中药臭灵丹中HTMF体外抗肿瘤机制研究
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摘要
目的：
本研究观察分离自臭灵丹(Laggera pterodonta)中的3,5-二羟基-6,7,3',4'-四甲氧基黄酮(3,5-hydroxy-6,7,3',4'-tetramethoxyflavone, HTMF)体外抗肿瘤活性及对正常细胞的细胞毒性,对其诱导肿瘤细胞凋亡的作用机制进行探讨,为将来开发抗肿瘤新药提供一些实验依据。
方法：
1 HTMF对正常细胞的细胞毒性及对肿瘤细胞增殖的影响
采用常规细胞培养方法,通过3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)法测定：HTMF对正常细胞(人脐静脉内皮细胞EVC304,非洲绿猴肾细胞Vero)的毒性,并计算出细胞的半数致死浓度CC50(50% Cytotoxicity Concentration)值；对肿瘤细胞(人肺癌细胞A549,肝癌细胞HepG2,喉表皮样癌细胞HEp-2和鼻咽癌细胞CNE)的增殖抑制率,并计算出细胞的半数致死浓度IC50 (50% Inhibitory Concentration)值；选择细胞增殖被显著抑制的肿瘤细胞(HEp-2和CNE)进行复筛,分析量效及时效关系；临床常用抗肿瘤药阿霉素(A driamycin, ADM)作为阳性对照,对HEp-2和CNE细胞的增殖抑制率：溶剂二甲亚砜(Dimethyl Sulfoxide, DMSO)对HEp-2, CNE, EVC304和Vero细胞的毒性。
2 HTMF对肿瘤细胞、细胞核形态变化及对细胞凋亡率的影响
预处理组给予不同浓度的HTMF,采用倒置显微镜从形态学上观察各组细胞的形态变化并拍照；Hoechst 33258染色,荧光显微镜下观察细胞核形态变化并拍照。流式细胞仪检测(flow cytometry, FCM) HEp-2和CNE细胞凋亡率的改变。
3 HTMF对凋亡蛋白Caspase-3/Caspase-9的影响
采用Western-blotting法分别检测不同浓度HTMF处理组及同一浓度不同时间段处理组中凋亡蛋白半胱氨酸天冬氨酸蛋白酶3 (Caspase-3)和半胱氨酸天冬氨酸蛋白酶9(Caspase-9)量的变化。
4 HTMF对线粒体膜电位(mitochondrial membrane potential,的影响
采用JC-1(一种广泛用于检测线粒体膜电位的荧光探针,通过荧光颜色的转变来观察线粒体膜电位的变化)染色,激光共聚焦显微镜下观察线粒体膜△Ψm的改变。罗丹明123(Rhodamine123,Rho123)染料染色,FCM检测线粒体膜AΨm值的变化。
结果：
1 MTT法结果显示HTMF作用48 h后A549、HepG2、HEp-2和CNE细胞的IC5o值分别为61.49、74.61、27.81和28.31μg/mL;EVC304和Vero细胞的CC50值分别56.49和87.64μg/mL。对HEp-2和CNE细胞进行复筛,发现其分别随着浓度和时间的增加细胞增殖抑制率增大,呈现剂量与效应及时间与效应双重依赖性关系(P< 0.05, P< 0.01). DMSO毒性结果显示不同实验组中所含不同体积分数的DMSO对HEp-2, CNE, EVC304和Vero细胞无细胞毒性(P>0.05)。阿霉素对HEp-2和CNE细胞抑制活性优于HTMF。
2细胞形态改变显示HTMF作用于HEp-2和CNE细胞48 h后,随着浓度的升高细胞变圆,体积缩小,贴壁能力下降,同细胞对照组相比,其细胞数及细胞密度均明显下降。Hoechst33258染色观察显示随HTMF浓度的增加细胞出现核固缩,核体积变小,分布不均匀；部分细胞核出现碎裂,呈现致密蓝色颗粒状荧光的凋亡小体。流式细胞仪检测显示HTMF作用于HEp-2和CNE细胞后,随浓度的增加,细胞凋亡率显著性增加(P<0.05,P<0.01)。
3 Western-blotting法结果显示经不同浓度的HTMF处理HEp-2和CNE细胞后,呈浓度依赖性激活凋亡蛋白Caspase、Caspase-9;经40μg/mL浓度的HTMF处理后,随着时间的延长,细胞中Caspase-3、Caspase-9呈时间依赖性被激活。差异均具有统计学意义(P<0.01)。
4 JC-1染色,激光共聚焦显微镜下观察显示荧光颜色分别随着浓度和时间的增加由红色向绿色转变,同时线粒体数目越来越少且分布变得越来越不均匀。Rho123染色,流式细胞仪检测结果显示线粒体膜△Ψm的平均荧光指数值分别随浓度及时间增加而下降(P<0.05,P<0.01)。
结论：
1 HTMF对HepG2, HEp-2, A549和CNE细胞均有不同程度的增殖抑制作用,其中对HEp-2和CNE细胞有显著性的抑增殖作用且呈现剂量、时间双重依赖性关系,而对正常细胞EVC304和Vero的细胞毒性相对较小。表明HTMF抗肿瘤具有选择性和相对高效低毒的特性。
2 HTMF可诱导细胞凋亡,显著抑制HEp-2和CNE细胞增殖,且细胞凋亡率呈浓度-效应依赖性关系。
3 HTMF诱导HEp-2和CNE细胞凋亡的通路中凋亡蛋白Caspase-3和Caspase-9呈浓度、时间双重依赖性被显著性激活。
4 HTMF可呈浓度和时间双重依赖性降低线粒体膜△Ψm,激活Caspase级联反应,通过线粒体途径诱导HEp-2和CNE细胞凋亡。
Objective:
This study was to observe the cytotoxicity on the normal cells and antiproliferative effects on the tumor cells in vitro of 3,5-hydroxy -6,7,3',4'-tetramethoxyflavone (HTMF) isolated from Laggera pterodonta. Moreover, the mechanism of inducting the tumor cell apoptosis was explored. Our results will provide the experiental basis for the exploitation of antitumor new drugs.
Methods:
1 Cytotoxicity of HTMF on the normal cells and the antiproliferative effects of HTMF on the tumor cells
Cells were cultured by the common cell culture method. The cytotoxicity on the normal cells (EVC304 and Vero) and inhibitory rate on the tumor cells (A549, HepG2, HEp-2 and CNE) were determined. The normal cell's CC50 (50% Cytotoxicity Concentration) and tumor cell's IC50 (50% Inhibitory Concentration) were accounted. Furthermore, the cells (HEp-2 and CNE) inhibited significantly by HTMF were chosed and quantity-effect and time-effect relationships of the inhibitory effect were analysed by MTT assay. The inhibitory rate of A driamycin (ADM) in clinical used commonly antineoplastic agent on HEp-2 and CNE cells and cytotoxicity of DMSO on HEp-2, CNE, EVC304 and Vero cells were also determined by MTT assay.
2 Effects of HTMF to changes of the cell and nuclear morphological characteristics, and cell apoptosis rate on the tumor cells
After the tumor cells were treated with different concentrations of HTMF, the changes of the cell morphological characteristics were observed under the inverted microscope and take a photograph. The changes of cell nuclear morphological characteristics were observed under the fluorescence microscope through the Hoechst 33258 staining and take a picture. The cell apoptosis rate on HEp-2 and CNE cells was displayed by flow cytometry (FCM).
3 Effects of HTMF to the expression of Caspase-3 and Caspase-9
The expressions of Caspase-3 and Caspase-9 were detected by Western-blotting with different concentrations or for different times with the same concertration treatments of HTMF.
4 Effects of HTMF to mitochondrial membrane potential (ΔΨm)
The mitochondrial membrane potential was analyzed by FCM with Rho 123 staining and by laser confocal microscope with JC-1 (a kind of fluorescent probe was used generally to detect mitochondrial membrane potential, and the changes of the mitochondrial membrane potential were observed by the changes of the flourescence color) staining.
Results:
1 MTT assay results showed that the IC50 values were 61.49,74.61,27.81 and 28.31μg/mL at 48 h treatments of HTMF on A549, HepG2, HEp-2 and CNE cells respectively, while CC50 values on EVC304 and Vero were 56.49 and 87.64μg/mL respectively. The inhibitory rate on HEp-2 and CNE cells increased with the increasing of concentration and time prolongation respectively, and showed up dose and time dependent manners (P< 0.05, P< 0.01). The cytotoxicity results exhibited DMSO with different volume percentagein in different groups had no cytotoxicity to HEp-2 and CNE cells (P> 0.05). The inhibitory effects of ADM were better than that of HTMF.
2 The changes of the cell morphological characteristics showed that the cells became round, the volume was shrinked, and the adherence capability was decreased. The cell population accounting and cell density were decreased significantly compaired with the control group with the increasing of concentration of HTMF after 48 h on HEp-2 and CNE cells. The cells showed karyopycnosis, nuclear volume shrinking, and maldistribution by Hoechst 33258 staining. A part of cell nuclears showed up to break into pieces, and to form some apoptotic body with thick white-blue fluorescent in Hoechst 33258 staining cells. The apoptosis rate is significantly increased with the increasing of concentration of HTMF on HEp-2 and CNE cells (P< 0.05, P < 0.01).
3 Western-blotting results showed that Caspase-3 and Caspase-9 were activated in dose dependent manners on HEp-2 and CNE cells with various concentrations of HTMF (P<0.01). After treated with 40μg/mL of HTMF, Caspase-3 and Caspase-9 were activated in time dependent manners on HEp-2 and CNE cells with time prolongation (P< 0.01).
4 Laser confocal microscope with JC-1 fluorescence staining showed that the flourescence color changed from red to green with the increasing of concentration and time prolongation, and at the same time mitochondria numbers became more and more less and maldistribution. FCM with Rho123 staining results showed that the number of the mean fluorecence index of the mitochondria decreased with the increasing of concentration and time prolongation, respectively (P< 0.05, P< 0.01).
Conclusion:
1 HTMF had different inhibitory effects on A549, HepG2, HEp-2 and CNE cells. Furthermore, HTMF had an significantly antiproliferative effects on HEp-2 and CNE cells in dose and time dependent manners, while exhibited low cytotoxicity to the two normal cells, Vero and EVC304. This showed that HTMF had the selectivity, high performance and low cytotoxicity on antitumor effect.
2 HTMF inhibited significantly the proliferation of HEp-2 and CNE cells by inducting the cell apoptosis. Furthermore, the cell apoptosis rate showed the dose dependent manner.
3 Caspase-3 and Caspase-9 were activated obviously in dose and time dependent manners in the pathway of the cell apoptosis induction by HTMF on HEp-2 and CNE cells.
4 HTMF decreased in mitochondrial membrane potential in dose and time dependent manners, activated caspase cascade reaction, and inducted the apoptosis of HEp-2 and CNE cells by the mitochondrial passway.
本研究观察分离自臭灵丹(Laggera pterodonta)中的3,5-二羟基-6,7,3',4'-四甲氧基黄酮(3,5-hydroxy-6,7,3',4'-tetramethoxyflavone, HTMF)体外抗肿瘤活性及对正常细胞的细胞毒性,对其诱导肿瘤细胞凋亡的作用机制进行探讨,为将来开发抗肿瘤新药提供一些实验依据。
方法：
1 HTMF对正常细胞的细胞毒性及对肿瘤细胞增殖的影响
采用常规细胞培养方法,通过3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)法测定：HTMF对正常细胞(人脐静脉内皮细胞EVC304,非洲绿猴肾细胞Vero)的毒性,并计算出细胞的半数致死浓度CC50(50% Cytotoxicity Concentration)值；对肿瘤细胞(人肺癌细胞A549,肝癌细胞HepG2,喉表皮样癌细胞HEp-2和鼻咽癌细胞CNE)的增殖抑制率,并计算出细胞的半数致死浓度IC50 (50% Inhibitory Concentration)值；选择细胞增殖被显著抑制的肿瘤细胞(HEp-2和CNE)进行复筛,分析量效及时效关系；临床常用抗肿瘤药阿霉素(A driamycin, ADM)作为阳性对照,对HEp-2和CNE细胞的增殖抑制率：溶剂二甲亚砜(Dimethyl Sulfoxide, DMSO)对HEp-2, CNE, EVC304和Vero细胞的毒性。
2 HTMF对肿瘤细胞、细胞核形态变化及对细胞凋亡率的影响
预处理组给予不同浓度的HTMF,采用倒置显微镜从形态学上观察各组细胞的形态变化并拍照；Hoechst 33258染色,荧光显微镜下观察细胞核形态变化并拍照。流式细胞仪检测(flow cytometry, FCM) HEp-2和CNE细胞凋亡率的改变。
3 HTMF对凋亡蛋白Caspase-3/Caspase-9的影响
采用Western-blotting法分别检测不同浓度HTMF处理组及同一浓度不同时间段处理组中凋亡蛋白半胱氨酸天冬氨酸蛋白酶3 (Caspase-3)和半胱氨酸天冬氨酸蛋白酶9(Caspase-9)量的变化。
4 HTMF对线粒体膜电位(mitochondrial membrane potential,的影响
采用JC-1(一种广泛用于检测线粒体膜电位的荧光探针,通过荧光颜色的转变来观察线粒体膜电位的变化)染色,激光共聚焦显微镜下观察线粒体膜△Ψm的改变。罗丹明123(Rhodamine123,Rho123)染料染色,FCM检测线粒体膜AΨm值的变化。
结果：
1 MTT法结果显示HTMF作用48 h后A549、HepG2、HEp-2和CNE细胞的IC5o值分别为61.49、74.61、27.81和28.31μg/mL;EVC304和Vero细胞的CC50值分别56.49和87.64μg/mL。对HEp-2和CNE细胞进行复筛,发现其分别随着浓度和时间的增加细胞增殖抑制率增大,呈现剂量与效应及时间与效应双重依赖性关系(P< 0.05, P< 0.01). DMSO毒性结果显示不同实验组中所含不同体积分数的DMSO对HEp-2, CNE, EVC304和Vero细胞无细胞毒性(P>0.05)。阿霉素对HEp-2和CNE细胞抑制活性优于HTMF。
2细胞形态改变显示HTMF作用于HEp-2和CNE细胞48 h后,随着浓度的升高细胞变圆,体积缩小,贴壁能力下降,同细胞对照组相比,其细胞数及细胞密度均明显下降。Hoechst33258染色观察显示随HTMF浓度的增加细胞出现核固缩,核体积变小,分布不均匀；部分细胞核出现碎裂,呈现致密蓝色颗粒状荧光的凋亡小体。流式细胞仪检测显示HTMF作用于HEp-2和CNE细胞后,随浓度的增加,细胞凋亡率显著性增加(P<0.05,P<0.01)。
3 Western-blotting法结果显示经不同浓度的HTMF处理HEp-2和CNE细胞后,呈浓度依赖性激活凋亡蛋白Caspase、Caspase-9;经40μg/mL浓度的HTMF处理后,随着时间的延长,细胞中Caspase-3、Caspase-9呈时间依赖性被激活。差异均具有统计学意义(P<0.01)。
4 JC-1染色,激光共聚焦显微镜下观察显示荧光颜色分别随着浓度和时间的增加由红色向绿色转变,同时线粒体数目越来越少且分布变得越来越不均匀。Rho123染色,流式细胞仪检测结果显示线粒体膜△Ψm的平均荧光指数值分别随浓度及时间增加而下降(P<0.05,P<0.01)。
结论：
1 HTMF对HepG2, HEp-2, A549和CNE细胞均有不同程度的增殖抑制作用,其中对HEp-2和CNE细胞有显著性的抑增殖作用且呈现剂量、时间双重依赖性关系,而对正常细胞EVC304和Vero的细胞毒性相对较小。表明HTMF抗肿瘤具有选择性和相对高效低毒的特性。
2 HTMF可诱导细胞凋亡,显著抑制HEp-2和CNE细胞增殖,且细胞凋亡率呈浓度-效应依赖性关系。
3 HTMF诱导HEp-2和CNE细胞凋亡的通路中凋亡蛋白Caspase-3和Caspase-9呈浓度、时间双重依赖性被显著性激活。
4 HTMF可呈浓度和时间双重依赖性降低线粒体膜△Ψm,激活Caspase级联反应,通过线粒体途径诱导HEp-2和CNE细胞凋亡。
Objective:
This study was to observe the cytotoxicity on the normal cells and antiproliferative effects on the tumor cells in vitro of 3,5-hydroxy -6,7,3',4'-tetramethoxyflavone (HTMF) isolated from Laggera pterodonta. Moreover, the mechanism of inducting the tumor cell apoptosis was explored. Our results will provide the experiental basis for the exploitation of antitumor new drugs.
Methods:
1 Cytotoxicity of HTMF on the normal cells and the antiproliferative effects of HTMF on the tumor cells
Cells were cultured by the common cell culture method. The cytotoxicity on the normal cells (EVC304 and Vero) and inhibitory rate on the tumor cells (A549, HepG2, HEp-2 and CNE) were determined. The normal cell's CC50 (50% Cytotoxicity Concentration) and tumor cell's IC50 (50% Inhibitory Concentration) were accounted. Furthermore, the cells (HEp-2 and CNE) inhibited significantly by HTMF were chosed and quantity-effect and time-effect relationships of the inhibitory effect were analysed by MTT assay. The inhibitory rate of A driamycin (ADM) in clinical used commonly antineoplastic agent on HEp-2 and CNE cells and cytotoxicity of DMSO on HEp-2, CNE, EVC304 and Vero cells were also determined by MTT assay.
2 Effects of HTMF to changes of the cell and nuclear morphological characteristics, and cell apoptosis rate on the tumor cells
After the tumor cells were treated with different concentrations of HTMF, the changes of the cell morphological characteristics were observed under the inverted microscope and take a photograph. The changes of cell nuclear morphological characteristics were observed under the fluorescence microscope through the Hoechst 33258 staining and take a picture. The cell apoptosis rate on HEp-2 and CNE cells was displayed by flow cytometry (FCM).
3 Effects of HTMF to the expression of Caspase-3 and Caspase-9
The expressions of Caspase-3 and Caspase-9 were detected by Western-blotting with different concentrations or for different times with the same concertration treatments of HTMF.
4 Effects of HTMF to mitochondrial membrane potential (ΔΨm)
The mitochondrial membrane potential was analyzed by FCM with Rho 123 staining and by laser confocal microscope with JC-1 (a kind of fluorescent probe was used generally to detect mitochondrial membrane potential, and the changes of the mitochondrial membrane potential were observed by the changes of the flourescence color) staining.
Results:
1 MTT assay results showed that the IC50 values were 61.49,74.61,27.81 and 28.31μg/mL at 48 h treatments of HTMF on A549, HepG2, HEp-2 and CNE cells respectively, while CC50 values on EVC304 and Vero were 56.49 and 87.64μg/mL respectively. The inhibitory rate on HEp-2 and CNE cells increased with the increasing of concentration and time prolongation respectively, and showed up dose and time dependent manners (P< 0.05, P< 0.01). The cytotoxicity results exhibited DMSO with different volume percentagein in different groups had no cytotoxicity to HEp-2 and CNE cells (P> 0.05). The inhibitory effects of ADM were better than that of HTMF.
2 The changes of the cell morphological characteristics showed that the cells became round, the volume was shrinked, and the adherence capability was decreased. The cell population accounting and cell density were decreased significantly compaired with the control group with the increasing of concentration of HTMF after 48 h on HEp-2 and CNE cells. The cells showed karyopycnosis, nuclear volume shrinking, and maldistribution by Hoechst 33258 staining. A part of cell nuclears showed up to break into pieces, and to form some apoptotic body with thick white-blue fluorescent in Hoechst 33258 staining cells. The apoptosis rate is significantly increased with the increasing of concentration of HTMF on HEp-2 and CNE cells (P< 0.05, P < 0.01).
3 Western-blotting results showed that Caspase-3 and Caspase-9 were activated in dose dependent manners on HEp-2 and CNE cells with various concentrations of HTMF (P<0.01). After treated with 40μg/mL of HTMF, Caspase-3 and Caspase-9 were activated in time dependent manners on HEp-2 and CNE cells with time prolongation (P< 0.01).
4 Laser confocal microscope with JC-1 fluorescence staining showed that the flourescence color changed from red to green with the increasing of concentration and time prolongation, and at the same time mitochondria numbers became more and more less and maldistribution. FCM with Rho123 staining results showed that the number of the mean fluorecence index of the mitochondria decreased with the increasing of concentration and time prolongation, respectively (P< 0.05, P< 0.01).
Conclusion:
1 HTMF had different inhibitory effects on A549, HepG2, HEp-2 and CNE cells. Furthermore, HTMF had an significantly antiproliferative effects on HEp-2 and CNE cells in dose and time dependent manners, while exhibited low cytotoxicity to the two normal cells, Vero and EVC304. This showed that HTMF had the selectivity, high performance and low cytotoxicity on antitumor effect.
2 HTMF inhibited significantly the proliferation of HEp-2 and CNE cells by inducting the cell apoptosis. Furthermore, the cell apoptosis rate showed the dose dependent manner.
3 Caspase-3 and Caspase-9 were activated obviously in dose and time dependent manners in the pathway of the cell apoptosis induction by HTMF on HEp-2 and CNE cells.
4 HTMF decreased in mitochondrial membrane potential in dose and time dependent manners, activated caspase cascade reaction, and inducted the apoptosis of HEp-2 and CNE cells by the mitochondrial passway.
引文
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