title: Tyrosine kinase receptor inhibitor-targeted combined chemotherapy for metastatic bladder cancer.
abstract: Overexpression of hypoxia-inducible factor-1 alpha is noted during the invasive and metastatic process of transitional cell carcinoma. It will upregulate vascular endothelial growth factor (VEGF) and drive proliferation, invasiveness, metastasis, and antiapoptotic ability of cancer cells. We proposed that tyrosine kinase receptor inhibitor, sunitinib malate-(Sutent; Pfizer Inc., Taiwan), combined with chemotherapeutic drug may present synergistic cytotoxic enhancement to transitional cell carcinoma cells with subsequent inhibition of their cellular behaviors, including proliferation, invasiveness, and metastatic activity. The contents of VEGF-A in mouse bladder tumor cells (MBT-2) and culture medium were detected by quantification-polymerase chain reaction and Western blot individually. The inhibitory concentrations of various chemotherapeutic drugs, sunitinib, and their combination treatment in MBT-2 were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Microchamber transmembrane migration assay was applied in evaluation of the inhibitory effects of different dosages of sunitinib and combination treatment on tumor cells. The cell cycle and apoptosis were analyzed after combination therapy by flow cytometry. Variation in apoptotic pathway was elucidated by Western blot using specific antibodies with cleaved PARP and caspase-3. Metastatic animal model mimicked by tail vein injection of MBT-2 cells was used to evaluate the treatment efficiency in tumor weight and survival rate. The mRNA and protein level of VEGF-A in MBT-2 cells increased by 70% at 48 hours interval under hypoxia stress condition. In MTT assay, MBT-2 cells had shown the highest sensitivity to epirubicin. Sunitinib combined with epirubicin had shown a synergistic cytotoxic effect to MBT-2 cells. Sunitinib and its combination with epirubicin showed significant inhibition on MBT-2 cells migration in microchambers. G2/M phase arrest and increased subG1 in cell cycle was seen in the epirubicin and sunitinib combination treatment group. The activation of apoptosis pathway was confirmed by increased cleaved caspase-3 and cleaved PARP in MBT-2 cells. In tail vein tumor inoculation C3H mice model, epirubicin alone and sunitinib combination therapy decreased tumor growth in lungs with marginal effect. Sunitinib and epirubicin combination had shown a synergistic cytotoxic effect and inhibited cell migration ability in MBT-2 cells. The combination can induce cell cycle arrest at G2/M phase and increase subG1 cells. Metastatic animal study also showed that sunitinib combined with epirubicin has a marginal effect on inhibition of tumor growth of lungs. The tyrosine kinase receptor inhibitor-targeted combined chemotherapy regimen may provide as a new treatment modality for advanced bladder cancer in the future.
authors.affiliation: Graduate Institute of Pathology, National Defense Medical Center, Taipei, Taiwan.
authors.forename: Chia-Lun
authors.initials: CL
authors.lastname: Wu
authors.forename: Szu-Yuan
authors.initials: SY
authors.lastname: Ping
authors.forename: Cheng-Ping
authors.initials: CP
authors.lastname: Yu
authors.forename: Dah-Shyong
authors.initials: DS
authors.lastname: Yu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D015251:Epirubicin / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D009362:Neoplasm Metastasis; D011758:Pyrroles / Q000494:pharmacology / Q000627:therapeutic use; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D000077210:Sunitinib; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D007211:Indoles; D011758:Pyrroles; D042461:Vascular Endothelial Growth Factor A; D015251:Epirubicin; D020794:Receptor Protein-Tyrosine Kinases; D000077210:Sunitinib