberine was more dramatic in U2OS-neo cells than in U2OS-mtp53 cells,suggesting that berberine-induced apoptosis was also dependent on the function of p53 in osteosarcoma cells.
3.To gain further insights into the mechanism by which the activation of p53 induces apoptosis,we analyzed the mRNA levels of p53 target genes involved in apoptosis.The mRNA levels of pro-apoptotic genes BAX,PUMA and FAS were all increased in U2OS-neo cells after berberine treatment.Similarly treated U2OS-mtp53 cells,on the other hand,showed no significant increase.
4.When we used Annexin V-PI assay to test apoptotic cells in U2OS cells treated with neferine.We didn't observe an increase in the percentage of apoptotic cells in neferine treated cells compared to control cells.
These results indicated that berberine induced apoptosis both in osteosarcoma and normal osteoblast cells.The induction of apoptosis was less pronounced in the non-neoplastic cells than neoplastic cells.Berberine induced apoptosis by p53 dependent and p53-independent pathways.Neferine didn't cause apoptosis in U2OS cells.
PART FOUR Investigation of upstream factors of cell cycle arrest and apoptosis in osteosarcoma cells treated with berberine
The results in the previous two parts showed that p53 played an important role in the inhibitory effect of berberine on human osteosarcoma cells.Berberine up regulated p53 both at mRNA and protein level.The level of phosphorylated p53 was also elevated in a dose- dependent manner.We speculated that DNA damage might be the upstream factor that activated p53 and caused cell cycle arrest and apoptosis.In this part,western blotting and immunofluorescence staining ofγ-H2AX were performed on berberine-treated osteosarcoma cells.In addition,micronucleus assay was used to evaluate whether berberine can cause the genomic instability.
1.Phosphorylation of H2AX was determined by immunofluorescence staining and western blotting in berberine treated U2OS cells.Compared to untreated cells,berberine treatment led to an increased phosphorylation of H2AX in a dose-dependent manner.Foci ofγ-H2AX were abundant in the nuclei of most cells exposed to berberine.
2.The foci ofγ-H2AX were less abundant in HCO cells than in U2OS cells. Berberine was accumulated to a less extent in HCO cells than in U2OS cells, as reflected by the fluorescence intensity in the nuclei.
3.The frequency of U2OS cells containing micronuclei treated with berberine was increased in a dose-dependent manner.
4.Neferine treatment induced no detectable phosphorylation of H2AX as determined by immunofluorescence staining.
These results suggest that berberine causes DNA damage in U2OS cells.The accumulation ofγ-H2AX in nuclei and the formation of micronuclei of U2OS cells indicate that berberine can inflict genomic lesions.However,unlike berberine, neferine did not cause DNA damage in U2OS cells.Thus,the mechanisms underlying their inhibitory effect on human osteosarcoma cells differ greatly between berberine and neferine.
小檗碱也叫黄连素,是一种异喹啉类生物碱,是中药黄连的主要成分。研究表明:小檗碱在体外对肝癌、肺癌、食道癌、胃癌、前列腺癌等肿瘤细胞具有明显的抑制效果。小檗碱可以导致肿瘤细胞周期停滞、诱导肿瘤细胞凋亡;并且可以抑制肿瘤的侵袭和转移,阻止肿瘤新生血管形成。小檗碱在体内也有明显的抗肿瘤作用。然而小檗碱是如何导致肿瘤细胞周期停滞并引起细胞凋亡的,有哪些上游分子和信号通路参与该过程的启动,小檗碱作用肿瘤细胞的直接靶点是什么现在还不清楚。
甲基莲心碱是从睡莲科植物莲成熟种子的绿色胚芽即莲子心中提取的一种双苄基异喹啉类生物碱。具有降压、抗心律失常、抗血小板聚集、抗氧化等药理作用。近年来研究发现甲基莲心碱对肿瘤细胞有明显的化疗增敏作用,并能克服肿瘤细胞的多耐药性。但甲基莲心碱自身对肿瘤细胞是否有抑制作用目前未见报道。
为深入研究小檗碱及甲基莲心碱的抗肿瘤作用及其分子机制,本课题进行了以下四个方面的工作:
第一部分小檗碱和甲基莲心碱对成骨肉瘤细胞和正常成骨细胞生长的抑制作用
为了检测小檗碱及甲基莲心碱对成骨肉瘤细胞和正常成骨细胞的抑制作用,我们选用了人成骨肉瘤细胞U2OS、Saos-2、HOS和正常成骨细胞HCO进行实验。通过形态学观察、四唑盐比色法(MTT)、BrdU掺入法分析小檗碱及甲基莲心碱对成骨肉瘤细胞及正常成骨细胞的抑制作用。
1、形态学观察和MTT法检测发现:
(1)小檗碱及甲基莲心碱对人成骨肉瘤细胞U2OS具有明显的抑制作用并呈剂量依赖性和时间依赖性。
(2)对比分析小檗碱和甲基莲心碱对U2OS的抑制作用发现,甲基莲心碱具有更显著的抑制作用,如药物处理24h后分析,4μmol/L甲基莲心碱处理的细胞存活率为50.6%,而135μmol/L小檗碱处理的细胞存活率为75.7%。
2、采用BrdU掺入法检测小檗碱处理后U2OS细胞的增殖情况发现,U2OS细胞中处于增殖期的细胞数目随药物浓度的升高而逐渐减少,与对照细胞相比有显著差异。该结果进一步证实了小檗碱对U2OS细胞的抑制作用。
3、MTT法检测小檗碱及甲基莲心碱对人成骨肉瘤细胞Saos-2、HOS和正常成骨细胞HCO生长的抑制作用时发现:
(1)小檗碱及甲基莲心碱对成骨肉瘤细胞均有抑制作用并呈明显的剂量依赖性和时间依赖性。
(2)小檗碱及甲基莲心碱对正常成骨细胞HCO的毒性明显低于肿瘤细胞,低浓度的甲基莲心碱(1,2,3,4μmol/L)对正常成骨细胞不仅没有抑制作用,而且能促进正常成骨细胞的存活。
以上结果表明小檗碱及甲基莲心碱对成骨肉瘤细胞的生长具有明显的抑制作用;而且对多种成骨肉瘤细胞均有抑制作用,具有普遍性;更重要的是两种生物碱对肿瘤细胞具有较好的杀伤作用,而对正常细胞毒性较小。因此深入研究两种药物的抗肿瘤机制具有重要意义。
第二部分小檗碱和甲基莲心碱对成骨肉瘤细胞和正常成骨细胞细胞周期的影响及其作用机制
导致细胞周期停滞是抗肿瘤药物抑制肿瘤细胞生长的重要机制之一。因此,我们采用流式细胞仪、Real-time PCR及Western Blotting等方法研究小檗碱和甲基莲心碱对成骨肉瘤细胞细胞周期的影响及其作用机制。
1、PI染色流式细胞仪检测细胞周期发现:
(1)U2OS(p53野生型)和正常成骨细胞HCO经小檗碱处理后G1期细胞随药物浓度升高而逐渐增多,S期细胞随药物浓度升高而逐渐减少,小檗碱使U2OS和HCO细胞停滞于G1期,研究结果还显示50μg/ml(135μmol/L)的小檗碱使U2OS细胞出现G2/M期的累积;Saos-2(p53缺陷型)细胞经小檗碱处理后G1期和S期细胞没有规律性变化,G2/M期细胞随药物浓度升高而逐渐增多,小檗碱使Saos-2停滞于G2/M期。
(2)U2OS(p53野生型)和Saos-2(p53缺陷型)细胞经甲基莲心碱处理后G1期细胞随药物浓度升高而逐渐增多,S期细胞随药物浓度升高而逐渐减少,甲基莲心碱处理后使U2OS和Saos-2细胞停滞于G1期。
2、Western Blotting和Real-time PCR方法检测两种药物处理后p53基因及与G1期停滞相关蛋白的变化发现:
(1)小檗碱处理下调U2OS细胞cyclinD1、cyclin E的表达,而上调p53、p21和p27的表达;小檗碱可以从mRNA和蛋白质两个水平来激活p53。
(2)甲基莲心碱引起U2OS细胞G1期停滞是通过上调p21的表达而下调Cyclin E的表达实现的,cyclin D1和p53的表达没有规律性改变。
3、降低U2OS细胞p53蛋白活性对小檗碱引起U2OS细胞G1期停滞的影响。
稳定转染p53突变载体降低U2OS细胞内源性p53蛋白的活性,并用流式细胞仪检测细胞周期变化发现:转染空载体的细胞随药物浓度升高G1期细胞逐渐增多,S期细胞逐渐减少;转染mtp53载体的细胞,G1期细胞逐渐增多、S期细胞逐渐减少的趋势被明显削弱。
以上结果显示,小檗碱诱导U2OS细胞G1期停滞并下调cyclin D1、cyclinE的表达,而上调p53、p21和p27的表达:小檗碱可以从mRNA和蛋白质两个水平来激活p53。p53参与了小檗碱引起的成骨肉瘤细胞的G1期停滞,G2/M期的停滞与p53无关。甲基莲心碱引起成骨肉瘤细胞G1期停滞是通过上调p21的表达而下调CyclinE的表达来实现的,与p53无关。
第三部分小檗碱和甲基莲心碱对成骨肉瘤细胞凋亡的影响
化疗药物抗肿瘤的另一重要机制是诱导肿瘤细胞凋亡。因此,本部分采用Annexin V-PI双染法、TUNEL法检测了小檗碱及甲基莲心碱处理后成骨肉瘤细胞的凋亡情况;并用Real-time PCR法检测了U2OS-neo和U2OS-mtp53细胞经小檗碱处理后p53下游与凋亡相关基因的表达情况。
1、形态学观察及TUNEL法检测小檗碱对成骨肉瘤细胞及正常成骨细胞凋亡的影响。结果显示小檗碱能够诱导成骨肉瘤细胞和正常成骨细胞发生凋亡;凋亡率随小檗碱浓度的升高而增加;相同浓度的小檗碱处理后正常细胞的凋亡率低于肿瘤细胞,p53缺陷细胞Saos-2在低浓度时凋亡率与正常细胞没有显著差异。
2、TUNEL法检测小檗碱处理U2OS-mtp53和U2OS-neo细胞的凋亡。小檗碱可以诱导U2OS-mtp53细胞及U2OS-neo细胞凋亡,U2OS-mtp53细胞的凋亡率明显低于同样浓度小檗碱处理的U2OS-neo细胞。该结果初步说明p53参与小檗碱引起的U2OS细胞凋亡。
3、Real-time PCR检测p53下游凋亡相关基因的表达。
为了进一步证明p53参与了小檗碱引起的U2OS细胞凋亡,我们用小檗碱处理U2OS-mtp53和U2OS-neo细胞,检测p53下游与凋亡相关基因mRNA的表达水平。U2OS-neo细胞经小檗碱处理后,p53下游与凋亡相关的基因BAX、PUMA和FAS的mRNA表达显著上升,并呈剂量依赖性;U2OS-mtp53细胞经小檗碱处理后,BAX、PUMA和FAS的mRNA表达水平没有明显变化。该结果进一步证明p53参与小檗碱引起的U2OS细胞凋亡。
4、Annexin V-PI双染法检测甲基莲心碱对U2OS细胞凋亡的影响。结果显示甲基莲心碱处理后细胞的凋亡率与对照组细胞相比没有明显差别,说明甲基莲心碱对U2OS细胞凋亡没有明显诱导作用。
以上结果表明小檗碱诱导成骨肉瘤细胞和正常成骨细胞凋亡,相同浓度的小檗碱引起正常细胞凋亡率低于肿瘤细胞。p53通过激活下游与凋亡相关基因BAX、PUMA和FAS的表达而参与小檗碱诱导的U2OS细胞凋亡。诱导凋亡不是甲基莲心碱抑制成骨肉瘤细胞生长的机制。
第四部分初步探讨小檗碱引起肿瘤细胞周期停滞和细胞凋亡的上游因素
前期实验结果证实,p53在小檗碱抑制成骨肉瘤细胞生长过程中发挥了重要作用,参与了小檗碱引起的细胞周期停滞和细胞凋亡。小檗碱升高p53的磷酸化水平并上调p53 mRNA表达水平。那么p53又是怎样被激活的?我们推测DNA损伤可能是小檗碱激活p53并引起细胞周期停滞和细胞凋亡的上游因素。本部分采用免疫荧光结合Western Blotting方法检测小檗碱处理U2OS细胞后γ-H2AX的累积;并用微核率检测U2OS细胞基因组的不稳定性。结果如下:
1、免疫荧光和Western Blotting检测小檗碱处理U2OS细胞后γ-H2AX的累积。免疫荧光结果显示小檗碱可以导致γ-H2AX在细胞内累积,随药物浓度的升高γ-H2AX阳性细胞增多,焦点更多更亮。Western Blotting结果显示,随小檗碱浓度的升高,磷酸化H2AX的表达水平逐渐增强。
2、相同浓度的小檗碱引起正常成骨细胞HCO中γ-H2AX累积要低于U2OS细胞。相同浓度的小檗碱处理后在U2OS细胞中的荧光累积要比HCO细胞强,说明与HCO细胞相比小檗碱更容易进入U2OS细胞。
3、微核计数发现,小檗碱处理后随药物浓度的升高,U2OS细胞微核率与对照细胞相比有显著上升趋势。
4、免疫荧光检测甲基莲心碱处理U2OS细胞γ-H2AX的累积。甲基莲心碱处理U2OS细胞未发现典型的γ-H2AX焦点出现,说明甲基莲心碱不能导致γ-H2AX在细胞内的累积。
以上结果表明,小檗碱处理U2OS细胞引起γ-H2AX的累积和微核率的上升,说明导致DNA损伤是小檗碱激活成骨肉细胞p53并引起细胞周期停滞和细胞凋亡的始动因素。甲基莲心碱不能导致U2OS细胞γ-H2AX的累积。甲基莲心碱抑制成骨肉瘤细胞生长的机制与小檗碱是不同的。
Osteosarcoma is the most common type of bone cancer in children and adolescents,although cancer in young people is rare in general.Most osteosarcomas develop between the ages of 10 and 25 when the bones are rapidly growing.Since osteosarcomas are usually resistant to ionizing radiation,its treatment primarily relies on the combination of chemotherapy and surgical removal of the tumor. Chemotherapy plays a very important role in cancer treatment.However,multidrug resistance and toxicity greatly limit its application.Great efforts have been devoted to the search for new cancer chemopreventive agents from natural products.
Berberine,an isoquinoline alkaloid component in several Chinese herbs including Huanglian,has been shown to have antimicrobial,anti-inflammatory,anti-diabetic and anti-angiogenesis and cholesterol-lowering effects.A large number of studies also showed that berberine possesses anti-tumor activity,against cancer cells established from cervical,esophageal,oral,colonic,prostate cancers,leukemia,melanoma and glioblastoma.Many studies showed that berberine inhibits tumor cell growth by inducing cell cycle arrest and/or apoptosis.Some in vitro studies showed that berberine could inhibit tumor cell adhesion,migration and invasion.Recent studies also showed the anti-tumor activity of berberine in vivo.However,there are many unanswered questions such as follows.How does berberine initiate the cascade that eventually leads to cell cycle arrest and/or apoptosis? Why does berberine induce some cancer cells arrest at G1 phase and other cancer cells at G2/M phase? What is the direct target of berberine in tumor cells?
Neferine(Nef) is a bis-benzylisoquinoline alkaloid isolated from the green seed embryo of Nelumbo nucifera Gaertn.Neferine has been shown to have anti-inflammation,anti-oxidant,anti-hypertensive,anti-arrhythmic anti-platelet aggregation effects.Recently,neferine has been found to be a potent agent for reversing Adriamycin resistance of tumor cells in vitro.Neferine can also enhance the anti-tumor effect of adriamycin on osteosarcoma cells in vitro.However,it is not known whether neferine has direct anti-tumor effect.
We evaluated the anti-tumor effects of berberine and neferine on osteosarcoma cells and characterized the mechanisms underlying the anti-tumor effect of these two natural products.
PART ONE The inhibitory effects of berberine and neferine on the proliferation of human osteosarcoma cells and normal osteoblast cells.
We analyzed the inhibitory effects of berberine and neferine on osteosarcoma cell lines U2OS,Saos-2,HOS and normal osteoblast HCO by morphological observation, MTT and BrdU assays.
1.Analysis of the growth-inhibitory effects of berberine and neferine on U2OS cells by MTT assay.
(1) Berberine and neferine significantly inhibited growth of U2OS cells in a time- and dose-dependent manner.
(2) Neferine showed a more potent inhibitory effect than berberine.Such as the viability of U2OS cells after 24 hours was 50.6%with 4μmol/L neferine and 75.7%with 135μmol/L berberine.
2.Berberine reduced the proportions of cells in S phase in a dose-dependent manner,as measured by BrdU incorporation assay..
3.Inhibitory effects of berberine and neferine on additional human osteosarcoma cells and normal osteoblast cells by MTT assay.
(1) Both berberine and neferine exhibited significant inhibitory effects in a time- and dose- dependent manner on osteosarcoma cell lines.
(2) Normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells to berberine and neferine.Neferine promoted HCO cell survival atlowdose(1,2,3,4μmol/L).
The results in this part suggested that berberine and neferine exhibited a dose-dependent growth-inhibitory on osteosarcoma cells.However,normal osteoblast cells HCO appeared to be more resistant than the osteosarcoma cells.Neferine showed a more potent inhibitory effect than berberine.
PART TWO Cell cycle analysis of human osteosarcoma cells in response to berberine and neferine
Induction of cell cycle arrest is one of the most important mechanisms of antitumor drugs.In this part,we studied the effect ofberberine and neferine on cell cycle progression and the underlying mechanism by flow cytometry,Real-time PCR and Western Blottinging.
1.U2OS,Saos-2 and HCO cells were analyzed for their cell cycle distribution by flow eytometry treated with berberine and neferine.
(1) In U2OS and HCO cells(p53 wild type) treated with berberine,the percentages of cells in G1 phase were increased at the expense of cells in S phase.While there was no change in the percentage of cells in G2/M at lower doses,the percentage of cells at G2/M was markedly increased at the highest dose tested,50μg/ml.The p53-deficient Saos-2 cells preceded into S phase in the presence of berberine.However,a great proportion of cells were arrested in G2/M.These results suggest that berberine arrests cells at G1 and/or at G2/M,depending on the concentration of berberine and on the cells it is applied to.
(2) In U2OS(p53 wild type) and Saos-2(p53-deficient) cells treated with neferine,the percentages of both two cell lines in G1 phase were increased in dose dependent manner at the expense of cells in S phase.
2.Responses of p53 and cell cycle effectors/regulators to berberine and neferine treatment.
(1) Berberine treatment resulted in a marked decrease in the expression of cyclin E in a dose-dependent manner.The expression level of cyclin D1 decreased only at the high concentration(50μg/ml).Berberine treatment resulted in a dose-dependent enhancement of p53 in protein and mRNA level.The level of phosphorylated p53 at serine 15 was also elevated in a dose-dependent manner.Berberine treatment resulted in a dose-dependent enhancement of p21WAF1/CIP1 and p27KIP1 protein level compared with control cells.
(2) Neferine caused G1 cycle arrest accompanied by an up regulation of p21WAF1/CIP1 and a down regulation of Cyclin E.There were no obvious changes of cyclin D1 and p53.Importantly,we observed that the up regulation of p21WAF1/CIP1 by neferine is independent on functional p53.
3.Cell cycle arrest at G1,but not G2/M,depends on functional p53 treated with berberine.Berberine treatment of U2OS-neo cells showed an accumulation at the G1 phase.U2OS-mtp53 cells,however,experienced no obvious changes. In contrast to the berberine-induced reduction of S phase cells in U2OS-neo, the percentage of U2OS-mtp53 cells at S phase was not significantly decreased upon berberine treatment.
The results above suggested that berberine induced U2OS cells arrest at G1 phase through down regulation of Cyclin E,cyclin D1 and up regulation of p21WAF1/CIP1 and p27KIP1.Cell cycle arrest at G1 in U2OS cells,but not G2/M,depends on functional p53.Neferine induced G1 arrest both in U2OS and Saos-2 cells.Neferine caused p53 independent G1 cycle arrest which was accompanied by up regulation of p21WAF 1/CIP1 and down regulation of Cyclin E in U2OS cells.
PART THREE Induction of apoptosis in osteosarcoma cells by berberine and neferine
In addition to cell cycle arrest,apoptosis has also been reported to be responsible for inhibition of tumor cell proliferation.In this part,we studied the effect of berberine and neferine on human osteosarcoma and normal osteoblast cells by Annexin V-PI and TUNEL assay.And we also analyzed the mRNA levels of p53 target genes involved in apoptosis in U2OS-neo and U2OS-mtp53 cells.
1.U2OS,HOS,Saos-2 and HCO cells were analyzed by TUNEL assay.After berberine treatment for 48h,the percentage of TUNEL-positive cells was increased in a dose-dependent manner in all the cell lines tested.Interestingly, the induction of apoptosis was less pronounced in the non-neoplastic cells HCO.There was no obvious difference of apoptotic cells between HCO and Saos-2 at low dose.
2.The induction of apoptosis by berberine was more dramatic in U2OS-neo cells than in U2OS-mtp53 cells,suggesting that berberine-induced apoptosis was also dependent on the function of p53 in osteosarcoma cells.
3.To gain further insights into the mechanism by which the activation of p53 induces apoptosis,we analyzed the mRNA levels of p53 target genes involved in apoptosis.The mRNA levels of pro-apoptotic genes BAX,PUMA and FAS were all increased in U2OS-neo cells after berberine treatment.Similarly treated U2OS-mtp53 cells,on the other hand,showed no significant increase.
4.When we used Annexin V-PI assay to test apoptotic cells in U2OS cells treated with neferine.We didn't observe an increase in the percentage of apoptotic cells in neferine treated cells compared to control cells.
These results indicated that berberine induced apoptosis both in osteosarcoma and normal osteoblast cells.The induction of apoptosis was less pronounced in the non-neoplastic cells than neoplastic cells.Berberine induced apoptosis by p53 dependent and p53-independent pathways.Neferine didn't cause apoptosis in U2OS cells.
PART FOUR Investigation of upstream factors of cell cycle arrest and apoptosis in osteosarcoma cells treated with berberine
The results in the previous two parts showed that p53 played an important role in the inhibitory effect of berberine on human osteosarcoma cells.Berberine up regulated p53 both at mRNA and protein level.The level of phosphorylated p53 was also elevated in a dose- dependent manner.We speculated that DNA damage might be the upstream factor that activated p53 and caused cell cycle arrest and apoptosis.In this part,western blotting and immunofluorescence staining ofγ-H2AX were performed on berberine-treated osteosarcoma cells.In addition,micronucleus assay was used to evaluate whether berberine can cause the genomic instability.
1.Phosphorylation of H2AX was determined by immunofluorescence staining and western blotting in berberine treated U2OS cells.Compared to untreated cells,berberine treatment led to an increased phosphorylation of H2AX in a dose-dependent manner.Foci ofγ-H2AX were abundant in the nuclei of most cells exposed to berberine.
2.The foci ofγ-H2AX were less abundant in HCO cells than in U2OS cells. Berberine was accumulated to a less extent in HCO cells than in U2OS cells, as reflected by the fluorescence intensity in the nuclei.
3.The frequency of U2OS cells containing micronuclei treated with berberine was increased in a dose-dependent manner.
4.Neferine treatment induced no detectable phosphorylation of H2AX as determined by immunofluorescence staining.
These results suggest that berberine causes DNA damage in U2OS cells.The accumulation ofγ-H2AX in nuclei and the formation of micronuclei of U2OS cells indicate that berberine can inflict genomic lesions.However,unlike berberine, neferine did not cause DNA damage in U2OS cells.Thus,the mechanisms underlying their inhibitory effect on human osteosarcoma cells differ greatly between berberine and neferine.
引文
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