Inhibition of bladder cancer cell growth. 235JB-V and KU7 cells were treated with gefitinib (A), betulinic acid (B), or curcumin (C) for 72 h in the presence or absence of EGF (100 ng/ml), and the effects of the treatments on cell proliferation were determined as described in the Materials and Methods. Results are expressed as means ± SE for at least 3 replicate experiments per treatment group, and a significant (P < 0.05) decrease in cell proliferation is indicated by an asterisk.

BA and curcumin decrease EGFR expression in bladder cancer cells. BA (A) and curcumin (B) decrease EGFR mRNA levels. Cells were treated with DMSO or different concentrations of BA or curcumin for 24 h, and EGFR mRNA levels were determined by RT-PCR as described in the Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations for each treatment group and significantly (P < 0.05) decreased expression (relative to DMSO) is indicated (*). C. Decreased EGFR promoter activity. Cells were transfected with PER6 and treated with DMSO, BA or curcumin, and luciferase activity was determined as described in the Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations and significant (P < 0.05) downregulation relative to DMSO is indicated (*). D. Gel shift assay. Nuclear extracts from cells treated with DMSO, BA or curcumin were incubated with a ³²P-labeled GC-rich probe derived from the EGFR promoter, and gel mobility shift assays were determined as described in the Materials and Methods. Competition assays used 100-fold less of unlabeled oligonucleotide.

Sp protein knockdown decreases EGFR-dependent PI3K signaling and induces autophagy. A. Effects of iSp on kinases and autophagy-related proteins and quantitation of p-Akt and LC3. Whole cell lysates from bladder cancer cells transfected with iSp were analyzed by western blots as described in the Materials and Methods. Quantitation of phospho-Akt and LC3 proteins in cells transfected with iSp was determined as a % of Lamin/β-actin protein ratios from 3 replicate western blot analyses as indicated in and described in the Materials and Methods. Results are expressed as means ± SE and significant (P < 0.05) increases or decreases in the iSp transfected compared to the iLamin transfected groups are indicated (*). B. Induction of punctuate green fluorescence in 253JB-V and KU7 cells transfected with GFP-LC3. Cells cotransfected with iSp (or iLamin as control) and GFP-LC3 were treated with DMSO, BA or curcumin for 18 h and the fluorescence associated with GFP-LC3 was determined as described in the Materials and Methods. Punctae per cell were quantitated and results are expressed as means ± SE for 3 replicate determinations. Significant (P < 0.05) induction of punctae is indicated (*). C. Induction of autophagy. Induced acridine orange staining in 253JB-V and KU7 cells. Cells were treated with DMSO (untreated), 10 μM BA, or 40 μM curcumin for 18 h, and detection of autophagic vacuoles by acridine orange staining was determined as described in the Materials and Methods. Observations were typical of replicate experiments. D. Model for the effects of BA, curcumin and iSp in bladder cancer cells.

Regulation of EGFR in bladder cancer cells is Sp-dependent. Downregulation of Sp1, Sp3, Sp4 and EGFR proteins in bladder cancer cells transfected with iLamin (control), small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), Sp4 (iSp4) (A) or iSp (combined iSp1, iSp3 and iSp4) (B). Cells were transfected with the appropriate oligonucleotide and after 72 h, whole cell lysates were obtained and analyzed by western blots as outlined in the Materials and Methods. Similar results were observed in replicate (2–3) experiments. C. Effects of iSp1, iSp3 and iSp4 on EGFR mRNA levels. 253JB-V and KU7 cells were transfected with iSp1, iSp3, iSp4 or iLamin (control) and mRNA was isolated from these cells and EGFR mRNA was determined by real-time PCR as described in the Materials and Methods. Results are expressed as means ± SE for 3 separate experiments and significantly (p < 0.05) decreased EGFR mRNA levels (relative to iLamin set at 1.0) are indicated (*). D. Effects of iSp on EGFR promoter expression. Cells were transfected with PER6 and iSp or iLamin (non-specific), and luciferase activity was determined as described in the Materials and Methods. Results are expressed as means ± SE for 3 replicate determinations and significantly (P < 0.05) decreased activity after Sp knockdown is indicated (*).

BA and curcumin decrease EGFR and Sp proteins and Sp-dependent genes. Compound-induced repression of EGFR (A) and other Sp-dependent proteins (B) in 253JB-V and KU7 cells. Cells were treated with DMSO or different concentrations of the compounds for 48 h, and whole cell lysates were analyzed by western blots as described in the Materials and Methods. C. Compound-induced repression of Sp1, Sp3 and Sp4 proteins in 253JB-V and KU7 cells. Protein expression was determined as outlined above in (A). Results (A–D) were observed in replicate experiments (at least 3).

Modulation of putative EGFR-dependent responses. Effects of BA and curcumin on EGFR-dependent effects in 253JB-V (A) and KU7 (B) cells compared to effects of gefitinib (C) in both cell lines. Cells were treated with DMSO or different concentrations of BA, curcumin or gefitinib for 48 h, and whole cell lysates were analyzed by western blot analysis as described in the Materials and Methods. Similar results were observed in replicate (2) experiments.