阿托伐他汀增加乳腺癌细胞对多柔比星敏感性的研究

doi:10.3877/cma.j.issn.1674-0807.2021.01.006

目的

探讨他汀类药物增加乳腺癌细胞对多柔比星敏感性的机制。

方法

(1)用不同浓度的多柔比星(0、0.01、0.02、0.04、0.08、0.16、0.32、0.64、1.28 μg/ml)与0、2 μmol/L的阿托伐他汀联合处理MDA-MB-231细胞，通过细胞计数检测试剂盒(CCK-8)检测450 nm波长下的吸光度值，从而计算细胞活力。(2)分别用0.3 μg/ml多柔比星、2 μmol/L阿托伐他汀单药及两药联合处理MDA-MB-231细胞，并以未经任何药物处理的细胞作为对照组，通过Hoechst染色检测MDA-MB-231细胞凋亡情况，通过细胞划痕实验、Transwell实验检测细胞迁移及侵袭能力，通过Western blot检测MDA-MB-231细胞中caspase 3、caspase 9、3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR)、甾醇调节元件结合蛋白转录因子2(SREBP2)及低密度脂蛋白受体(LDLR)的表达。细胞活力比较采用析因分析，未凋亡细胞数、划痕面积百分比、穿膜细胞数及不同蛋白表达等指标的多组比较采用单因素方差分析，两两比较采用LSD法。

结果

(1)与多柔比星单药相比，阿托伐他汀与多柔比星联合应用可以显著抑制MDA-MB-231细胞活力(F＝243.043, P<0.001)；不同浓度多柔比星组细胞活力比较，差异有统计学意义( F＝1 803.617, P<0.001)；两个因素存在交互作用(F＝21.030, P<0.001)。(2)各组的未凋亡细胞数比较，差异具有统计学意义(对照组：94.00±4.24：阿托伐他汀组：57.00±1.41，多柔比星组：34.50±2.12，阿托伐他汀+多柔比星组：19.00±2.83，F＝261.021，P<0.001)，两两比较结果显示，组间差异均有统计学意义(P均<0.050)。各组间的划痕面积百分比比较，差异具有统计学意义[对照组：(28.94±3.59)%，阿托伐他汀组：(31.00±2.99)%，多柔比星组：(40.16±2.38)%，阿托伐他汀+多柔比星组：(60.86±3.60)%，F＝42.080，P<0.050]。与对照组、阿托伐他汀组及多柔比星组相比，阿托伐他汀+多柔比星组划痕面积均增加(P均<0.050)。各组穿膜细胞数比较，差异具有统计学意义(对照组：101.20±14.55，阿托伐他汀组：75.80±7.33，多柔比星组：32.40±4.78，阿托伐他汀+多柔比星组：8.80±2.50，F＝118.031，P<0.001)，两两比较结果显示差异均具有统计学意义(P均<0.050)。各组MDA-MB-231细胞中凋亡相关蛋白caspase 3、caspase 9的表达比较，差异均有统计学意义(F＝128.854、247.530，P均<0.001)。与多柔比星组相比，阿托伐他汀+多柔比星组caspase 3、caspase 9蛋白的表达显著增加(P均<0.050)。4组MDA-MB-231细胞中HMGCR、SREBP2、LDLR蛋白的表达量比较，差异均具有统计学意义(F＝183.193、227.470、586.087，P均<0.001)。两两比较结果显示，与对照组比较，阿托伐他汀+多柔比星组中HMGCR表达明显升高，SREBP2、LDLR表达明显降低(P均<0.050)；与对照组相比，多柔比星组HMGCR的表达无明显改变(P>0.050)，SREBP2、LDLR的表达明显降低(P均<0.050)，阿托伐他汀组HMGCR表达高于对照组(P<0.050)；与多柔比星组相比，阿托伐他汀+多柔比星组HMGCR表达增加(P<0.050)，SREBP2的表达降低(P<0.050), LDLR的表达无明显变化(P>0.050)。

结论

阿托伐他汀通过抑制HMGCR表达，减少细胞内胆固醇的合成，使细胞更依赖于外源性胆固醇的摄取；多柔比星通过抑制LDLR表达减少外源性胆固醇的摄取；因此，当两药联用后由于细胞内胆固醇合成减少，使细胞更依赖于外源性胆固醇的摄取，对多柔比星更加敏感。

关键词: 乳腺肿瘤, 胆固醇, 多柔比星, 阿托伐他汀

Objective

To explore the mechanism of statins increasing the sensitivity of breast cancer cells to doxorubicin.

Methods

(1)Doxorubicin at gradient concentrations (0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28 μg/ml) and atorvastatin (0, 2 μmol/L) were used to treat MDA-MB- 231 cells, respectively. The optical density at 450 nm wavelength was detected by the cell counting detection kit (CCK-8) to calculate the cell viability. (2) MDA-MB-231 cells were treated with 0.3 μg/ml doxorubicin, 2 μmol/L atorvastatin and the combination of both and the cells without any drug treatment served as control. The apoptosis of MDA-MB-231 cells was detected after Hoechst staining. The cell migration and invasion abilities were measured by cell scratch test and Transwell test. The expression of caspase 3, caspase 9, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), sterol regulatory element binding protein transcription factor 2 (SREBP2) and low-density lipoprotein receptor (LDLR) in MDA-MB-231 cells were determined by Western blot. The cell viability was compared using factor analysis. The number of survival cells, percentage of scratch area and number of cells penetrating membranes and expression of different proteins were compared among multiple groups using one-way analysis of variance and the LSD method was used for pairwise comparison.

Results

(1) Compared with cells treated by doxorubicin alone, the combination of atorvastatin and doxorubicin significantly inhibited the viability of MDA-MB-231 cells (F=243.043, P<0.001); the viability of cells treated by different concentrations of doxorubicin presented a significant difference (F=1 803.617, P<0.001); there was an interaction between the two factors (F=21.030, P<0.001). (2) The number of survival cells presented a significant difference among four groups (control group: 94.00±4.24: atorvastatin group: 57.00±1.41, doxorubicin group: 34.50±2.12, atorvastatin+ doxorubicin group: 19.00±2.83, F=261.021, P<0.001). Pairwise comparison showed that the difference between groups was statistically significant (all P<0.050). The percentage of scratch area presented a significant difference among four groups [control group: (28.94±3.59)%, atorvastatin group: (31.00±2.99)%, doxorubicin group: (40.16±2.38)%, atorvastatin+ doxorubicin group: (60.86±3.60)%, F=42.080, P<0.050]. The scratch area of atorvastatin+ doxorubicin group was significantly increased compared with control group, atorvastatin group and doxorubicin group (all P<0.050). The number of cells penetrating membranes presented a significant difference among four groups (control group: 101.20±14.55, atorvastatin group: 75.80±7.33, doxorubicin group: 32.40±4.78, atorvastatin + doxorubicin group: 8.80±2.50, F=118.031, P<0.001). Pairwise comparison showed that the difference between groups was statistically significant (all P<0.050). After treatment with different drugs, the expressions of caspase 3 and caspase 9 (apoptosis-related proteins) in MDA-MB-231 cells presented a significant difference among four groups (F=128.854, 247.530, both P<0.001). Compared with doxorubicin group, the expression of caspase 3 and caspase 9 in atorvastatin+ doxorubicin group was significantly increased (both P<0.050). The expression of HMGCR, SREBP2 and LDLR in MDA-MB-231 cells presented a significant difference among four groups (F=183.193, 227.470, 586.087, all P<0.001). Pairwise comparison showed that compared with control group, HMGCR expression in atorvastatin+ doxorubicin group was significantly increased (P<0.050), while the expression of SREBP2 and LDLR was significantly decreased (both P<0.050); there was no significant difference in HMGCR expression between control group and doxorubicin group(P>0.050), while the expression of SREBP2 and LDLR in doxorubicin group was significantly lower than that in control group(both P<0.050); HMGCR expression in atorvastatin group was significantly higher than that in control group(P<0.050); compared with doxorubicin group, atorvastatin+ doxorubicin group presented significantly higher expression of HMGCR and lower expression of SREBP2 (both P<0.050), while LDLR expression presented no significant difference (P>0.050).

Conclusions

Atorvastatin reduces the synthesis of intracellular cholesterol by inhibiting HMGCR expression, making MDA-MB-231 cells more dependent on the uptake of exogenous cholesterol; doxorubicin reduces the uptake of exogenous cholesterol by inhibiting LDLR expression. If two drugs are used in combination, MDA-MB-231 cells are more dependent on the uptake of exogenous cholesterol due to reduced synthesis intracellular cholesterol, so they are more sensitive to doxorubicin.

Key words: Breast neoplasms, Cholesterol, Doxorubicin, Atorvastatin

表1 不同浓度多柔比星联合阿托伐他汀处理后的MDA-MB-231细胞活力比较(%)

阿托伐他汀	多柔比星
阿托伐他汀	0 μg/ml	0.02 μg/ml	0.04 μg/ml	0.08 μg/ml	0.16 μg/ml	0.32 μg/ml	0.64 μg/ml	1.28 μg/ml
0 μmol/L	100.00±1.33	96.73±1.54	88.16±1.27	81.52±1.63	56.95±2.17	41.31±1.33	30.02±0.03	16.56±1.06
2 μmol/L	95.97±2.06	91.25±1.24	82.84±0.56	68.33±1.31	37.38±1.36	26.98±1.11	22.98±2.55	18.86±1.78

表1 不同浓度多柔比星联合阿托伐他汀处理后的MDA-MB-231细胞活力比较(%)

阿托伐他汀	多柔比星
阿托伐他汀	0 μg/ml	0.02 μg/ml	0.04 μg/ml	0.08 μg/ml	0.16 μg/ml	0.32 μg/ml	0.64 μg/ml	1.28 μg/ml
0 μmol/L	100.00±1.33	96.73±1.54	88.16±1.27	81.52±1.63	56.95±2.17	41.31±1.33	30.02±0.03	16.56±1.06
2 μmol/L	95.97±2.06	91.25±1.24	82.84±0.56	68.33±1.31	37.38±1.36	26.98±1.11	22.98±2.55	18.86±1.78

图1 不同药物处理后MDA-MB-231乳腺癌细胞凋亡情况(Hoechst　×20)　a～d图分别所示对照组、阿托伐他汀组、多柔比星组及阿托伐他汀+多柔比星组细胞凋亡情况

图2 光镜下观察不同药物处理后MDA-MB-231乳腺癌细胞形态的变化(×10)　a～d图分别所示对照组、阿托伐他汀组、多柔比星组及阿托伐他汀+多柔比星组细胞形态变化

图3 Western blot检测不同药物处理后MDA-MB-231乳腺癌细胞中caspase 3、caspase 9的表达

表2 不同药物处理后MDA-MB-231乳腺癌细胞中caspase 3、caspase 9蛋白的表达

组别	样本量	caspase 3	caspase 9
对照组	3	0.29±0.02	0.14±0.02
阿托伐他汀组	3	0.49±0.04^a	0.13±0.02
多柔比星组	3	0.68±0.01^ab	0.49±0.04^ab
阿托伐他汀+多柔比星组	3	0.82±0.04^abc	0.57±0.02^abc
F值		128.854	247.53
P值		<0.001	<0.001

表2 不同药物处理后MDA-MB-231乳腺癌细胞中caspase 3、caspase 9蛋白的表达

组别	样本量	caspase 3	caspase 9
对照组	3	0.29±0.02	0.14±0.02
阿托伐他汀组	3	0.49±0.04^a	0.13±0.02
多柔比星组	3	0.68±0.01^ab	0.49±0.04^ab
阿托伐他汀+多柔比星组	3	0.82±0.04^abc	0.57±0.02^abc
F值		128.854	247.53
P值		<0.001	<0.001

图4 不同药物处理后MDA-MB-231乳腺癌细胞划痕实验结果(×20)　a～d图分别所示对照组、阿托伐他汀组、多柔比星组及阿托伐他汀+多柔比星组细胞划痕实验结果

图5 不同药物处理后MDA-MB-231乳腺癌细胞Transwell实验结果(结晶紫　×20)　a～d图分别所示对照组、阿托伐他汀组、多柔比星组及阿托伐他汀+多柔比星组细胞Transwell实验结果

图6 不同药物处理后MDA-MB-231乳腺癌细胞中胆固醇代谢相关蛋白表达

表3 不同药物处理后MDA-MB-231乳腺癌细胞中HMGCR、SREBP2、LDLR蛋白的表达

组别	样本量	HMGCR	SREBP2	LDLR
对照组	3	0.38±0.03	1.03±0.02	0.85±0.06
阿托伐他汀组	3	0.79±0.01^a	0.84±0.07	0.93±0.01
多柔比星组	3	0.33±0.01^b	0.38±0.01^ab	0.04±0.01^ab
阿托伐他汀+多柔比星组	3	0.70±0.03^ac	0.26±0.01^abc	0.05±0.02^ab
F值		183.193	227.470	586.087
P值		<0.001	<0.001	<0.001

表3 不同药物处理后MDA-MB-231乳腺癌细胞中HMGCR、SREBP2、LDLR蛋白的表达

组别	样本量	HMGCR	SREBP2	LDLR
对照组	3	0.38±0.03	1.03±0.02	0.85±0.06
阿托伐他汀组	3	0.79±0.01^a	0.84±0.07	0.93±0.01
多柔比星组	3	0.33±0.01^b	0.38±0.01^ab	0.04±0.01^ab
阿托伐他汀+多柔比星组	3	0.70±0.03^ac	0.26±0.01^abc	0.05±0.02^ab
F值		183.193	227.470	586.087
P值		<0.001	<0.001	<0.001

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Statins increase sensitivity of breast cancer cells to doxorubicin

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Statins increase sensitivity of breast cancer cells to doxorubicin