The epidermal growth factor receptor is overexpressed or constitutively active in bladder and other cancer cell lines and tumors, and expression of this gene/gene product can be regulated by multiple factors including Sp transcription factors (25
). The role of Sp proteins in regulating EGFR expression in bladder cancer cells was investigated by RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) or a cocktail of iSp1, iSp3 and iSp4 combined (iSp) which simultaneously decrease expression of all three transcription factors as previously described (10
). illustrates knockdown of the individual Sp proteins in both bladder cancer cell lines and the results show that only iSp1 and iSp3 significantly decrease EGFR protein levels. demonstrates that transfection of 253JB-V and KU7 cells with the iSp cocktail decreased Sp1, Sp3 and Sp4 and EGFR protein levels confirming that expression of this receptor tyrosine kinase is Sp-dependent in bladder cancer cells and this is consistent with the multiple GC-rich Sp binding sites identified in the EGFR promoter (25
). However, results of RNA interference which knocks down individual Sp proteins show that EGFR is primarily regulated by Sp1 and Sp3 (). Moreover, in a parallel experiment, we also show by RNA interference that knockdown of Sp1 or Sp3 but not Sp4 decreased EGFR mRNA levels (). We also investigated the effects of Sp knockdown in 253JB-V and KU7 cells cotransfected with PER6-luc and iSp or non-specific iLamin (). Knockdown of Sp proteins significantly decreased luciferase activity demonstrating that expression of the EGFR promoter was also Sp-dependent.
FIGURE 1 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 (more ...)
In previous studies, we reported that BA and curcumin inhibit growth and decrease Sp1, Sp3, Sp4 and Sp-dependent genes in prostate and bladder cancer cells, respectively (9
). Since EGFR plays an important role in the growth of bladder cancer cells, we hypothesized that the anticarcinogenic activity of BA and curcumin may be due, in part, to downregulation of EGFR and other Sp-dependent genes. Results illustrated in show that both BA and curcumin inhibited basal and EGF-induced growth of 253JB-V and KU7 bladder cancer cell growth, whereas gefitinib, the clinically used EGFR tyrosine kinase, inhibitor inhibited basal and EGF-induced proliferation of 253JB-V but not KU7 cells. The differential gefitinib-responsiveness of these bladder cancer cell lines has previously been reported (27
FIGURE 2 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 (more ...)
Results in show that after treatment of 253JB-V and KU7 cells with BA or curcumin for 48 h, there was a decrease in EGFR protein expression and this was accompanied by a parallel decrease in other Sp-dependent genes/proteins, namely VEGF and survivin, and also induction of cleaved PARP (). Moreover, treatment of 253JB-V and KU7 cells with BA or curcumin for 48 h also decreased expression of Sp1, Sp3 and Sp4 proteins (). These results confirm that like curcumin (10
), BA also decreased Sp proteins and Sp-dependent proteins in bladder cancer cells and show for the first time that both BA and curcumin decrease expression of EGFR protein, an important target for bladder cancer chemotherapy (17
). In contrast, the tyrosine kinase inhibitor gefitinib did not affect EGFR or Sp protein expression in these cell lines (data not shown) and decreased growth of 253JB-V but not KU7 cells corresponding to the reported gefitinib-responsiveness and -nonresponsiveness of these cell lines (27
FIGURE 3 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 (more ...)
Treatment of 253JB-V and KU7 cells with 5 or 10 μM BA for 24 h also significantly decreased EGFR mRNA levels (). Moreover, treatment with 25 or 40 μM curcumin also significantly decreased EGFR mRNA levels in both bladder cancer cell lines (). BA- and curcumin-dependent inhibition of EGFR transcription was further investigated in bladder cancer cells transfected with PER6-luc, a construct which contains the −771 to −16 region of the EGFR promoter and many of the proximal GC-rich sites are located in this region of the promoter (). Both compounds significantly decreased luciferase activity in 253JB-V and KU7 cells and these results confirm that curcumin and BA inhibit EGFR transcription. The effects of BA on downregulation of luciferase activity in KU7 required relatively high concentrations, suggesting that for BA additional cis-elements may also be important. In contrast, gefitinib did not affect EGFR transcription in these cell lines (data not shown). The EGFR promoter contains GC-rich Sp binding sites and we used the EGFR oligonucleotide containing the −112 to −77 GC-rich EGFR sequence in EMSA assays to investigate the effects of BA and curcumin on Sp protein binding to the EGFR promoter (). Incubation of nuclear extracts from 253JB-V cells with the GC-rich oligonucleotide formed a retarded band complex, whereas decreased binding was observed using extracts from cells treated with 20 μM BA or 40 μM curcumin. Competition with unlabeled EGFR or a consensus GC-rich oligonucleotide decreased retarded band formation, and antibody experiments showed immunodepletion with Sp1 and Sp3 antibodies, and similar results were noted with Sp4 antibodies. Supershifted bands were not observed. Similar results were obtained using nuclear extracts from KU7 cells, thus confirming that BA- and curcumin-dependent downregulation of Sp1, Sp3 and Sp4 proteins decreases Sp binding to the GC-rich region of the EGFR promoter.
FIGURE 4 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 (more ...)
EGFR regulates multiple genes and pathways through activation of downstream kinases such as PI3-K and MAPK. summarizes the effects of BA and curcumin on Akt/phospho-Akt and MAPK/phospho-MAPK expression in 253JB-V cells. Cells treated with 10 or 15 μM BA and 25 μM curcumin decreased constitutive phospho-MAPK expression; however, the same concentrations of BA also decreased levels of MAPK protein, whereas curcumin had minimal effects on MAPK protein levels. Both BA and curcumin also decreased phospho-Akt in 253JB-V cells and this was accompanied by decreased Akt protein. In KU7 cells, BA and curcumin increased levels of phospho-MAPK but did not affect MAPK protein and both compounds decreased phospho-Akt and Akt protein expression (). Thus, in the gefitinib-resistant cells, BA and curcumin inhibited the PI3-K and not the MAPK signaling pathways.
FIGURE 5 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 (more ...)
EGFR also enhances cancer cell survival by inhibition of autophagic cell death in breast cancer cells through stabilization of the sodium/glucose cotransporter 1 (SGLT1) and this response is independent of the kinase activity of this receptor (28
). Results in also demonstrate that after treatment of 253JB-V and KU7 cells with BA or curcumin, there was a decrease in SGLT1 protein expression in both cell lines. Moreover, this was also accompanied by induction of LC3 which is a protein biomarker of autophagy (29
). Thus, knockdown of EGFR in bladder cancer cells after treatment with curcumin or BA inhibited both EGFR kinase-dependent (PI3-K) and kinase-independent (SGLT downregulation and LC3 induction) survival pathways. Gefitinib also decreases expression of phospho-Akt and phospho-MAPK in 253JB-V cells () which is consistent with inhibition of EGFR tyrosine kinase activity by this compound. However, in KU7 cells, gefitinib did not affect expression of phospho-Akt and induced phospho-MAPK which is consistent with previous studies showing that this cell line is gefitinib-resistant (21
). Gefitinib did not affect SGLT or LC3 protein expression in either cell line which is in contrast to the effects of BA and curcumin ().
Knockdown of Sp proteins by RNA interference in KU7 and 253JB-V cells decreased EGFR expression (); however, the effects of Sp knockdown on EGFR kinase-dependent and -independent pathways has not previously been investigated. Results in show that in 253JB-V and KU7 cells transfected with iSp, phospho-MAPK, MAPK or Akt expression was not changed, whereas phospho-Akt protein levels were decreased and this was similar to the effects of curcumin and BA on phospho-Akt in these cell lines, suggesting that this response is EGFR-dependent (). SGLT expression was not affected, whereas LC3 was induced in 253JB-V and KU7 cells transfected with iSp (). Induction of LC3, a protein biomarker for autophagy was observed after ablation of EGFR by treatment of 253JB-V and KU7 cells with curcumin and BA () or transfection with iSp (), and this was observed in a recent study showing that downregulation of EGFR (by RNA interference) induced autophagy in prostate and breast cancer cells (28
). This was further confirmed in 253JB-V and KU7 cells transfected with the GFP-LC3 construct. In untreated cells, there was a diffuse pattern of green fluorescence throughout the cells (); however, after treatment with 10 μM BA and 40 μM curcumin or transfection with iSp, a punctate fluorescent staining was observed and this is characteristic of cells undergoing autophagy (30
). Quantitation of the number of punctae per cell showed that BA, curcumin and iSp significantly increased punctae formation in both cell lines.
FIGURE 6 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 (more ...)
Further confirmation that BA, curcumin and Sp knockdown by RNA interference induced autophagy in 253JB-V and KU7 cells is illustrated in . Compared to DMSO (untreated controls), BA, curcumin and iSp induced acridine orange staining which is consistent with formation of acidic autophagic vacuoles (autophagolysomes) which are characteristically observed in autophagic cells (31
). These results clearly demonstrate that BA- and curcumin-dependent downregulation of EGFR and this results in the loss of EGFR-dependent kinase activity (decreased phospho-Akt) and upregulation of LC3 and autophagy which are also EGFR-regulated (suppressed) responses () and these effects are also observed after knockdown of Sp1, Sp3 and Sp4 by RNA interference. BA and curcumin also affect other genes and responses in bladder and other cancer cell lines that are due to loss of Sp proteins and to Sp-independent compound-specific responses ().