Curcumin potentiates the antitumor effects of gemcitabine in an orthotopic model of human bladder cancer through suppression of proliferative and angiogenic biomarkers

Abstract

Little progress has been made in the last three decades in the treatment of bladder cancer. Novel agents that are nontoxic and can improve the current standard of care of this disease are urgently needed. Curcumin, a component of Curcuma longa (also called turmeric), is one such agent that has been shown to suppress pathways linked to oncogenesis, including cell survival, proliferation, invasion and angiogenesis. We investigated whether curcumin has potential to improve the current therapy for bladder cancer, using an orthotopic mouse model. Curcumin potentiated the apoptotic effects of gemcitabine against human bladder cancer 253JBV cells in culture. Electrophoretic mobility shift assay revealed that curcumin also suppressed the gemcitabine-induced activation of the cell survival transcription factor NF-kappaB. In an orthotopic mouse model, bioluminescence imaging revealed that while curcumin alone significantly reduced the bladder tumor volume, maximum reduction was observed when curcumin was used in combination with gemcitabine (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone). Curcumin also significantly decreased the proliferation marker Ki-67 and microvessel density (CD31) (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone), but maximum reduction occurred when it was combined with gemcitabine (P<0.01 versus vehicle; P<0.01 versus gemcitabine alone). Curcumin abolished the constitutive activation of NF-kappaB in the tumor tissue; induced apoptosis, and decreased cyclin D1, VEGF, COX-2, c-myc and Bcl-2 expression in the bladder cancer tissue. Overall our results suggest that curcumin alone exhibits significant antitumor effects against human bladder cancer and it further potentiates the effects of gemictabine, possibly through the modulation of NF-kappaB signaling pathway.

Figure 1. Curcumin potentiates the effects of gemcitabine against human bladder cancer cells in vitro

(A) 253JBV cells (5000 cells per 0.1 mL) were incubated with indicated concentrations of curcumin and gemcitabine at 37°C for 48 h and then cell viability was examined using the 3-(4, 5-dimethylthiazol -2- yl)- 2,5-diphenyltetrazolium bromide reagent. Data are the representative of three independent experiments. Columns, mean (n=3); bars, SE. *, P < 0.001, compared with untreated cells. (B) Curcumin potentiates the cytotoxic effects of gemcitabine as determined by the Live/Dead assay. Cells were treated with curcumin (5 μM), gemcitabine (100 nM), or the combination. After 24 h incubation, cells were stained with the assay reagents and cell viability was determined under a fluorescence microscope. Percentages, apoptotic bladder cancer cells. Values are mean of triplicates. (C) Curcumin potentiates the cell cycle arrest in human bladder cancer cells. 253JBV (1 × 10⁶) cells were incubated with curcumin (5 μM), gemcitabine (100 nM) or both for 48 h, then stained with propidium iodide and analyzed using FACS analysis. Percentage of Sub G₀–G₁ apoptosis in each group was plotted. Data are the representative of three independent experiments. Columns, mean (n = 3); bars, SE. (D) Curcumin potentiate the gemcitabine-induced apoptosis in bladder cancer cells. 253JBV (1 × 10⁶) cells were incubated with curcumin (5 μM), gemcitabine (100 nM) or both for 48 h. Thereafter, cells were incubated with anti-Annexin V antibody with FITC and analyzed with a flow cytometry for early apoptotic events. Columns, mean (n = 3); bars, SE. (E) Curcumin potentiates the effect of gemcitabine on the expression of gene products linked to tumor cell survival, inflammatory, proliferation and angiogenesis. 253JBV (0.5 × 10⁶) cells were incubated with curcumin (5 μM), gemcitabine (100 nM) or both for 48 h. Whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis using the indicated proteins. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading.

Figure 2. Gemcitabine induces NF-κB activation in human bladder cancer cells and curcumin inhibits it

(A) Bladder cancer cells (253JBV cells, 1 × 10⁶) were treated with gemcitabine 100 μM for indicated time intervals. Nuclear extracts were prepared and then analyzed for NF-κB activation using EMSA. Fold activation is indicated. (B) Bladder cancer cells (253JBV cells, 1 × 10⁶) were exposed to gemcitabine 100 μM for 8 h and then treated with different concentrations of curcumin for 4 h. Nuclear extracts were prepared and were analyzed for NF-κB activation using EMSA. CV (%) indicates cell viability of the cells. (C) NF-κB induced by gemcitabine is composed of p65 and p50 subunits. Nuclear extracts from untreated 253JBV cells or 100 M gemcitabine-treated were incubated with the indicated antibodies, an unlabeled NF-κB oligonucleotide probe, or a mutant oligonucleotide (Mutant oligo) probe. They were then assayed for NF-κB activation by EMSA.

Figure 3. Curcumin potentiates the antitumor effects of gemcitabine against human bladder cancer growth in nude mice

(A) Schematic representation of experiment protocol described in materials and methods. Animals were divided into four groups. (a) untreated control (corn oil, 100 μL daily); (b) curcumin alone (1 g/kg), once daily, orally; (c) gemcitabine alone (25 mg/kg), thrice weekly, i.p.; and (d) combination of curcumin (1 g/kg), once daily, orally and gemcitabine (25 mg/kg), thrice weekly, i.p. (B) (Left) Bioluminescence IVIS images of orthotopically implanted bladder tumor in live, anesthetized mice. (Right) Measurements of photons per second depicting the tumor volumes of mice using the IVIS imaging at indicated time intervals (n = 8). Points, mean; Bars, SE. *, P < 0.01, vehicle versus Cur + Gem; **, P < 0.0001, gemcitabine versus Cur + Gem. (C) Tumor weights measured on the last day of the experiment. (n = 8); Bars, SE. P < 0.001, vehicle versus Cur + Gem; P < 0.05, gemcitabine versus Cur + Gem.

Figure 4. Curcumin potentiates the effect of gemcitabine by inhibiting markers of proliferation (Ki-67) and microvessel density (CD-31)

(A) Immunohistochemical analysis of proliferation marker Ki-67 in bladder tumors indicates the inhibition of cell proliferation in curcumin alone and in combination with gemcitabine treated mice. (B) Quantification of Ki-67⁺ cells as described in materials and methods. Columns, mean (n = 3); bars, SE. (C) Immunohistochemical analysis of CD31 for microvessel density indicates inhibition of angiogenesis by curcumin alone and in combination with gemcitabine treated mice. (D) D. Quantification of CD31⁺ for microvessel density as described in materials and methods. Columns, mean (n = 3); bars, SE.

Figure 5. Curcumin potentiates the effect of gemcitabine by inducing apoptosis and inhibiting cyclin D1, COX-2, and VEGF

(A) TUNEL assay showed that Curcumin potentiates the effect of gemcitabine by inducing apoptosis in mouse bladder tissues. (B) Immunohistochemical analysis of Cyclin D1. The percentage inhibition cyclin D1 by curcumin alone and in combination with gemcitabine is indicated. (C) Immunohistochemical analysis of COX2. The percentage inhibition COX-2 by curcumin alone and in combination with gemcitabine is indicated. (D) Immunohistochemical analysis of VEGF. The percentage inhibition VEGF by curcumin alone and in combination with gemcitabine is indicated.

Figure 6. Curcumin inhibits NF-κB and NF-κB regulated gene products in tumor tissue

(A) Immunohistochemical analysis of nuclear p65. The percentage inhibition nuclear p65 by curcumin alone and in combination with gemcitabine is indicated. (B) Detection of NF-κB by DNA binding in orthotopic tumor tissue samples. (C) Western blot showing that curcumin inhibits the expression of NF-κB dependent gene products cyclin D1, COX-2, VEGF, c-myc, and Bcl-2 in human bladder tumors.