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Background <p>Bladder cancer remains a major urologic malignancy with substantial recurrence and progression risk, underscoring the need for mechanism-informed therapeutic candidates. Ginkgetin, a biflavonoid derived from <i>Ginkgo biloba</i> leaves, has shown antitumor potential in several cancer settings, yet its key signaling axis and actionable molecular node in bladder cancer have not been systematically defined.</p> Methods <p>We evaluated ginkgetin across multiple bladder cancer cell lines (5637, T24, HT-1376, J82) and normal urothelial cells (SV-HUC-1) using viability assays and IC₅₀ estimation. Antitumor phenotypes were assessed by colony formation, wound-healing migration assays, EMT marker profiling, and Annexin V/PI flow cytometry. Network pharmacology and RNA-seq were integrated to prioritize enriched pathways, followed by western blot validation of PI3K/AKT/mTOR phosphorylation. An insulin reactivation (“rescue”) strategy was used to functionally test pathway dependence. Transcriptome-derived candidates were further examined by RT–qPCR and STEAP2 overexpression to probe node-level involvement. In addition, molecular docking and 100-ns molecular dynamics simulations were performed to characterize ligand–target binding stability.</p> Results <p>Ginkgetin suppressed bladder cancer cell viability in a time- and dose-dependent manner at low micromolar concentrations, while normal urothelial cells required markedly higher exposures. Functionally, ginkgetin reduced clonogenic survival, inhibited migration, and shifted EMT features toward an epithelial phenotype. Apoptosis increased in parallel, accompanied by a pro-apoptotic protein signature. Multi-omics and network analyses converged on PI3K–Akt signaling, and experimental validation showed that ginkgetin primarily dampened pathway output by reducing PI3K/AKT/mTOR phosphorylation rather than total protein abundance. Insulin-mediated reactivation partially reversed phosphorylation suppression and attenuated apoptosis-related shifts, supporting a functional link between axis inactivation and apoptotic tendency. STEAP2 was consistently downregulated after treatment, and STEAP2 overexpression partially counteracted apoptosis-associated changes.</p> Conclusion <p>These findings support a coherent “phenotype–pathway–node” model in which ginkgetin inhibits malignant phenotypes and promotes apoptosis in bladder cancer cells, associated with reduced PI3K/AKT/mTOR activity and STEAP2 downregulation. The PI3K/AKT/mTOR axis and STEAP2 emerge as testable mechanistic entry points for further translational validation.</p>

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STEAP2-associated modulation of PI3K/AKT/mTOR signaling contributes to ginkgetin-induced apoptosis in bladder cancer cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月1日

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  • Pengze Wu,
  • Lin Chen,
  • Jin Yang,
  • Zhengkang Liang,
  • Xiaofeng Yin,
  • Shaowen Zhou,
  • Zhilin Deng,
  • Yafei Yang

摘要

Background

Bladder cancer remains a major urologic malignancy with substantial recurrence and progression risk, underscoring the need for mechanism-informed therapeutic candidates. Ginkgetin, a biflavonoid derived from Ginkgo biloba leaves, has shown antitumor potential in several cancer settings, yet its key signaling axis and actionable molecular node in bladder cancer have not been systematically defined.

Methods

We evaluated ginkgetin across multiple bladder cancer cell lines (5637, T24, HT-1376, J82) and normal urothelial cells (SV-HUC-1) using viability assays and IC₅₀ estimation. Antitumor phenotypes were assessed by colony formation, wound-healing migration assays, EMT marker profiling, and Annexin V/PI flow cytometry. Network pharmacology and RNA-seq were integrated to prioritize enriched pathways, followed by western blot validation of PI3K/AKT/mTOR phosphorylation. An insulin reactivation (“rescue”) strategy was used to functionally test pathway dependence. Transcriptome-derived candidates were further examined by RT–qPCR and STEAP2 overexpression to probe node-level involvement. In addition, molecular docking and 100-ns molecular dynamics simulations were performed to characterize ligand–target binding stability.

Results

Ginkgetin suppressed bladder cancer cell viability in a time- and dose-dependent manner at low micromolar concentrations, while normal urothelial cells required markedly higher exposures. Functionally, ginkgetin reduced clonogenic survival, inhibited migration, and shifted EMT features toward an epithelial phenotype. Apoptosis increased in parallel, accompanied by a pro-apoptotic protein signature. Multi-omics and network analyses converged on PI3K–Akt signaling, and experimental validation showed that ginkgetin primarily dampened pathway output by reducing PI3K/AKT/mTOR phosphorylation rather than total protein abundance. Insulin-mediated reactivation partially reversed phosphorylation suppression and attenuated apoptosis-related shifts, supporting a functional link between axis inactivation and apoptotic tendency. STEAP2 was consistently downregulated after treatment, and STEAP2 overexpression partially counteracted apoptosis-associated changes.

Conclusion

These findings support a coherent “phenotype–pathway–node” model in which ginkgetin inhibits malignant phenotypes and promotes apoptosis in bladder cancer cells, associated with reduced PI3K/AKT/mTOR activity and STEAP2 downregulation. The PI3K/AKT/mTOR axis and STEAP2 emerge as testable mechanistic entry points for further translational validation.