Anthracycline-based chemotherapy, such as doxorubicin (Dox), while effective against many solid tumors, is not widely used for head and neck cancers. In this study, we evaluated the efficacy of Dox, and its derivative AD198 in human, canine, and feline oral squamous cell carcinomas cells (OSCC) in vitro. Dox and AD198 had significant an anti-proliferative effect on human, canine, and feline OSCC cells in dose-dependent manner. AD198 inhibited cell proliferation more effectively than Dox in tested OSCC cells. In the human oral squamous cell carcinoma SCC25 cells, Dox and AD198 increased the production of reactive oxygen species and subsequently increased apoptosis through activation of caspase signaling pathway. Dox and AD198 increased activation of AKT, ERK1/2, and p38 MAPK signaling pathways in tested OSCC cells by dose-dependent manner. The efficacy of Dox and AD198 treatments in inhibition of cell proliferation was increased in tested OSCC when combined with PI3K/AKT inhibitor, LY294002 treatment. Inhibition of PI3K/AKT reduced Dox- and AD198-induced activation of ERK1/2 and further increased Dox- and AD198-induced phosphorylation of p38 MAPK in OSCC. Our results suggest that the anthracycline therapies, such as Dox or AD198, can be more effective for treatment of OSCC when combined with inhibitors of the PI3K/AKT pathway.

PurposeReceptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with highly expressed receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR) and c-Kit receptor. Resistance to targeted therapies is partially due to the activation of alternative pro-survival signaling pathways, including cyclooxygenase (COX)-2. In this study, we validated the effects of two RTKIs, axitinib and AB1010, in combination with COX inhibitors on the V-akt murine thymoma oncogene homolog 1 (Akt) and COX-2 signaling pathways in bladder cancer cells.MethodsThe expression of several RTKs and their downstream signaling targets was analyzed by Western blot (WB) analysis in human and canine bladder transitional cell carcinoma (TCC) cell lines. The effects of RTKIs and COX inhibitors in bladder TCC cells were assessed by MTS for cell viability, by Caspase-3/7 and Annexin V assay for apoptosis, by WB analysis for detection of COX-2 and Akt signaling pathways, and by enzyme-linked immunosorbent assay for detection of prostaglandin E2 (PGE2) levels.ResultsAll tested TCC cells expressed the c-Kit and PDGFRα receptors, except human 5637 cells that had low RTKs expression. In addition, all tested cells expressed COX-1, COX-2, Akt, extracellular signal regulated kinases 1/2, and nuclear factor kappa-light-chain-enhance of activated B cells proteins, except human UM-UC-3 cells, where no COX-2 expression was detected by WB analysis. Both RTKIs inhibited cell viability and increased apoptosis in a dose-dependent manner in tested bladder TCC cells, which positively correlated with their expression levels of the PDGFRα and c-Kit receptors. RTKIs increased the expression of COX-2 in h-5637 and K9TCC#1Lillie cells. Co-treatment of indomethacin inhibited AB1010-induced COX-2 expression leading to an additive effect in inhibition of cell viability and PGE2 production in tested TCC cells.ConclusionCo-treatment of RTKIs with indomethacin inhibited cell viability and AB1010-induced COX-2 expression resulting in decreased PGE2 production in tested TCC cells. Thus, COX inhibition may further potentiate RTKIs therapies in bladder cancer.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo . The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro . The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro . In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo . In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

Due to doxorubicin (Dox) cardiotoxicity, the next generation of novel non-cardiotoxic anthracyclines, including AD 312 and AD 198, were synthesized and validated. In this study, we assessed the efficacy and mechanisms of anthracyclines-induced apoptosis and inhibition of cell viability in human bladder cancer cells expressing wild-type (wt) p53 (RT4 and SW780) and mutated (mt) p53 (UM-UC-3, 5637, T-24, J82, and TCCSUP) protein. Anthracyclines inhibited cell viability in tested TCC cells, but were less effective in mt-p53 TCC cells, especially in the drug-resistant J82 and TCCSUP cells. Anthracyclines upregulated the expression of wt p53 protein in RT4 and SW780 cells, but had no effect on expression of mt p53 protein in UM-UC-3, 5637, T-24, J82, and TCCSUP cells. The anthracyclines activated caspase 3/7 and cleavage of PARP in wt-p53 RT4 and SW780 cells, and mt-p53 5637, UM-UC-3, and T-24, but not in mt-p53 J82 and TCCSUP cells. The anthracyclines-induced cleavage of PARP was blocked by p53 siRNA in wt-p53 RT4 cells. Co-treatment of AD 198 with PRIMA-1 significantly inhibited cell viability of mt-p53 J82 cells, but had no effect in wt-p53 RT4 cells. AD 198 blocked c-myc expression in mt-p53 UM-UC-3, 5637, T-24, and J82 cells, however no expression of c-myc was detected in wt-p53 RT4 and SW780 cells. In conclusion, our results demonstrated that the anthracycline-induced resistance in bladder cancer cells positively correlated with TP53 mutations in the tetramerization domain in J82 and TCCSUP cells. Further, AD 312 and AD 198 are promising chemotherapeutic drugs for bladder cancer, especially in combination with PRIMA-1.