Effects of metformin alone or in combination with compound C on proliferation and cyclinD1 expression in mouse breast cancer 4T1 cells

doi:10.3877/cma. j. issn.1674-0807.2014.04.007

Abstract

Abstract:

Objective

To evaluate the effects of metformin alone or in combination with AMPK inhibitor compound C on proliferation and cyclin D1 expression in mouse breast cancer 4T1 cells and explore the possible role of AMPK pathway in the antitumor mechanism of metformin.

Methods

The mouse breast cancer 4T1 cells were cultured in vitro until the logarithm growth period, and then treated with 2.5, 5, 10, 20,40 mmol/L metformin and 20 μmol/L compound C respectively and 20 μmol/L compound C combined with 10 mmol/L metformin for 24,48,72 h. CCK-8 method was used to study the effects of metformin with different concentrations,20 μmol/L compound C+10 mmol/L metformin, 20 μmol/L compound C on cell proliferation.The expression of cyclinD1 was detected with Western blot method. Multiple group comparison was performed by F test, pairwise comparison by q test, analysis of variance for repeated measurement data before and after treatment.

Results

CCK-8 test showed that in vitro cell culture, at 24, 48 h after 2.5 mmol/L metformin treatment, there was no significant difference in cell survival compared with control group(24 h:F=2.87,P=0.129;48 h:F=1.63, P=0.290), but a significant difference was observed at 72 h (F=6.40, P=0.000).at 24,48 and 72 h after 5,10,20,40 mmol/L metformin treatment, the proliferation of mouse breast cancer 4T1 cells was significantly inhibited by metformin in a dose-dependent and time-dependent manner(F=332.89,363.54,352.11, all P=0.000). Meanwhile in compound C group and 10 mmol/L metformin plus compound C group, cell survival showed no significant difference compared with the control group(F=1.08, P=2.283;F=1.92,P=0.050). The results in Western blot analysis, the expression of cyclin D1 decreased with the increase of metformin concentration (F=54.41, 61.69, 75.84, all P=0.000), but showed no significant difference(F=1.87, P=0.190) between 10 mmol/L metformin plus compound C group and control group.

Conclusions

Metformin can inhibit the cell proliferation of mouse breast cancer 4T1 cells, but compound C can antagonize the inhibition of metformin. Metformin can decrease cyclin D1 protein expression by activating AMPK pathway in mouse breast cancer 4T1 cells.

Key words: Metformin, Breast neoplasms, 4T1 cells, Cyclin D1

Hongquan Du, Lili Huang, Aihua Jia, Qunlong Jiang, Guangzhen Zhang, Jie Bai. Effects of metformin alone or in combination with compound C on proliferation and cyclinD1 expression in mouse breast cancer 4T1 cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2014, 08(04): 258-265.

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References 23

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组别	存活率			合计	F值	P值
组别	24 h	48 h	72 h	合计	F值	P值
组2	1	1	1	1	0	1
组3	0.97±0.04	0.86±0.05	0.81±0.06	0.88±0.08	41.55	0.000
组4	0.65±0.03	0.64±0.04	0.59±0.03	0.63±0.04	5.21	0.008
组5	0.57±0.02	0.54±0.01	0.52±0.01	0.55±0.02	3.77	0.028
组6	0.53±0.01	0.47±0.01	0.47±0.01	0.49±0.03	9.87	0.000
组7	0.50±0.02	0.40±0.01	0.40±0.01	0.43±0.05	21.05	0.000
合计	0.70±0.02	0.65±0.03	0.63±0.03	0.66±0.05	273.79^a	0.000^a
F值	332.89	363.54	352.11	2193.07^a	30.01^b
P值	0.000	0.000	0.000	0.000^a	0.000^b

组别	存活率			合计	F值	P值
组别	24 h	48 h	72 h	合计	F值	P值
组2	1	1	1	1	0	1
组3	0.97±0.04	0.86±0.05	0.81±0.06	0.88±0.08	41.55	0.000
组4	0.65±0.03	0.64±0.04	0.59±0.03	0.63±0.04	5.21	0.008
组5	0.57±0.02	0.54±0.01	0.52±0.01	0.55±0.02	3.77	0.028
组6	0.53±0.01	0.47±0.01	0.47±0.01	0.49±0.03	9.87	0.000
组7	0.50±0.02	0.40±0.01	0.40±0.01	0.43±0.05	21.05	0.000
合计	0.70±0.02	0.65±0.03	0.63±0.03	0.66±0.05	273.79^a	0.000^a
F值	332.89	363.54	352.11	2193.07^a	30.01^b
P值	0.000	0.000	0.000	0.000^a	0.000^b

组别	存活率			合计	F值	P值
组别	24 h	48 h	72 h	合计	F值	P值
组2	1	1	1	1	0	1
组8	0.99±0.03	0.99±0.03	0.95±0.04	0.98±0.04	1.03	0.366
组9	0.97±0.03	0.96±0.01	0.92±0.15	0.95±0.09	1.64	0.205
组10	0.63±0.01	0.58±0.05	0.47±0.02	0.56±0.07	12.30	0.000
合计	0.86±0.17	0.85±0.19	0.78±0.24	0.83±0.06	28.43^a	0.000^a
F值	60.38	76.47	112.40	866.72^{^a}	7.88^b
P值	0.000	0.000	0.000	0.000^a	0.000^b

组别	存活率			合计	F值	P值
组别	24 h	48 h	72 h	合计	F值	P值
组2	1	1	1	1	0	1
组8	0.99±0.03	0.99±0.03	0.95±0.04	0.98±0.04	1.03	0.366
组9	0.97±0.03	0.96±0.01	0.92±0.15	0.95±0.09	1.64	0.205
组10	0.63±0.01	0.58±0.05	0.47±0.02	0.56±0.07	12.30	0.000
合计	0.86±0.17	0.85±0.19	0.78±0.24	0.83±0.06	28.43^a	0.000^a
F值	60.38	76.47	112.40	866.72^{^a}	7.88^b
P值	0.000	0.000	0.000	0.000^a	0.000^b

组别	cyclin D1相对表达量			合计	F值	P值
组别	24 h	48 h	72 h	合计	F值	P值
组2	0.74±0.04	0.70±0.01	0.69±0.01	0.71±0.03	4.63	0.020
组4	0.64±0.01	0.60±0.01	0.54±0.01	0.60±0.04	16.84	0.000
组5	0.58±0.01	0.53±0.03	0.48±0.01	0.53±0.05	11.23	0.000
组6	0.53±0.02	0.49±0.01	0.45±0.01	0.49±0.04	17.41	0.000
合计	0.62±0.08	0.58±0.09	0.54±0.09	0.58±0.04	191.00^a	0.000^a
F值	54.41	61.69	75.84	732.39^a	4.38^b
P值	0.000	0.000	0.000	0.000^a	0.000^b

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