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. 2020 Mar 4;13:1909-1919.
doi: 10.2147/OTT.S241632. eCollection 2020.

Berberine Reverses Doxorubicin Resistance by Inhibiting Autophagy Through the PTEN/Akt/mTOR Signaling Pathway in Breast Cancer

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Free PMC article

Berberine Reverses Doxorubicin Resistance by Inhibiting Autophagy Through the PTEN/Akt/mTOR Signaling Pathway in Breast Cancer

Ye Wang et al. Onco Targets Ther. .
Free PMC article

Abstract

Purpose: Berberine (BBR), a traditional Chinese medicine, has been shown effects on inhibiting cancer development. Autophagy-mediated resistance plays an important role in cancer progression; therefore, regulation of autophagy is a novel therapeutic strategy for cancer treatment. However, effects of BBR on autophagy-mediated resistance have not been reported.

Methods: MCF-7 breast cancer cells and the doxorubicin (ADR)-resistant MCF-7 cells (MCF-7/ADR) were used for analyses. Western blotting was conducted to evaluate protein expression; MTT, colony formation, and EdU assays were conducted to assess cell proliferation; transmission electron microscopy was used to monitor autophagy levels; and a xenograft tumor model was established to assess the effects of BBR on reversing doxorubicin resistance.

Results: We confirmed that BBR, recently identified as a suppressor of autophagy, inhibits autophagosome formation in MCF-7/ADR cells. Treatment with BBR blocked the accumulation of the autophagy-associated protein LC3II, resulting in cellular accumulation of p62, reduced cell proliferation, and reversal of doxorubicin resistance. Mechanistically, we found that BBR inhibited autophagy by modulating the PTEN/Akt/mTOR signaling pathway. In vivo, our study showed that BBR exerts clear anti-tumor effects.

Conclusion: The results of this study suggest that BBR reverses doxorubicin resistance in breast cancer cells by inhibiting autophagy. This finding highlights the potential clinical application of BBR in the treatment of breast cancer.

Keywords: ADR; PTEN; autophagy; berberine; breast cancer; chemoresistance.

Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Doxorubicin (ADR) resistance is related to a high level of autophagy in ADR-resistant MCF-7 (MCF-7/ADR) cells. (A) MCF-7 and MCF-7/ADR cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and then post-fixed in 1% OsO4 for 2 h at 4°C. The cells were dehydrated via a graded ethanol series and embedded in LR White resin. The solidified blocks were cut into ultrathin sections and stained with uranyl acetate and lead citrate. Samples were observed under a transmission electron microscope. MCF-7/ADR cells showed higher autophagosome levels than MCF-7 cells. Yellow arrows indicate Autophagosome. (B) Total protein was extracted using a combination of RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% sodium deoxycholate, 200 mM NaF, 200 mM PMSF, 1.0% NP40, and 1 mM EDTA), PMSF, and phosphatase inhibitors. Lysates (10 uL) were subjected to SDS–PAGE and then transferred to PVDF membranes. Membranes were analyzed using ImageJ software. Western blotting analysis showed that MCF-7/ADR cells exhibit higher expression of the autophagy-associated protein LC3II/I, and lower expression of p62 protein, than MCF-7 cells. (C) MCF-7/ADR cells were treated with the autophagy inhibitor 3-MA (5 mM). Western blotting analysis showed that 3-MA treatment inhibited the expression of LC3II/I and MDR1. (D) MCF-7/ADR cells were treated with ADR, 3-MA, or a combination of both. MTT was added after 48 h. After 4 h of incubation, the MTT solution was discarded and DMSO was added for 10 min with slow shaking. Absorbance was measured at 550 nm using a spectrophotometer. The results showed that 3-MA treatment increased the sensitivity of MCF-7/ADR cells to ADR. N/A, not significant; ***p<0.001 (Student’s t-test).
Figure 2
Figure 2
Berberine (BBR) reverses doxorubicin (ADR) resistance in ADR-resistant MCF-7 (MCF-7/ADR) cells. (A) MTT analysis showed that combined treatment with BBR and ADR inhibited cell growth in a dose-dependent manner in MCF-7/ADR cells. (B) MCF-7/ADR cells were treated with BBR and/or ADR. Western blotting analysis showed that BBR treatment inhibited MDR1 expression in these ADR-resistant cells (BBR, 100 uM; ADR, 0.517 uM). (C) MCF-7/ADR cells were treated with BBR and/or ADR. MTT analysis showed that BBR treatment increased the sensitivity of the cells to ADR (BBR, 100 uM; ADR, 0.517 uM). N/A, not significant; ***p<0.001 (Student’s t-test).
Figure 3
Figure 3
Berberine (BBR)-mediated reversal of doxorubicin (ADR) resistance is associated with inhibition of autophagy. (A) ADR-resistant MCF-7 (MCF-7/ADR) cells were treated with BBR and/or ADR. Combined ADR and BBR treatment significantly decreased the number of autophagosomes in the MCF-7/ADR cells (BBR, 100 µM; ADR, 0.517 µM). Yellow arrows indicate Autophagosome. (B) MCF-7/ADR cells were treated with BBR and/or ADR. Western blotting analysis showed that combined ADR and BBR treatment led to a significant decrease in the expression of LC3I/II and an increase in that of p62 in MCF-7/ADR cells (BBR, 100 µM; ADR, 0.517 µM). (C) After treatment with rapamycin, MCF-7/ADR cells were then treated with BBR and/or ADR. In the presence of rapamycin, BBR treatment induced no significant change in the activity of MCF-7/ADR cells (BBR, 100 µM; ADR, 0.517 µM; rapamycin, 50 nM). N/A, not significant; ***p<0.001 (Student’s t-test).
Figure 4
Figure 4
Berberine (BBR) inhibits autophagy and reverses doxorubicin (ADR) resistance through inhibition of the PTEN/Akt/mTOR signaling pathway. (A) ADR-resistant MCF-7 (MCF-7/ADR) cells were treated with BBR and/or ADR. Western blotting analysis showed that BBR treatment led to a significant decrease in PTEN expression and increased the activation of Akt and mTOR in MCF-7ADR cells (BBR, 100 µM; ADR, 0.517 µM). (B) MCF-7/ADR cells were treated with BBR and/or transfected with siPTEN. This combined siPTEN and BBR treatment significantly reduced the autophagosome number in MCF-7/ADR cells (BBR, 100 µM). Yellow arrows indicate Autophagosome. (C) MCF-7/ADR cells were treated with BBR and/or transfected with siPTEN. The results showed that the combination of siPTEN and BBR treatment increased Akt and mTOR activation (BBR, 100 µM). (D) MCF-7/ADR cells were treated with BBR and/or transfected with PTEN-expressing plasmids. The results showed that PTEN overexpression combined with BBR treatment induced no significant changes in autophagosome number in MCF-7/ADR cells (BBR, 100 µM). (E) MCF-7/ADR cells were treated with BBR and/or transfected with PTEN-expressing plasmids. Western blotting analysis showed that PTEN overexpression combined with BBR treatment did not lead to significant changes in the levels of phosphorylated Akt and mTOR (BBR, 100 µM). (F) Cells overexpressing PTEN or PTEN-knockdown cells were treated with BBR and/or ADR. Analysis by MTT assay showed that BBR treatment significantly inhibited the activity of PTEN-knockdown cells, whereas no significant effects were observed in PTEN-overexpressing breast cancer cells (BBR, 100 µM; ADR, 0.517 µM). N/A, not significant; ***p<0.001 (Student’s t-test).
Figure 5
Figure 5
Berberine (BBR) reverses doxorubicin (ADR) resistance in vivo. (A) ADR-resistant MCF-7 (MCF-7/ADR) cells were subcutaneously injected into BALB/C nude female mice to establish a xenograft tumor model. (B) A combination of BBR and ADR significantly inhibited tumor growth in vivo. (C) Tumor volumes were measured every 3 days. The results showed that BBR treatment significantly inhibited tumor growth in vivo. (D) Body weight was measured every 3 days. The results showed that BBR and/or ADR treatment did not affect body weight in vivo. (E) Western blotting analysis of extracted tumor tissues showed that BBR treatment inhibited the expression of PTEN and LC3II/I and increased Akt phosphorylation levels (BBR, 10 mg/kg; ADR, 4 mg/kg). N/A, not significant; ***p<0.001 (Student’s t-test).
Figure 6
Figure 6
Berberine reverses doxorubicin resistance by inhibiting autophagy through the PTEN/Akt/mTOR signaling pathway in breast cancer. BBR inhibits autophagy, therefore, reversing ADR resistance by inhibiting the PTEN/Akt/mTOR signaling pathway. Akt phosphorylation results in mTORC1 activation and mTORC1 activation has inhibited effects on the PI3K/Akt signaling pathway. A positive regulation exists between the PI3K signaling and mTORC2. Activation of mTORC1 downregulates autophagy, and Akt phosphorylation by mTORC2 also contributes to inhibition of autophagy.

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