Effect of isorhamnetin on carbonic anhydrase IX expression and tumorigenesis of bladder cancer by activating PPARγ/PTEN/AKT pathway.

BACKGROUND: To clarify the research prospect and mechanism analysis of isorhamnetin as a therapeutic drug for bladder cancer. METHODS: Firstly, the effects of different concentrations of isorhamnetin on the expression of PPARγ/PTEN/Akt pathway protein, CA9, PPARγ, PTEN and AKT protein were discussed by western blot. The effects of isorhamnetin on the growth of bladder cells were also analyzed. Secondly, we verified whether the effect of isorhamnetin on CA9 was related to PPARγ/PTEN/Akt pathway by western blot, and the mechanism of isorhamnetin on the growth of bladder cells is related to this pathway by CCK8, cell cycle and ball formation experiment. Further, nude mouse model of subcutaneous tumor transplantation was constructed to analyze the effects of isorhamnetin, PPAR and PTEN on 5637 cell tumorigenesis and the effects of isorhamnetin on tumorigenesis and CA9 expression through PPARγ/PTEN/Akt pathway. RESULTS: Isorhamnetin inhibited the development of bladder cancer, and regulated the expression of PPAR, PTEN, AKT, CA9. Isorhamnetin inhibits cell proliferation and the transition of cells from G0/G1 phase to S phase, and tumor sphere formation. Carbonic anhydrase IX is a potential downstream molecule of PPARγ/PTEN/AKT pathway. Overexpression of PPARγ and PTEN inhibited expression of CA9 in bladder cancer cells and tumor tissues. Isorhamnetin reduced CA9 expression in bladder cancer via PPARγ/PTEN/AKT pathway, thereby inhibiting bladder cancer tumorigenicity. CONCLUSION: Isorhamnetin has the potential to become a therapeutic drug for bladder cancer, whose antitumor mechanism is related to PPARγ/PTEN/AKT pathway. Isorhamnetin reduced CA9 expression in bladder cancer via PPARγ/PTEN/AKT pathway, thereby inhibiting bladder cancer tumorigenicity.

Carbonic Anhydrase IX Controls Vulnerability to Ferroptosis in Gefitinib-Resistant Lung Cancer.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI, such as gefitinib) in lung cancer continues to be a major problem. Recent studies have shown the promise of ferroptosis-inducing therapy in EGFR-TKI resistant cancer, but have not been translated into clinical benefits. Here, we identified carbonic anhydrase IX (CA9) was upregulated in gefitinib-resistant lung cancer. Then we measured the cell viability, intracellular reactive oxygen species (ROS) levels, and labile iron levels after the treatment of ferroptosis inducer erastin. We found that CA9 confers resistance to ferroptosis-inducing drugs. Mechanistically, CA9 is involved in the inhibition of transferrin endocytosis and the stabilization of ferritin, leading to resistance to ferroptosis. Targeting CA9 promotes iron uptake and release, thus triggering gefitinib-resistant cell ferroptosis. Notably, CA9 inhibitor enhances the ferroptosis-inducing effect of cisplatin on gefitinib-resistant cells, thus eliminating resistant cells in heterogeneous tumor tissues. Taken together, CA9-targeting therapy is a promising approach to improve the therapeutic effect of gefitinib-resistant lung cancer by inducing ferroptosis.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, a P300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrate or propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

Effective inhibition of tumor in vivo with a novel DNA vaccine targeting chimeric G250.

OBJECTIVE: To develop a promising approach for tumor immunotherapy with G250 antigen-based DNA vaccine and to investigate its anti-tumor response in mice with renal cell carcinoma. MATERIALS AND METHODS: G250 derived from human, monkey and mouse were prepared by PCR. The heterogeneous chimeric G250 gene was obtained by integrating different gene fragments of three species. Then, the chimeric G250 was inserted into a eukaryotic expression plasmid pVAX1-IRES-GM/B7 to obtain DNA vaccine (named pVAX1-tG250-GM/B7) which could express chimeric G250 antigen and immune adjuvants simultaneously. By transfecting into Cos7 cells, the expression of chimeric G250 antigen was tested using flow cytometry and immunofluorescence assay. The immunological response and protection against tumor were evaluated in vivo. RESULTS: Recombinant plasmid DNA vaccine was constructed successfully through identification of PCR and gene sequencing. The chimeric G250 antigen was well expressed in Cos7 cells. A strong immune response can be detected through ELISPOT and ELISA induced by pVAX1-tG250-GM/B7. The mice vaccinated with pVAX1-tG250-GM/B7, balb/c showed significant inhibition of tumor and a longer time of survival compared with control group. CONCLUSIONS: The experimental results of this study exhibited that the DNA vaccine based on heterogeneous chimeric antigen can produce efficient anti-tumor effect in vivo and they represent a promising strategy for tumor immunotherapy.

Combining DNA Vaccine and AIM2 in H1 Nanoparticles Exert Anti-Renal Carcinoma Effects via Enhancing Tumor-Specific Multi-functional CD8+ T-cell Responses.

Renal carcinoma presents a rapid progression in patients with high metastasis with no effective therapeutic strategy. In this study, we designed a folate-grafted PEI600-CyD (H1) nanoparticle-mediated DNA vaccine containing an adjuvant of absent in melanoma 2 (AIM2) and a tumor-specific antigen of carbonic anhydrase IX (CAIX) for renal carcinoma therapy. Mice bearing subcutaneous human CAIX (hCAIX)-Renca tumor were intramuscularly immunized with H1-pAIM2/pCAIX, H1-pCAIX, H1-pAIM2, or Mock vaccine, respectively. The tumor growth of hCAIX-Renca was significantly inhibited in H1-pAIM2/pCAIX vaccine group compared with the control group. The vaccine activated CAIX-specific CD8+ T-cell proliferation and CTL responses, and enhanced the induction of multi-functional CD8+ T cells (expressing TNF-α, IL-2, and IFN-γ). CD8+ T-cell depletion resulted in the loss of anti-tumor activity of H1-pAIM2/pCAIX vaccine, suggesting that the efficacy of the vaccine was dependent on CD8+ T-cell responses. Lung metastasis of renal carcinoma was also suppressed by H1-pAIM2/pCAIX vaccine treatment accompanied with the increased percentages of CAIX-specific multi-functional CD8+ T cells in the spleen, tumor, and bronchoalveolar lavage as compared with H1-pCAIX vaccine. Similarly, the vaccine enhanced CAIX-specific CD8+ T-cell proliferation and CTL responses. Therefore, these results indicated that H1-pAIM2/pCAIX vaccine exhibits the therapeutic efficacy of anti-renal carcinoma by enhancing tumor-specific multi-functional CD8+ T-cell responses. This vaccine strategy could be a potential and promising approach for the therapy of primary solid or metastasis tumors.