illustrate the concentration-dependent effects of 5, 10 and 25 µM curcumin on proliferation of 253JB-V and KU7 bladder cancer cells over a 6 day period with change of media and treatment with DMSO (control) or curcumin every 48 hr. Proliferation of both 253JB-V and KU7 cells was inhibited by curcumin. However, the pattern of inhibition was slightly different; 5 and 10 µM curcumin significantly inhibited 253JB-V cells after treatment for 4 and 6 days, whereas 10 µM (but not 5 µM) curcumin significantly inhibited KU7 cell proliferation after treatment for 2, 4 and 6 days. The highest concentration of curcumin (25 µM) was cytotoxic to both cell lines, and similar results were previously reported on the cytotoxicity of curcumin in KU7 and RT4V6 bladder cancer cells (17
). The effects of curcumin on the distribution of 253JB-V and KU7 cells in G0
, S and M phases of the cell cycle are illustrated in , respectively. Curcumin (5 – 25 µM) increased the percentage of 253JB-V cells in G0
and decreased the percentage in S and G2
/M phases, although the effects on G2
/M were concentration-dependent and variable. In contrast, in KU7 cells, minimal effects were observed after treatment with 5 or 10 µM curcumin, whereas 25 µM curcumin decreased the percentage of cells in G0
and increased the percentage in S and G2
/M phases (). These results demonstrate that curcumin-induced changes in the distribution of 253JB-V and KU7 cells in different phases of the cycle were dependent on cell context.
Figure 1 Curcumin inhibits bladder cancer cell growth and modulates the cell cycle. Inhibition of 253JB-V (A) and KU7 (B) cell growth. Cells were treated with DMSO (solvent control), 5, 10 or 25 µM curcumin and the effects of cell growth were determined (more ...)
The effects of curcumin on selected proteins involved in cell cycle progression are summarized in . Curcumin decreased expression of cyclin D1 in both 253JB-V and KU7 cells (), and the cyclin-dependent kinase inhibitors p21 and p27 were also decreased. The loss of cyclin D1 expression in 253JB-V cells treated with curcumin was consistent with the inhibition of G0
to S phase progression in the cell line (). The cyclin-dependent kinase inhibitors p21 and p27 were also decreased in both cell lines by curcumin, but at different concentrations. p21 was decreased by 25 – 50 and 10 – 50 µM curcumin in 253JB-V and KU7 cells, respectively, whereas 25 – 50 µM curcumin decreased p27 expression in both cell lines. The role of these responses in mediating the distribution of bladder cancer cells in different phases of the cell cycle () is unclear. Previous studies showed that curcumin induced apoptosis in bladder cancer cells (17
) and, in pancreatic tumors, curcumin suppressed proliferation and inhibited angiogenesis (13
). Results in demonstrate that curcumin induced PARP cleavage in 253JB-V and KU7 cells, and this was also accompanied by downregulation of the antiapoptotic gene survivin
. Decreased survivin and increased PARP cleavage were initially observed at curcumin concentrations of 10 and 25 µM in 253JB-V and KU7 cells, respectively, and maximal responses were observed in cells treated with 25 and 50 µM curcumin. In addition, curcumin also decreased expression of two angiogenic proteins, VEGF and VEGFR1, in both cell lines (). The proapoptotic activity of curcumin was also confirmed in a TUNEL assay () where 40 µM curcumin induced increased TUNEL staining in 253JB-V and KU7 cells, whereas minimal effects were observed in the solvent (DMSO)-treated cells.
Figure 2 Curcumin modulates expression of cell cycle, survival and angiongenic proteins and induces apoptosis. (A) Decreased expression of p27 and p21 proteins. 253JB-V and KU7 cells were treated with DMSO, 5, 10, 25, or 50 µM curcumin for 24 hr, and whole (more ...)
Figure 5 Effects of curcumin and Sp knockdown on NFκB. (A) Curcumin decreases NFκB promoter activity in 253JB-V and KU7 cells. 253JB-V and KU7 cells were transfected with pNFκB-luc treated with DMSO, 10, 25 or 40 µM curcumin, and (more ...)
Results in show that after treatment of 253JB-V and KU7 cells with curcumin (5 – 50 µM) for 24 hr, there was a concentration-dependent decrease in the expression of Sp1, Sp3 and Sp4 proteins. Curcumin-induced downregulation of Sp proteins was dependent on the concentration of curcumin, Sp protein (i.e. Sp1, Sp3 or Sp4), and cell context. However, in both 253JB-V and KU7 cells, decreased expression of all three proteins was observed after treatment with 10 µM and higher concentrations of curcumin for 24 hr. Since curcumin decreased expression of Sp1, Sp3, Sp4 and Sp-dependent survivin, VEGF and VEGFR1 proteins (, ), we also investigated the effects of curcumin on luciferase activity in 253JB-V and KU7 cells transfected with constructs containing GC-rich promoters that bind Sp proteins. Luciferase activity was decreased in 253JB-V and KU7 cells treated with 10 – 40 µM curcumin and transfected with GC-rich Sp1For4 and Sp3For5 constructs containing the −751 to −20 and −417 to −38 regions of the Sp1 and Sp3 gene promoters, respectively (31
) (linked to the luciferase gene) (). In addition, curcumin (10 – 40 µM) also decreased luciferase activity in 253JB-V and KU7 cells transfected with GC-rich pVEGF and pSurvivin constructs that contain the −2018 to +50 and −269 to +49 GC-rich regions of the VEGF and survivin gene promoters, respectively (). Thus, like tolfenamic acid and betulinic acid, curcumin-induced downregulation of Sp1, Sp3 and Sp4 not only decreased expression of Sp-regulated proteins such as VEGF and survivin (), but also decreased transactivation in 253JB-V and KU7 cells transfected with pVEGF and pSurvivin constructs containing GC-rich promoter inserts.
Figure 3 Effects of curcumin on Sp proteins and Sp-dependent transactivation. Curcumin decreases Sp proteins in 253JB-V (A) and KU7 (B) bladder cancer cells. Cells were treated with DMSO, 5, 10, 25 or 50 µM curcumin for 24 hr and whole cell lysates were (more ...)
Previous studies show that tolfenamic and betulinic acids induce proteasome-dependent degradation of Sp1, Sp3 and Sp4 in pancreatic and prostate cancer cells (24
), and summarize the effects of the proteasome inhibitor MG132 on curcumin-induced downregulation of these proteins. In KU7 cells, MG132 inhibited curcumin-induced downregulation of Sp1, Sp3 and Sp4 proteins, and similar effects were observed in 253JB-V cells. However, in the latter bladder cancer cell line, MG132 alone also decreased Sp protein expression and this response was most pronounced for Sp1. Other proteasome inhibitors such as lactacystin and gliotoxin gave similar results and, in combination with curcumin, high cytotoxicity was observed in 253JB-V cells (data not shown). Nevertheless, it was apparent that MG132 plus curcumin blocked Sp protein downregulation in both cell lines, suggesting that curcumin, like betulinic and tolfenamic acids (24
), induced proteasome-dependent downregulation of Sp1, Sp3 and Sp4 proteins in 253JB-V and KU7 cells. summarizes the effects of MG132 on curcumin-dependent decreased luciferase activity in 253JB-V and KU7 cells transfected with pSp1For4 and pSp3For5. In 253JB-V cells, MG132 only partially reversed downregulation of activity by 25 µM curcumin, whereas in KU7 cells, MG132 completely inhibited the effects of curcumin on luciferase activity. The relative effectiveness of MG132 as an inhibitor of curcumin-dependent downregulation of luciferase activity in cells transfected with pVEGF or pSurvivin also differed in 253JB-V and KU7 cells (). MG132 reversed the effects of curcumin in KU7 cells (80 – 100%) but was less efficient in 253JB-V cells. This may be due, in part, to the cytoxocity of MG132 alone and in combination with curcumin in 253JB-V cells. These results demonstrate that curcumin primarily induced proteasome-dependent degradation of Sp1, Sp3 and Sp4 in the bladder cancer cells, and similar effects have been observed for tolfenamic acid and betulinic acid in pancreatic and prostate cancer cells (24
Figure 4 MG132 inhibition of curcumin-induced effects on Sp proteins and Sp-dependent transactivation. MG132 inhibits curcumin-dependent downregulation of Sp proteins in 253JB-V (A) and KU7 (B) cells. Bladder cancer cells were treated with DMSO alone, 25 µM (more ...)
Previous studies in bladder and other cancer cell lines report that the broad spectrum of anticancer activities of curcumin have been associated, in part, with decreased NFκB-dependent activity (3
). Results illustrated in show that 10 – 40 µM curcumin decreased luciferase activity in 253JB-V and KU7 cells transfected with pNFκB, a construct containing 5 tandem NFκB response elements that regulate a luciferase reporter gene. Thus, curcumin decreases NFκB-dependent transactivation in 253JB-V and KU7 cell lines. Since curcumin also decreases expression of Sp1, Sp3 and Sp4 in bladder cancer cells (), we investigated the effects of Sp protein knockdown on luciferase activity in cells transfected with pNFκB-luc (). 253JB-V and KU7 cells were transfected with iSp which is a cocktail of small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) (33
). The results showed that transfection with iSp significantly inhibited NFκB-dependent activity and this was not directly related to a decrease in p65 or p50 protein levels after transfection with iSp (). There was a minimal but not significant decrease in these proteins over replicated experiments, suggesting that decreased NFκB-dependent transactivation by iSp was not dependent on decreased expression of p65 or p50. We also investigated the effects of iSp1, iSp3 and iSp4 on p65 and p50 protein expression in 253JB-V and KU7 cells (Supplement 1
). In the former cell line, iSp3 and iSp4 (but not iSp1) slightly decreased p65 protein levels, whereas none of the small inhibitory RNAs affected p65 or p50 expression in KU7 cells and p50 expression in 253JB-V cells. Results in show that curcumin also decreased expression of VEGF and survivin which are regulated by both NFκB and Sp transcription factors (28
). shows that curcumin decreased expression of cyclin D1 and the antiapoptotic protein bcl-2 in 253JB-V and KU7 cells, and both of these genes are also regulated by NKκB and Sp transcription factors (38
). It is possible that curcumin-induced downregulation of Sp proteins may directly affect NFκB since both p65 and p50 are Sp-dependent genes in some cell lines (34
). However, treatment of 253JB-V and KU7 cells with DMSO, 10, 25 or 40 µM curcumin for 24 hr did not affect nuclear p65 expression, whereas nuclear p50 protein levels were decreased only at the higher (40 µM) concentration () and 25 µM curcumin, which decreases Sp protein levels, did not affect p65 or p50 expression. Thus, curcumin-mediated inhibition of NFκB-dependent gene expression is not due to direct effects on p65 or p50.
A close inspection of many NFκB-regulated genes such as cyclin D1, bcl-2, survivin and VEGF indicates that these genes also contain multiple GC-rich promoter sequences and are coregulated by Sp proteins in many cell lines (24
). Results in show that in 253JB-V and KU7 cells transfected with iLamin (control) or the iSp cocktail (containing iSp1, iSp3 and iSp4), there was a significant decrease in levels of bcl-2, cyclin D1, survivin and VEGF and this correlated with the effects of curcumin on these same proteins ( and ). In contrast, combined knockdown of p65 and p50 by RNA interference () did not affect expression of all these proteins. Only VEGF and bcl-2 proteins were decreased by p65/p50 knockdown, suggesting that basal expression of cyclin D1 and survivin were NFκB-independent in the bladder cancer cells. illustrates gel mobility shift assays in which nuclear extracts from 253JB-V and KU7 cells bound a 32
P-labeled GC-rich oligonucleotide to form Sp-DNA retarded bands as previously characterized in other cell lines (27
). Treatment with 25 or 40 µM curcumin decreased retarded band intensities and coincubation with unlabeled wild-type GC-rich or mutant oligonucleotides decreased or did not affect retarded band intensities, respectively (). Supershift experiments with Sp1 antibodies did not give a defined supershifted (SS) antibody-protein complex but a diffuse band. However, the Sp1 band intensity was decreased due to immunodepletion. We also determined the effects of curcumin on NFκB-DNA binding in a gel mobility shift assay using a consensus NFκB response element and extracts from cells, and the results showed that curcumin decreased retarded band intensities associated with the NFκB-DNA complex (Supplement 2
). These data confirm that curcumin decreases Sp proteins and Sp-DNA complex formation and the NFκB-DNA retarded band and this correlated with decreased expression of NFκB regulated proteins which are also coregulated by Sp transcription factors. These factors suggest that curcumin-dependent downregulation of Sp proteins contributes to decreased expression of several NFκB-dependent proteins which are coregulated by both Sp and NFκB transcription factors (24
Figure 6 Role of Sp protein and NFκB on protein expression and the effects of curcumin on Sp-DNA binding and bladder tumor growth. Effects of iSp and iLamin (A) and small inhibitory RNAs for p65 (ip65) and p50 (ip50) [combined (B)] on genes regulated by (more ...)
We also investigated the antitumorigenic activity of curcumin in athymic nude mice bearing KU7 cells as xenografts. At a dose of 50 mg/kg/d, curcumin significantly decreased tumor volumes and tumor weights (). Moreover, Western blot analysis of tumor lysates from three control and three treated mice show that Sp1, Sp3 and Sp4 protein expression is decreased in the latter group. These results complement the in vitro studies and demonstrate that curcumin-induced effects on Sp proteins also plays a role in the mechanism of action of this compound in bladder cancer cells.