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Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway

  • Authors:
    • Huili Feng
    • Lixin He
    • Talha Umar
    • Haipeng Wang
    • Yanling Gao
    • Gongyi Chen
    • Panfeng Sun
    • Ling Yin
    • Wenbin Zhao
    • Huifang Lu
    • Ganzhen Deng
    • Changwei Qiu
  • View Affiliations / Copyright

    Affiliations: School of Pet Science and Technology, Henan Vocational College of Agriculture, Zhengzhou, Henan 451450, P.R. China, Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China
    Copyright: © Feng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 131
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    Published online on: May 14, 2026
       https://doi.org/10.3892/or.2026.9136
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Abstract

Breast cancer remains one of the leading causes of cancer‑related mortality among women globally, necessitating the exploration of novel therapeutic strategies. The present study investigated the combined effects of Ivermectin (IVM) and metformin (MET) on the human mammary tumor cell line MCF‑7, with a focus on their mechanisms of action. Cell Counting Kit‑8 assays were employed to evaluate proliferative activity, while Transwell migration and scratch assays assessed invasive and migratory capabilities. Transcriptomic analysis identified differentially expressed genes and key signalling pathways, while flow cytometry quantified reactive oxygen species (ROS) levels. Autophagosome formation was visualized using transmission electron microscopy. Western blotting and immunofluorescence staining were employed to detect changes in the expression of key proteins. Results demonstrated that combined IVM and MET intervention significantly inhibited MCF‑7 cell viability after 24 h, showing concentration‑dependent reductions in proliferation, migration and invasiveness. Transcriptomic analysis revealed a significant enrichment of the PI3K/AKT/mTOR signaling pathway, accompanied by decreased expression of thrombospondin‑1 (THBS1). Western blotting revealed that the combination therapy reduced phosphorylation levels of phosphorylated (p‑)PI3K, p‑AKT and p‑mTOR. Through the use of THBS1 overexpression plasmids, the present study validated its positive regulatory role in the PI3K/AKT/mTOR pathway, demonstrating that THBS1 mediates phosphorylation within this signaling cascade. Flow cytometry analysis demonstrated that the IVM combined with MET treatment group exhibited significantly elevated intracellular ROS levels compared with the single‑drug therapy group, triggering oxidative stress responses. After clearing ROS using N‑acetyl‑L‑cysteine, the PI3K/AKT/mTOR signaling pathway was reactivated. Additionally, transmission electron microscopy revealed extensive autophagosome formation within tumor cells of the IVM‑MET combination group. The results indicate that IVM‑MET inhibits PI3K/AKT/mTOR pathway phosphorylation through the accumulation of ROS and the downregulation of THBS1 expression, thereby activating autophagy programs and ultimately leading to tumor cell death.
View Figures

Figure 1

Effects of IVM, MET and the
combination of the two drugs on the toxicity and proliferation of
MCF-7 cells. (A) Cell viability assays after treatment of MCF-7
cells with IVM. (B) Cell viability assays after treatment of MCF-7
cells with MET. (C) CCK-8 assay evaluating cell viability in MCF-7
cells treated with PBS, IVM, MET and IVM + MET at 0, 12, 24, 48 and
72 h; (D) Comparison of MCF-7 cell viability after co-treatment
with 2, 4 µM and 6 mMdoses of IVM combined with 2, 4 and 6 mmol/l
doses of MET using the CCK-8 method. CCK-8, Cell Counting Kit-8;
IVM, ivermectin; MET, metformin, MCF-7, human mammary tumor cell
line; Ctrl, control. All data were measured as mean ± SEM by three
independent experiments. *P<0.05.

Figure 2

IVM combined with MET inhibits the
invasive and migratory ability of MCF-7 cells. (A) Representative
Transwell images and (B) quantification of the invasive ability of
MCF-7 cells. (C) Representative scratch images and (D)
quantification of the migration ability in [Ctrl, IVM (6 µM), MET
(6 mM), and IVM + MET] groups of MCF-7 cells. IVM, ivermectin; MET,
metformin, MCF-7, human mammary tumor cell line; Ctrl, control. All
data were measured as mean ± SEM by three independent experiments.
**P<0.01.

Figure 3

Western blotting detection of THBS1
protein expression in MCF-7 cells. (A) Representative western
blotting image shows the expression of THBS1 protein in MCF-7 cells
was downregulated. The molecular weights of THBS1 and β-actin are
indicated as 180 and 42 kDa, respectively. (B) Semi-quantification
of the western blotting results. IVM, ivermectin (6 µM); MET,
metformin (6 mM); MCF-7, human mammary tumor cell line; Ctrl,
control; +, with treatment. All data were measured as mean ± SEM by
three independent experiments. **P<0.01.

Figure 4

IVM combined with MET reduces the
phosphorylation levels of proteins in the PI3K/AKT/mTOR pathway.
(A) Representative images of western blotting and, (B) The
expression levels of PI3K, AKT, mTOR and their phosphorylated
proteins in MCF-7 cells treated with PBS, IVM, and IVM + MET. IVM,
ivermectin; MET, metformin, MCF-7, human mammary tumor cell line;
Ctrl, control; p, phosphorylated. All data were measured as mean ±
SEM by three independent experiments. *P<0.05, **P<0.01.

Figure 5

Overexpression of THBS1 enhances the
phosphorylation level of proteins in the PI3K/AKT/mTOR pathway. (A)
Schematic diagram of THBS1 overexpression plasmid construction. (B)
Detection of THBS1 CDS region amplification by agarose gel
electrophoresis. (C) Representative western blotting images and (D)
semi-quantification of THBS1 overexpression efficiency in MCF-7
cells. MCF-7, human mammary tumor cell line. All data were measured
as mean ± SEM by three independent experiments. **P<0.01.

Figure 6

Autophagy induction in MCF-7 cells by
IVM combined with MET. (A) Representative immunofluorescence images
showing the expression and localization of the autophagosome marker
LC3B (red) in MCF-7 cells treated with PBS (Ctrl), IVM (6 µM), MET
(6 mM) or IVM + MET for 24 h. Nuclei are stained with DAPI (blue).
The pronounced increase in LC3B puncta formation (indicated by
arrows) in the IVM + MET-treated group indicates autophagosome
accumulation, a hallmark of autophagy induction. Scale bar, 200 µm.
(B) Representative immunofluorescence images showing the expression
and localization of the autophagic flux marker p62/SQSTM1 (red) in
MCF-7 cells under the same treatment conditions as in (A). Nuclei
are stained with DAPI (blue). The marked reduction in p62
fluorescence intensity in the IVM + MET-treated group reflects
enhanced autophagic degradation, confirming active autophagic flux.
Scale bar, 200 µm. (C) Representative transmission electron
microscopy images of MCF-7 cells following 24 h of treatment.
Images are shown at increasing magnifications with scale bars of 2
µm (top), 1 µm (middle), and 500 nm (bottom) for each treatment
group (Ctrl, IVM, MET and IVM + MET). Numerous double-membrane
autophagosomes (indicated by red arrows) are evident in the IVM +
MET-treated cells, providing ultrastructural confirmation of
autophagy activation. (D) Western blot analysis of key
autophagy-related proteins (LC3B-I/II, Beclin1, p62 and Bcl-2) in
MCF-7 cells treated as indicated for 24 h. β-actin serves as the
loading control. (E) Densitometric semi-quantification of the
protein levels. (F) Cell viability assessment by Cell Counting
Kit-8 assay. Pre-treatment with 3-MA significantly rescued the loss
of cell viability induced by IVM + MET, confirming the functional
contribution of autophagy to the cytotoxic effect. IVM, ivermectin;
MET, metformin, MCF-7, human mammary tumor cell line; Ctrl,
control. All data were measured as mean ± SEM by three independent
experiments. *P<0.05, **P<0.01.

Figure 7

IVM combined with MET promotes the
accumulation of ROS in MCF-7 cells. (A) Representative flow
cytometry plot and (B) quantification of ROS expression levels in
MCF-7 cells after treatment with different experimental groups. PC,
Ctrl, IVM, MET and IVM + MET groups are shown. (C) Representative
flow cytometry plot and (D) quantification of ROS expression levels
in MCF-7 cells after treatment with NAC. PC, Ctrl, IVM, MET, and
IVM + MET groups are shown. (E) Representative western blotting
images and (F) semi-quantification of p-PI3K, p-AKT and p-mTOR
protein expression levels in MCF-7 cells after treatment with
different experimental groups; (G) Representative western blotting
images and (H) semi-quantification of autophagy-related protein
expression levels in MCF-7 cells treated with PBS, IVM, MET and IVM
+ MET. ROS, reactive oxygen species; p, phosphorylated; IVM,
ivermectin; MET, metformin, MCF-7, human mammary tumor cell line;
Ctrl, control; PC, positive control. All data were measured as mean
± SEM by three independent experiments. *P<0.05,
**P<0.01.
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Copy and paste a formatted citation
Spandidos Publications style
Feng H, He L, Umar T, Wang H, Gao Y, Chen G, Sun P, Yin L, Zhao W, Lu H, Lu H, et al: Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway. Oncol Rep 56: 131, 2026.
APA
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G. ... Qiu, C. (2026). Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway. Oncology Reports, 56, 131. https://doi.org/10.3892/or.2026.9136
MLA
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G., Sun, P., Yin, L., Zhao, W., Lu, H., Deng, G., Qiu, C."Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway". Oncology Reports 56.1 (2026): 131.
Chicago
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G., Sun, P., Yin, L., Zhao, W., Lu, H., Deng, G., Qiu, C."Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway". Oncology Reports 56, no. 1 (2026): 131. https://doi.org/10.3892/or.2026.9136
Copy and paste a formatted citation
x
Spandidos Publications style
Feng H, He L, Umar T, Wang H, Gao Y, Chen G, Sun P, Yin L, Zhao W, Lu H, Lu H, et al: Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway. Oncol Rep 56: 131, 2026.
APA
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G. ... Qiu, C. (2026). Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway. Oncology Reports, 56, 131. https://doi.org/10.3892/or.2026.9136
MLA
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G., Sun, P., Yin, L., Zhao, W., Lu, H., Deng, G., Qiu, C."Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway". Oncology Reports 56.1 (2026): 131.
Chicago
Feng, H., He, L., Umar, T., Wang, H., Gao, Y., Chen, G., Sun, P., Yin, L., Zhao, W., Lu, H., Deng, G., Qiu, C."Combination of ivermectin and metformin promotes autophagy in MCF‑7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway". Oncology Reports 56, no. 1 (2026): 131. https://doi.org/10.3892/or.2026.9136
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