败酱有效部位PHEB抗肿瘤作用及其作用机理的研究
摘要
目的
①从败酱中分离纯化得到抗肿瘤的有效部位PHEB,并明确其成分组成。
②通过体内外抗肿瘤实验,明确PHEB的抗肿瘤作用。
③考察PHEB诱导肿瘤细胞凋亡,探讨其抗肿瘤作用的机理。
方法
①采用硅胶柱层析分离制备PHEB,并采用薄层层析(TLC)和高效液相色谱法(HPLC)分析PHEB的化学组成。②采用MTT比色法,结合生长曲线以及集落形成实验考察PHEB对MCF-7、COLO-205、PC-3、SGC-7901、HepG2、C6、NB4和B16肿瘤细胞的体外增殖抑制能力。③采用S-180肉瘤和H-22肝癌荷瘤小鼠模型,观察PHEB对各模型小鼠移植瘤抑制作用。④通过倒置显微镜观察细胞的凋亡现象；AO染色荧光显微镜观察凋亡小体的形成；流式细胞术(FCM)检测各组细胞周期变化及细胞凋亡,进一步明确PHEB诱导PC-3细胞调亡的作用。
结果
①从败酱醇提物中分离精制得到PHEB78g (4.0‰)。
②MTT比色法表明PHEB对人源乳腺癌细胞MCF-7、人白血病癌细胞NB4、人前列腺癌细胞PC-3、人结肠癌细胞COLO-205、人胃癌细胞SGC-7901和人肝癌细胞HepG2;鼠源小鼠黑色素瘤细胞B16和大鼠脑胶质瘤细胞C6肿瘤细胞均有明显的抑制作用,IC50分别为3.47±0.5、8.40±0.5、10.0±0.4、12.3±0.9、13.9±1.3、24.7±2.6、33.5±3.7、41.2±3.5μg/ml,且具有明显的量-效关系；细胞生长曲线结果表明PHEB对COLO-205、SGC-7901肿瘤细胞的作用随着给药浓度的增加而增强,且在一定的剂量范围内随着给药时间的延长,其对两种肿瘤细胞的抑制作用增强,说明具有一定的量-效和时-效关系；克隆形成实验结果表明：随着PHEB浓度的增高,其抗肿瘤作用增强,亦呈一定的量-效关系。
③PHEB给药组对小鼠移植性S-180肉瘤和H-22肝癌瘤的抑制率随着给药浓度的升高而增加,有一定的量-效关系。60mg/kgPHEB剂量组腹腔注射(ip)给药对小鼠移植性S-180肉瘤和H-22肝癌抑瘤率分别为53.6±4.9%和57.2±2.9%,与对照组比较差异有显著性意义(P<0.01),表明PHEB对小鼠移植性肿瘤有一定的抑制作用。
④倒置显微镜观察到PHEB能使细胞密度明显降低,细胞浆固缩,体积缩小,细胞皱缩,部分悬浮,细胞碎片增多；吖啶橙(AO)染色荧光显微镜观察形态,镜下可见：PC-3肿瘤细胞在经PHEB作用后,可见明显的核断裂和核固缩,还有凋亡小体的形成,同时揭示,随着PHEB给药浓度的增加,凋亡的形态学特征愈发明显；FCM结果显示：PHEB可以诱导人前列腺细胞PC-3的凋亡,40μg/mlPHEB作用8h后AnnexinV单阳性即开始增多,至32h, AnnexinV及PI双阳性即坏死细胞也开始增多。随着药物浓度的提高,各组凋亡细胞所占比例也随之升高。且流式细胞术和荧光染色的结果保持一致。
结论
①PHEB体外对多种肿瘤细胞均有抑制作用,并有一定的细胞选择性。
②PHEB对小鼠移植性肿瘤S-180肉瘤和肝癌H-22均有明显抑制作用。
③在本研究中通过倒置显微镜发现随着给药浓度的升高,细胞凋亡数明显增多；通过荧光显微镜观察到PHEB可诱发PC-3肿瘤细胞的凋亡发生,通过吖啶橙(AO)荧光染色显示,PHEB诱发人前列腺癌细胞凋亡的形态特征是导致瘤细胞核断裂和核固缩,以及在瘤细胞内有凋亡小体形成；FCM观察到PHEB诱导PC-3肿瘤细胞凋亡作用呈时间、剂量依赖。
Objective
Firstly, this study mainly aims to find the tumor cells strains those could be significantly inhibited by PHEB according to the in vivo and in vitro extend experiments on the anti-tumor activity of the PHEB. Secondly, to study the induced apoptosis effects on tumor cells in vitro, as to search for the anti-tumor mechanism of PHEB.
Methods
①The sample PHEB was separated and purified by the column chromatography and the chemical composition of PHEB was analyzing by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).②The effects of PHEB on the proliferation of cytokines such as MCF-7、COLO-205、PC-3、SGC-7901、HepG2、C6、NB4 and B-16 were tested by MTT assay methods, growth curve methods and colony formation assay.③The inhibition ratio of the PHEB on different tumor cells were tested with S-180 sarcoma and H-22 liver tumor cells'model mice in vivo.④Under inverted microscope cell apoptosis were observed; Under fluoresence microscope apoptosis bodies were found; Cell cycle and cell apoptosis of tumor cells in each group was detected by flow cytometer. Inhibitive effect on tumor cells was compared between PC-3 and cisplatin.
Results
①78g PHEB was separated from extracts of Valerianceae which accounts for 4.0‰.
②PHEB had a certain inhibition effects on MCF-7、NB4、PC-3、COLO-205、SGC-7901、HepG2、B-16 and C6 tumor cells in vitro, and PHEB had better effects on MCF-7、NB4 and PC-3tumor cells than others good and had dose-effect relationship. the 50% inhibiting concentration (IC50) were 3.47±0.5、8.40±0.5、10.0±0.4、12.3±0.9、 13.9±1.3、24.7±2.6、33.5±3.7、41.2±3.5μg/ml respectively. The results of growth curve methods indicated that effects of PHEB on COLO-205 and SGC-7901 tumor cells in vitro enhanced as the concentration of PHEB increased in a certain time, moreover, it also enhanced as the dosing time increased in a certain range of PHEB concentration that shows time-effect relationship; colony forming assay showed that the anti-tumor effect enhanced as the concentration of PHEB increased, also showing a good amount-effect relationship.
③PHEB could significant inhibit the growth of S-180 sarcoma and H-22 liver tumor in model mice (with P< 0.01 while compared to control group), and the effects enhanced as the concentration of PHEB increased that showed good dose-effect relationship. The tumor growth inhibiting rate (IR) of PHEB on S-180 sarcoma and H-22 liver tumor were 53.6±4.9% and 57.2±2.9% respectively at the intraperitoneal injection 60 mg/kg in vivo (with P> 0.05 while compared to control group), indicating PHEB have a certain anti-tumor effect.
④The results of cell-morph observation under inverted microscope indicate that PHEB could significantly inhibit the growth of tumor cells, reduce density of tumor cells in culture media, meanwhile some of the tumor cells crimpled, some suspended, some decomposed into fragments in the culture media which with PHEB, and it indicated that the effects of PHEB on tumor cell were concentration depended. Observed by fluorescence microscope and found that PC-3 tumor cells were evident nuclear fragmentation and nuclear pyknosis, as well as the formation of apoptotic bodies, as the same time revealed that the role of time as the PHEB prolonged inhibition rate also increases. FCM results showed that:apoptosis could be induced in PC-3 by PHEB, the percentage of AnnexinV+PI- cells increased after being treated with 40μg/ml PHEB for 8h, both apoptotic and necrotic cells increased at 32h. Apoptotic cells were also identified by AO staining.
Conclusion
①PHEB could inhibit the growth of many kinds of tumors in vitro, and this profile of PHEB was selective to certain kinds of tumors.
②PHEB could obviously inhibit the growth of S-180 sarcoma and H-22 hepatoma tumor in model mice in vivo.
③In this study, with the concentration increased, the number of apoptotic cells evident increased through inverted microscopy; PHEB can induce apoptosis in PC-3 tumor cells observed by fluorescence microscopy, the morphological characteristics of apoptosis is the tumor cell nucleus and nuclear pyknosis fracture, as well as apoptotic body formation. Apoptosis induced by PHEB is time and dose dependent. AnnexinⅤand PI double staining is a specific、sensitive method for analyzing apoptotic cells.
①从败酱中分离纯化得到抗肿瘤的有效部位PHEB,并明确其成分组成。
②通过体内外抗肿瘤实验,明确PHEB的抗肿瘤作用。
③考察PHEB诱导肿瘤细胞凋亡,探讨其抗肿瘤作用的机理。
方法
①采用硅胶柱层析分离制备PHEB,并采用薄层层析(TLC)和高效液相色谱法(HPLC)分析PHEB的化学组成。②采用MTT比色法,结合生长曲线以及集落形成实验考察PHEB对MCF-7、COLO-205、PC-3、SGC-7901、HepG2、C6、NB4和B16肿瘤细胞的体外增殖抑制能力。③采用S-180肉瘤和H-22肝癌荷瘤小鼠模型,观察PHEB对各模型小鼠移植瘤抑制作用。④通过倒置显微镜观察细胞的凋亡现象；AO染色荧光显微镜观察凋亡小体的形成；流式细胞术(FCM)检测各组细胞周期变化及细胞凋亡,进一步明确PHEB诱导PC-3细胞调亡的作用。
结果
①从败酱醇提物中分离精制得到PHEB78g (4.0‰)。
②MTT比色法表明PHEB对人源乳腺癌细胞MCF-7、人白血病癌细胞NB4、人前列腺癌细胞PC-3、人结肠癌细胞COLO-205、人胃癌细胞SGC-7901和人肝癌细胞HepG2;鼠源小鼠黑色素瘤细胞B16和大鼠脑胶质瘤细胞C6肿瘤细胞均有明显的抑制作用,IC50分别为3.47±0.5、8.40±0.5、10.0±0.4、12.3±0.9、13.9±1.3、24.7±2.6、33.5±3.7、41.2±3.5μg/ml,且具有明显的量-效关系；细胞生长曲线结果表明PHEB对COLO-205、SGC-7901肿瘤细胞的作用随着给药浓度的增加而增强,且在一定的剂量范围内随着给药时间的延长,其对两种肿瘤细胞的抑制作用增强,说明具有一定的量-效和时-效关系；克隆形成实验结果表明：随着PHEB浓度的增高,其抗肿瘤作用增强,亦呈一定的量-效关系。
③PHEB给药组对小鼠移植性S-180肉瘤和H-22肝癌瘤的抑制率随着给药浓度的升高而增加,有一定的量-效关系。60mg/kgPHEB剂量组腹腔注射(ip)给药对小鼠移植性S-180肉瘤和H-22肝癌抑瘤率分别为53.6±4.9%和57.2±2.9%,与对照组比较差异有显著性意义(P<0.01),表明PHEB对小鼠移植性肿瘤有一定的抑制作用。
④倒置显微镜观察到PHEB能使细胞密度明显降低,细胞浆固缩,体积缩小,细胞皱缩,部分悬浮,细胞碎片增多；吖啶橙(AO)染色荧光显微镜观察形态,镜下可见：PC-3肿瘤细胞在经PHEB作用后,可见明显的核断裂和核固缩,还有凋亡小体的形成,同时揭示,随着PHEB给药浓度的增加,凋亡的形态学特征愈发明显；FCM结果显示：PHEB可以诱导人前列腺细胞PC-3的凋亡,40μg/mlPHEB作用8h后AnnexinV单阳性即开始增多,至32h, AnnexinV及PI双阳性即坏死细胞也开始增多。随着药物浓度的提高,各组凋亡细胞所占比例也随之升高。且流式细胞术和荧光染色的结果保持一致。
结论
①PHEB体外对多种肿瘤细胞均有抑制作用,并有一定的细胞选择性。
②PHEB对小鼠移植性肿瘤S-180肉瘤和肝癌H-22均有明显抑制作用。
③在本研究中通过倒置显微镜发现随着给药浓度的升高,细胞凋亡数明显增多；通过荧光显微镜观察到PHEB可诱发PC-3肿瘤细胞的凋亡发生,通过吖啶橙(AO)荧光染色显示,PHEB诱发人前列腺癌细胞凋亡的形态特征是导致瘤细胞核断裂和核固缩,以及在瘤细胞内有凋亡小体形成；FCM观察到PHEB诱导PC-3肿瘤细胞凋亡作用呈时间、剂量依赖。
Objective
Firstly, this study mainly aims to find the tumor cells strains those could be significantly inhibited by PHEB according to the in vivo and in vitro extend experiments on the anti-tumor activity of the PHEB. Secondly, to study the induced apoptosis effects on tumor cells in vitro, as to search for the anti-tumor mechanism of PHEB.
Methods
①The sample PHEB was separated and purified by the column chromatography and the chemical composition of PHEB was analyzing by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).②The effects of PHEB on the proliferation of cytokines such as MCF-7、COLO-205、PC-3、SGC-7901、HepG2、C6、NB4 and B-16 were tested by MTT assay methods, growth curve methods and colony formation assay.③The inhibition ratio of the PHEB on different tumor cells were tested with S-180 sarcoma and H-22 liver tumor cells'model mice in vivo.④Under inverted microscope cell apoptosis were observed; Under fluoresence microscope apoptosis bodies were found; Cell cycle and cell apoptosis of tumor cells in each group was detected by flow cytometer. Inhibitive effect on tumor cells was compared between PC-3 and cisplatin.
Results
①78g PHEB was separated from extracts of Valerianceae which accounts for 4.0‰.
②PHEB had a certain inhibition effects on MCF-7、NB4、PC-3、COLO-205、SGC-7901、HepG2、B-16 and C6 tumor cells in vitro, and PHEB had better effects on MCF-7、NB4 and PC-3tumor cells than others good and had dose-effect relationship. the 50% inhibiting concentration (IC50) were 3.47±0.5、8.40±0.5、10.0±0.4、12.3±0.9、 13.9±1.3、24.7±2.6、33.5±3.7、41.2±3.5μg/ml respectively. The results of growth curve methods indicated that effects of PHEB on COLO-205 and SGC-7901 tumor cells in vitro enhanced as the concentration of PHEB increased in a certain time, moreover, it also enhanced as the dosing time increased in a certain range of PHEB concentration that shows time-effect relationship; colony forming assay showed that the anti-tumor effect enhanced as the concentration of PHEB increased, also showing a good amount-effect relationship.
③PHEB could significant inhibit the growth of S-180 sarcoma and H-22 liver tumor in model mice (with P< 0.01 while compared to control group), and the effects enhanced as the concentration of PHEB increased that showed good dose-effect relationship. The tumor growth inhibiting rate (IR) of PHEB on S-180 sarcoma and H-22 liver tumor were 53.6±4.9% and 57.2±2.9% respectively at the intraperitoneal injection 60 mg/kg in vivo (with P> 0.05 while compared to control group), indicating PHEB have a certain anti-tumor effect.
④The results of cell-morph observation under inverted microscope indicate that PHEB could significantly inhibit the growth of tumor cells, reduce density of tumor cells in culture media, meanwhile some of the tumor cells crimpled, some suspended, some decomposed into fragments in the culture media which with PHEB, and it indicated that the effects of PHEB on tumor cell were concentration depended. Observed by fluorescence microscope and found that PC-3 tumor cells were evident nuclear fragmentation and nuclear pyknosis, as well as the formation of apoptotic bodies, as the same time revealed that the role of time as the PHEB prolonged inhibition rate also increases. FCM results showed that:apoptosis could be induced in PC-3 by PHEB, the percentage of AnnexinV+PI- cells increased after being treated with 40μg/ml PHEB for 8h, both apoptotic and necrotic cells increased at 32h. Apoptotic cells were also identified by AO staining.
Conclusion
①PHEB could inhibit the growth of many kinds of tumors in vitro, and this profile of PHEB was selective to certain kinds of tumors.
②PHEB could obviously inhibit the growth of S-180 sarcoma and H-22 hepatoma tumor in model mice in vivo.
③In this study, with the concentration increased, the number of apoptotic cells evident increased through inverted microscopy; PHEB can induce apoptosis in PC-3 tumor cells observed by fluorescence microscopy, the morphological characteristics of apoptosis is the tumor cell nucleus and nuclear pyknosis fracture, as well as apoptotic body formation. Apoptosis induced by PHEB is time and dose dependent. AnnexinⅤand PI double staining is a specific、sensitive method for analyzing apoptotic cells.
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[36]Lawry J. Detection of apoptosis by the TUNEL assay[J]. Methods Mol Med, 2004,88:183-190.
[37]Tolskaya EA, Romanova LI, Kolesnikova MS, et al. Apoptosis-inducing and apoptosis-preventing functions of poliovirus [J].1995,69(2):1181-1189.
[38]Satchell PG, Gutmann JL, Witherspoon DE. Apoptosis:an introduction for the endodontist [J]. International Endodontic,2003,36:237-245
[39]Schwartz L M, Ashwell J D. Met hods Cell Biology:Apoptosis [M]. USA: Academic Press,2001,66:492246
[40]许屏,梁玉,任怡敏,等。显示分代阶段不同的细胞的荧光染色法[J].解剖学报,1998,22(增刊)：501
[41]Span L P, Pennings AH, Vierwinden G, et al The dynamic process of apoptosis analyzed by flow cytometry using AnnexinV/propidium iodide and a modified in situ end abeling technique. Cytometry,2002,47 (1):24
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[1]World Health Orgnization. Facts about Cancer. WHO Media centre.2006
[2]Gene L, Bidwell, Izabela F, et al. Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin[J]. Biochemical pharmacology,2007, 73:620-631
[3]Casado P, Zuazua-Pillar P, ValleE D, et al. Vincristme regulates the Phosphorylation of the antiapoptotic protein HSP27 in breast cancer cells[J]. Cancer Letters,2007,247:273-282
[4]Patel D J, Phan A T, Kuryavyi V. Human telomere, oncogenic promoter and 5'-UTR G-quadrup lexes:diverse higher order DNA and RNA targets for cancer therapeutics Nucleic Acids Res,2007,35:7429-7455
[5]Fabricant D S, Farnsworth N R..The value of plants used in tradition-al medicine for drug discovery. Environ. Health Perspect,2001,109 (Suppl 1):69-75
[6]Verpoorte R. Pharmacognosy in the new millennium:leadfinding and biotechnology. J. Pharm. Pharmacol,2000,52:253-262
[7]张学敏.MTS法在肿瘤药敏实验中的研究进展[J].江西医学检验,2005,23(3)：284-285
[8]李铁军,李爱云,张晓峰.乳酸菌抗菌机理进展[J].微生物学通报,2002,29(3)：81-85
[9]D I JKHU IS A J, KLAPPE K, KAM PS W, et al Gangliosides do not affect ABC transporter function in human neuroblastoma cells[J]. Annual Review of B iochem istry,2006,75:1-39
[10]刘玉和,于春艳,王丹,等。白花蛇舌草提取物对人肝癌多药耐药细胞Bel-7402体外抗肿瘤活性及作用机制的实验研究[J].中华综合临床医学杂志,2005,7(4)：3-5。
[11]方蓉,李芳秋,武建国.M TT比色法的条件探讨[J].临床检验杂志,2003,21(1)：34-35
[12]Sargent JM. Taylor CG. Appraisal of MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. Br J Cancer,1989,60 (2):206
[13]鲁迎年,王秋波,等。MTT比色法在测定化疗药物敏感试验中的条件选择。青岛医学院学报,1997,33(3)：224-226
[14]Skehan P, et al. J Natl Cancer Inst,1990,82:1107.
[15]SKEHAN P. New Colorim etric cytotoxicity assay for anticancer drug screening[J]. J Nat Cancer Inst,1990,82 (11):7-12
[16]谭卫东,金红,罗第祥,等。抗肿瘤药物筛选中M TT和SRB法的比较[J].天然产物研究与开发,1999,11(3)：17-22
[17]SKEHAN P,STORENG R, SCUD IRTO D,et al New colorim etric cutotoxicity assay for anticancer drug screening[J]. J Nat Cancer Inst,1990,82:1 107
[18]赵俊萍,张惠丽.LDH释放法测定NK细胞活性对红细胞的影响[J].肿瘤研究与临床,1999,12(3)：203-204
[19]赵培荣,田爱琴,马湘玲,等. 山豆根对人食管癌细胞株(Eca-109)杀伤、抑制及脱氧氢酶类的影响[J].河南肿瘤学杂志,1998,11(2)：87.
[20]ASH I K C, HAD I A Y A lcoholic turmeric extract sim ultaneously activating murine lymphocytes and inducing apop tosis of Ehlrich ascitic carcinoma cells[J] International I munopharmacology,2005,5 (10):1574-1581
[21]GEORGE F, JOHN A. Timbrell. In vitro cytotoxicity assays:Comparison of LDH, neutral red, M TT and protein assay in hepatoma cell lines following exposure to cadmium chloride [J]. Toxicology Letters,2006,160:171-177
[22]卢虹,吴兴中。酸性磷酸酶法检测肿瘤细胞的增殖状态[J].中华检验医学杂志,2001,24(2)：84-86
[23]陈历排,周文,李志阳。用微量板ATP生物发光法检测肿瘤细胞对化疗药物的敏感性。肿瘤,2000,20(2)：103-105。
[24]梁永钜,潘启超,麦伟源,等。鼻咽癌体外药敏试验初步研究。癌症,1996,15(3)：216-218。
[25]梁永钜,周听熙,潘启超,等。肝癌体外药物敏感试验。肿瘤,2001,21(1)：17-18。
[26]张阳,石虹,王英丽,等。复方赤木煎剂及肝癌细胞克隆形成的实验研究[J].实用肿瘤学杂志,1999,13(3)：181-182