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. 2024 Oct 23:15:1494265.
doi: 10.3389/fphar.2024.1494265. eCollection 2024.

Synergistic effects of anlotinib and DDP on breast cancer: targeting the VEGF/JAK2/STAT3 axis

Hongmei Zhang #  1   2 , Chunling Liu #  3 , Ye Jin  2 , Zheng Wang  2 , Yi Guan  1 , Zhenxian Jia  1 , Tong Cui  4 , Zhi Zhang  5 , Xuemei Zhang  1   4
Affiliations

Synergistic effects of anlotinib and DDP on breast cancer: targeting the VEGF/JAK2/STAT3 axis

Hongmei Zhang et al. Front Pharmacol. .

Abstract

Background: Anlotinib, a highly selective inhibitor of VEGFR2, has demonstrated significant anti-tumor effects in various cancers. However, its potential synergistic effects with DDP (cisplatin) in breast cancer (BRCA) remain to be fully elucidated. This study aims to discover the therapeutic efficacy of anlotinib on BRCA, specifically the synergistic effects with DDP, and to elucidate the underlying molecular mechanisms.

Methods: BRCA cells were treated with anlotinib and/or DDP. The proliferation, migration and invasion capabilities of BRCA cells were evaluated using CCK-8 assays, cell cycle distribution, clone formation assays, wound healing assays and transwell assays. Cell apoptosis was detected by flow cytometry technique and Hoechst33342 fluorescence staining. The potential mechanism of anlotinib in the development of BRCA was predicted through bioinformatics analysis, and the mRNA or protein levels were subsequently quantified using qPCR, immunofuorescence and western blot. The anti-breast cancer efficacy of anlotinib was evaluated in vivo using a xenograft tumor model.

Results: Our findings reveal that increased VEGFA expression in BRCA patients is associated with poorer prognosis, underscoring the need for targeted therapeutic strategies. We also demonstrate that both anlotinib and DDP independently inhibit BRCA cell growth, migration, and invasion, while their combination exhibits a synergistic effect, significantly enhancing the inhibition of these oncogenic processes. This synergy is further evident through the induction of apoptosis and autophagy in BRCA cells. Mechanistically, anlotinib's effectiveness is linked to its inhibition of the JAK2/STAT3 pathway, a critical axis in BRCA progression. In vivo study further support these results, showing that anlotinib markedly inhibits tumor growth in xenografted mice.

Conclusion: This study confirms the efficacy of anlotinib or in combination with DDP and elucidates the mechanism behind anlotinib's effectiveness, highlighting its role in inhibiting the JAK2/STAT3 pathway.

Keywords: JAK2/STAT3; anlotinib; apoptosis; autophagy; breast cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
VEGFA expression and its relationship with different clinicopathological features in BRCA. (A) Pan-cancer analysis of VEGFA expression in tumor and adjacent normal tissues. VEGFA expression in (B) unpaired and (C) paired tumor and normal tissues. (D) Immunohistochemistry analysis of VEGFA protein. VEGFA expression in relation to (E) ER status, (F) PR status, (G) HER status and (H) pathologic stage. Data shown as mean ± SD. **P < 0.01, ***P < 0.001, ns: no significance.
FIGURE 2
FIGURE 2
Prognosis analysis of VEGFA expression in BRCA patients. Kaplan-Meier plotter of (A) Overall survival, (B) relapse free survival, (C) distant metastasis free survival, and (D) post-progression survival, (E) Forest plot between VEGFA expression and overall survival categorized by different ER, PR, HER2 status.
FIGURE 3
FIGURE 3
Anlotinib inhibit BRCA cells growth. Cell proliferation analysis by CCK8 assays in (A) MCF7 and (B) MDA-MB231 cells. (C) Effect of anlotinib on BRCA cell cycle distribution. (D) Subcutaneous tumor volumes comparison in nude treated with or without anlotinib. Tumor growth curves depicting (E) the volume and (F) weight of nude mice. Representative immunohistochemical image showcasing (G) HE stain and (H) Ki-67 in nude mice.
FIGURE 4
FIGURE 4
Combined effects of anlotinib and DDP on cell proliferation and migration of BRCA. The proliferation analysis by (A) CCK8 assays (A, B) and colony formation assays (C). Transwell assays examined the (D) migration and (E) invasion capabilities of BRCA cells (magnification: ×200). (F, G) Wound healing assays illustrated the migration of BRCA cells (magnification: × 40). Data shown as mean ± SD. **P < 0.01, ***p < 0.001.
FIGURE 5
FIGURE 5
Apoptosis detection in BRCA cells by combined treatment with anlotinib and DDP. (A) Flow cytometry analysis. (B) Hoechst33324 staining. Apoptotic bodies are marked with red arrows (magnification: ×200). (C) The mRNA levels of apoptosis-related genes. (D) Apoptosis-related proteins (BAX, BCL2, PARP1) detection by western blot. Data shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.
FIGURE 6
FIGURE 6
Immunofluorescence assay of apoptosis proteins in BRCA cells treated with anlotinib and/or DDP. The fluorescence intensity of (A) BAX and (B) BCL2 (magnification: ×200).
FIGURE 7
FIGURE 7
Induction of autophagy in BRCA cells by Anlotinib combined with DDP. (A–D) The expression of autophagy markers LC3B and SQSTM1/P62 in both unpaired and paired samples. Kaplan Meier Plotter of overall survival by (E) LC3B and (F) SQSTM1/P62 expression in BRCA patients. (G) Detection of key autophagy proteins (LC3B Ⅱ/Ⅰ, SQSTM1/P62) in BRCA cells. (H) The fluorescence intensity of P62 (magnification: ×200). Data shown as mean ± SD. ***p < 0.001.
FIGURE 8
FIGURE 8
Inhibition of the VEGF/JAK2/STAT3 signaling pathway by combined Anlotinib and DDP treatment. (A–C) The correlation analysis among VEGFR2, JAK2 and STAT3. (D) Evaluation of mRNA levels of core genes in the VEGF/JAK2/STAT3, PI3K/AKT pathway in BRCA cells. (E) Assessment of protein expression in VEGFR/JAK2/STAT3 signaling pathway. (F) The mechanism by which anlotinib combined with DDP played its anticancer effect in BRCA. Data shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by Hebei Provincial Department of Science and Technology Centrally Guided Local Development Fund Project (246Z7712G), the Applied and Basic Research Program from Tangshan Science and Technology Bureau (24130222C) and the Basic Scientific Research Funds for Provincial Universities of North China University of Science and Technology (JQN2023046).

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