Effects of Metformin on ALDH1 Expression in Pancreatic Cancer and Pancreatic Cancer Stem Cells
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摘要:目的
观察二甲双胍对胰腺癌PANC-1细胞和胰腺癌干细胞中ALDH1表达的影响，探讨其作用机制。
方法采用CCK-8和Western blot检测不同浓度二甲双胍对胰腺癌PANC-1细胞的增殖及ALDH1和p-mTOR蛋白表达的影响。Western blot、免疫荧光法及qRT-PCR检测二甲双胍、雷帕霉素以及联合用药组PANC-1细胞中ALDH1、4EBP1、p-mTOR蛋白及mRNA的表达。超低黏附培养板悬浮培养胰腺癌细胞后观察干细胞成球数目的改变以及Western blot检测干细胞ALDH1的表达；建立胰腺癌裸鼠移植瘤模型比较PANC-1细胞以及胰腺癌成球细胞的成瘤能力。
结果二甲双胍抑制胰腺癌PANC-1细胞的增殖，且呈时间剂量依赖性，最佳干预时间为48 h，IC50约为20 mmol/L；与对照组相比，二甲双胍、雷帕霉素和联合用药组ALDH1、4EBP1、p-mTOR的ALDH1的蛋白表达以及mRNA水平明显降低（P < 0.05）。胰腺癌成球细胞的体外成瘤能力以及ALDH1的表达比PANC-1细胞组显著增高；相比于对照组，二甲双胍、雷帕霉素和联合用药组胰腺癌细胞的成球能力以及胰腺癌干细胞中ALDH1的表达均显著降低（P < 0.05）。
结论二甲双胍以及雷帕霉素均能显著抑制胰腺癌PANC-1细胞和胰腺癌干细胞ALDH1的表达，其作用机制可能是通过抑制mTOR通路。
Abstract:ObjectiveTo investigate the effect of metformin on ALDH1 expression in pancreatic cancer cells and pancreatic cancer stem cells, and explore its mechanism.
MethodsPANC-1 cells were used to detect the inhibition rate and the expression levels of ALDH1 and p-mTOR by CCK-8 and Western blot after the intervention of metformin in different concentrations, respectively. Rapamycin is an inhibitor of mTOR; Western blot, immunofluorescence and qRT-PCR were employed to detect the protein expression and mRNA level of ALDH1, 4EBP1, p-mTOR among metformin group, rapamycin group and combination group. We used ultra low adhesion culture plate to culture PANC-1 cell, the number of spheroid body cells were observed and the expression of ALDH1 protein level were detected by Western blot in pancreatic cancer stem cells. The tumorigenicability between pancreatic cancer cells and spheroid body cells were compared by establishing nude mouse transplantation tumor model of pancreatic cancer.
ResultsMetformin could inhibit the proliferation of PANC1 along with the increase of treatment time and metformin concentrations, the optimal concentration and respond time of metformin were 20mmol/L and 48h; Compared with control group, the protein expression and mRNA level of ALDH1, 4EBP1 and p-mTOR among metformin group, rapamycin group and combination group were suprressed significantly (P < 0.05). The tumorigenic potential in spheroid body cells was significantly higher than that in parental cells; Compared with control group, the tumor sphere forming capability and the expression of ALDH1 in pancreatic cancer stem cells were inhibited among metformin group, rapamycin group and combination group (P < 0.05).
ConclusionMetformin and rapamycin significantly inhibit the expression of ALDH1 in pancreatic cancer PANC-1 cells and pancreatic cancer stem cells, and the mechanism may be through suprressing mTOR pathway.
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Key words:
- Metformin /
- Rapamycin /
- mTOR /
- ALDH1 /
- Pancreatic cancer stem cells
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图 1 Western blot检测不同浓度的二甲双胍对胰腺癌PANC-1细胞中ALDH与p-mTOR蛋白表达的影响
Figure 1 Effect of different concentrations of metformin on protein expression levels of ALDH1A1 and p-mTOR in PANC-1 cells evaluated by Western blot
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图 2 四组PANC-1细胞中4EBP1，ALDH1A1，p-mTOR蛋白的表达
Figure 2 Protein expression levels of 4EBP1, ALDH1A1, p-mTOR in four groups evaluated by Western blot
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图 3 四组PANC-1细胞中4EBP1, ALDH1A1, p-mTOR mRNA的表达
Figure 3 mRNA levels of 4EBP1, ALDH1A1, p-mTOR in four groups evaluated by qRT-PCR
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图 4 免疫荧光检测ALDH1A1在PANC-1细胞胞质中的表达 (×200)
Figure 4 Immunofluorescence detection of ALDH1A1 expressin in PANC-1 cytoplasm (×200)
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图 5 四组PANC-1细胞成球数目的比较 (×100)
Figure 5 Comparison of PANC-1 spheroid body cells number in four groups (×100)
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图 6 不同组成球细胞中ALDH1A1蛋白的表达
Figure 6 The expressions of ALDH1A1 in different groups of spheroid body cells
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图 7 不同组中裸鼠移植瘤体积的比较
Figure 7 The volume comparison of nude mice transplanted tumors in different groups
表 1 不同浓度二甲双胍对PANC-1细胞增殖的影响
Table 1 Effects of different concentrations of metformin on the proliferation of PANC-1 cells
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