Rapamycin promotes the anticancer action of dihydroartemisinin in breast cancer MDA-MB-231 cells by regulating expression of Atg7 and DAPK

Abstract

There is limited knowledge regarding the influence of autophagy on the anticancer effect of dihydroartemisinin (DHA). The present study aimed to investigate this influence within human breast cancer cells. Changes in cell viability, cell cycle distribution, apoptosis and associated genes were analyzed in MDA-MB-231 cells subjected to DHA following alteration in autophagy levels; the autophagy level was decreased following autophagy-related 7 (Atg7) knockdown or increased using rapamycin. The data indicated that rapamycin had the ability to notably enhance the anticancer effect of DHA on MDA-MB-231 cells. Autophagy induction may be key in mediating the anticancer effects of DHA, and rapamycin may regulate the death-associated protein kinase via the alteration of Atg7 expression, which would influence cell apoptosis. The present study presented a novel insight into enhancing the effectiveness of future treatment regimens for breast cancer using DHA.

Keywords: MDA-MB-231; autophagy; autophagy-related 7 small interfering RNA; breast cancer; dihydroartemisinin; rapamycin.

Figure 1.

Autophagy induction promoted the death of DHA-treated MDA-MB-231 breast cancer cells. Rapamycin inhibited proliferation and Atg7 deficiency promoted survival in DHA-treated MDA-MB-231 breast cancer cells. MDA-MB-231 cells were examined an MTT assay (6, 12, 24, 48 and 72 h), normalized to the cell viability of control-MDA-MB-231 cells. This figure indicated that Atg7 knockdown increased the viability of MDA-MB-231 breast cancer cells and rapamycin decreased the viability. This graph indicates that Atg7 may function as a promoter of the anticancer effect of DHA. These data indicated that Atg7 knockdown facilitated the survival of MDA-MB-231 breast cancer cells, whereas rapamycin treatment enhanced the DHA action on this breast cancer cell line. DHA, dihydroartemisinin; Atg7, autophagy-related 7. *P<0.05.

Figure 2.

Atg7 may regulate DAPK to inhibit proliferation of the MDA-MB-231 cells. The mRNA levels of Atg7 increased, and DAPK changed accordingly. Atg7 and DAPK gene expressions in the DHA-treated MDA-MB-231 cells at 24 h were determined by reverse transcription-quantitative polymerase chain reaction (β-actin was used as a reference). The expression levels of Atg7 and DAPK increased in the rapamycin group, and decreased in the Atg7-knockdown group. DHA, dihydroartemisinin; Atg7, autophagy-related 7; DAPK, death-associated protein kinase. *P<0.05.

Figure 3.

Rapamycin enhanced the pro-apoptotic effect of DHA in MDA-MB-231 cells. Cell cycle distribution and the proportion of apoptotic cells were detected using a flow cytometer at 24 and 48 h. (A) Cell numbers during the apoptotic phase (SubG₁) of the MDA-MB-231 cells at 24 h. (B) Cell numbers during the apoptotic phase (SubG₁) in the MDA-MB-231 cells at 48 h. Treatment with rapamycin notably promoted the effect of DHA on apoptosis at 24 and 48 h, compared with the Atg7 deficient group. (C) Cell cycle distribution and apoptosis graphs from flow cytometry. DHA, dihydroartemisinin; Atg7, autophagy-related 7; PI, propidium iodide. *P<0.05.