Curcumin circumvents chemoresistance in vitro and potentiates the effect of thalidomide and bortezomib against human multiple myeloma in nude mice model

Abstract

Curcumin (diferuloylmethane), a yellow pigment in turmeric, has been shown to inhibit the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor closely linked to chemoresistance in multiple myeloma cells. Whether curcumin can overcome chemoresistance and enhance the activity of thalidomide and bortezomib, used to treat patients with multiple myeloma, was investigated in vitro and in xenograft model in nude mice. Our results show that curcumin inhibited the proliferation of human multiple myeloma cells regardless of their sensitivity to dexamethasone, doxorubicin, or melphalan. Curcumin also potentiated the apoptotic effects of thalidomide and bortezomib by down-regulating the constitutive activation of NF-kappaB and Akt, and this correlated with the suppression of NF-kappaB-regulated gene products, including cyclin D1, Bcl-xL, Bcl-2, TRAF1, cIAP-1, XIAP, survivin, and vascular endothelial growth factor. Furthermore, in a nude mice model, we found that curcumin potentiated the antitumor effects of bortezomib (P<0.001, vehicle versus bortezomib+curcumin; P<0.001, bortezomib versus bortezomib+curcumin), and this correlated with suppression of Ki-67 (P<0.001 versus control), CD31 (P<0.001 versus vehicle), and vascular endothelial growth factor (P<0.001 versus vehicle) expression. Collectively, our results suggest that curcumin overcomes chemoresistance and sensitizes multiple myeloma cells to thalidomide and bortezomib by down-regulating NF-kappaB and NF-kappaB-regulated gene products.

Figure 1. Curcumin suppresses the proliferation of drug-resistant MM cells

(A) Structure of curcumin. (B) U266 cells (5 × 10³ cells) treated with various concentrations of curcumin, dexamethasone-sensitive (top-middle panel) and dexamethasone-resistant (top-right panel) MM.1 cells (5 × 10³ cells), doxorubicin-sensitive (middle-left panel) and doxorubicin-resistant (middle-right panel) RPMI-8266 cells (5 × 10³ cells), and melphalan-sensitive (bottom-right panel) and melphalan-resistant (bottom-right panel) RPMI-8266 cells were plated in triplicate, treated with 5 and 10 μM curcumin, and then subjected to MTT assay on days 2, 4, or 6 to analyze MM cell proliferation. Data are means ± SD for three experiments; ^**, P < 0.01; ^*, P < 0.05, vs. control.

Figure 2. Curcumin potentiates the apoptotic effect of bortezomib and of thalidomide

(A) (left) U266 cells (1 × 10⁶/mL) were treated with 25 μM curcumin, 20 nM bortezomib, or 10 μg/mL thalidomide alone or a combination of these two agents with curcumin for 24 hours at 37°C. Cells were stained with a Live/Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope. Percentage of apoptosis is indicated in the inset. (right) a graphic representation of the data with U266 cells. (B) U266 cells (1 × 10⁶/mL) were treated with 25 μM curcumin, 20 nM bortezomib, or 10 μg/mL thalidomide alone or a combination of these agents with curcumin for 12 hours at 37°C. Cells were incubated with anti-annexin V antibody conjugated with fluorescein isothiocyanate and then analyzed with a flow cytometery to detect early apoptotic effects. Data are means ± SD for three experiments; ^*, P < 0.001, vs. control as well as single agent. (C) U266 cells (1 × 10⁶/mL) were treated with 25 μM curcumin, 20 nM bortezomib, or 10 μg/mL thalidomide alone or a combination of these agents with curcumin for 12 hours at 37°C. Whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis using antibody against PARP. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. The results shown represent 3 independent experiments. (D) U266 cells (1 × 10⁶/mL) were treated with 5 μM curcumin or 10 nM bortezomib alone or a curcumin+bortezomib combination for 12 hours at 37°C and then tested for NF-κB activation by EMSA (left panel). U266 cells (1 × 10⁶/mL) were treated with 5 μM curcumin or 10 μg/mL thalidomide alone or a curcumin+thalidomide combination for 12 hours at 37°C and then tested for NF-κB activation by EMSA (right panel). Oct-1 EMSA served as a loading control. The results shown represent 3 independent experiments.

Figure 3. Curcumin suppresses the expression of antiapoptotic proteins in MM cells

(A) Curcumin suppresses Akt activation in U266 cells. U266 cells (2 × 10⁶/mL) were treated with 50 μM curcumin for the indicated times. 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 Akt antibody to show equal protein loading. (B) Curcumin inhibits the expression of antiapoptotic gene products. U266 cells (2 × 10⁶/mL) were treated with 50 μM curcumin for the indicated times. 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 Akt and β-actin antibody to show equal protein loading.

Figure 4. Curcumin potentiates the antitumor effects of bortezomib in myeloma tumor growth in nude mice induced by U266 cells

(A) Schematic representation of the experimental protocol as described in “Materials and Methods.” U266 cells (2×10⁶/mice) were injected subcutaneously into the left flank of the mice. The animals were randomized after 1 week of tumor cell injection into four groups based on tumor volume. Group I was treated with corn oil (100 μL; orally; daily) and saline (100 μL; orally; once a week), group II was treated with curcumin alone (1 g/kg daily; orally), group III was treated with bortezomib alone (0.25 mg/kg; orally; once a week), and group IV with a combination of curcumin (1 g/kg orally; daily) and bortezomib (0.25 mg/kg; orally; once a week) (n = 6). (B) The tumor diameters were measured at 5-day intervals with Vernier calipers, and the tumor volumes were calculated using the formula V = 2/3 πr³ (n = 5). (C, left panel) Necropsy photographs of mice bearing xenotransplanted MM. (C, right panel) Tumor volumes (mean ± SE) calculated using the formula V = 2/3 πr³ (n = 6) after tumor diameters were measured on the last day of the experiment at autopsy using Vernier calipers. (D) The tumor volume of mice at different time intervals.

Figure 5. Curcumin enhances the inhibitory effect of bortezomib on the activation of NF-κB and expression of the cell proliferation marker Ki-67 in MM tumor samples

(A) EMSA analysis revealed that curcumin inhibited NF-κB activation in nuclear extracts from animal tissue. (B, left panel) Immunohistochemical analysis of nuclear p65 in tumor tissues showed that curcumin alone and in combination with bortezomib inhibited NF-κB activation. (B, right panel) Quantification of NF-κB expression in MM tumor samples. Percentages indicate p65 nuclear positive cells. Samples from 3 animals in each treatment group were analyzed, and representative data are shown. (C, left panel) Immunohistochemical analysis of Ki-67⁺ cells in MM indicated that curcumin alone and in combination with bortezomib suppressed cell proliferation. Samples from 3 animals in each treatment group were analyzed, and representative data are shown. (C, right panel) Quantification of Ki-67 proliferation index as described in “Materials and Methods.” Values are represented as mean ± SE of triplicate.

Figure 6. Curcumin enhances effects of bortezomib to inhibit angiogenesis in MM tumor samples

(A, left panel) Immunohistochemical analysis of the microvessel density marker CD31 in MM tumor samples. Samples from 3 animals in each treatment group were analyzed, and representative data are shown. (A, right panel) Quantification of CD31⁺ microvessel density as described in “Materials and Methods.” Values are represented as mean ± SE of triplicate. (B, left panel) Immunohistochemical analysis of VEGF in MM tumor samples revealed that curcumin alone and in combination with bortezomib suppresses angiogenesis. Samples from 3 animals in each treatment group were analyzed, and representative data are shown. (B, right panel) Quantification of VEGF as described in “Materials and Methods.” Values are represented as mean ± SE of triplicate.