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

(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.

(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).

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.