Although chemotherapy used as a primary therapy for many cancers is to cause massive cell death, there are some residual cancer cells that remain alive. Gene therapy can target cancer cells with specific genetic defect(s) which provides an attractive regimen to eradicate those cells. Transitional cell carcinoma (TCC) of bladder is considered as an ideal model for evaluating the combination of chemotherapy and adenovirus-based gene therapy. However, it is known that coxsackie and adenovirus receptor (CAR), first identified as a high affinity adenovirus receptor, is often down regulated in human TCC cells. Therefore, the objective of this study is to screen any chemotherapeutic agent that can not only kill TCC cells but also upregulate CAR expression in cancer cells. We have used two TCC cell lines (i.e., T24 and TCC-SUP) expressing low endogenous levels of CAR protein. Several techniques and assays employed in this study were: (1) real-time RT-PCR for determining the steady-state levels of CAR mRNA; (2) western blot analysis for determining the CAR protein expression; (3) β-gal assay for measuring the transgene expression in target cells infected with β-gal adenovirus; (4) crystal violet assay for determining the effect of chemotherapeutic agents or virus on TCC; (5) in vitro histone deacetylase (HDAC) activity assay for determining the total HDAC activity in the nuclear extract of cells. One known mechanism of down-regulation of CAR gene in TCC cells is due to the decreased histone acetylation associated with the promoter region of this gene. In this study, we have screened a chemical library and discovered an anti-allergic agent, Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) as a potential candidate of CAR gene inducer. In general, Tranilast alone is a weak CAR gene inducer. By combining Tranilast with a low dose of FK228 (an HDAC inhibitor), we observed a synergistic effect on CAR mRNA expression in both T24 and TCC-SUP cells. Based on the structure of Tranilast, we further identified an analog (i.e., 2, 3-dimethoxycinnamoyl azide [DMCA]), when combined with FK228, has a more potent effect than the combination of Tranilast and FK228 on both CAR gene expression and virus uptake in TCC cells, which is consistent with the increased suppression of HDAC activity in treated cells. To determine the therapeutic effect on TCC cells, we observed that either Tranilast or DMCA alone did not cause any obvious cytotoxic effect. However, in combination with a low dosage of FK228, DMCA can significantly potentiate the growth inhibitory effect of FK228 on both T24 and TCC-SUP cells. In summary, our data indicate that DMCA in combination with FK228 can be used as a primary chemotherapeutic regimen for TCC and, with the elevation of CAR expression, a subsequent adenovirus gene therapy can then be used as an adjuvant therapy for any remaining cancer cells.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]