Previous experiments have shown that curcumin or cisplatin treatment suppresses growth of head and neck squamous cell carcinoma (HNSCC). To study the potential cooperative effect of both agents, two HNSCC cell lines were treated with curcumin or cisplatin alone or in combination. In vivo studies consisted of intravenous tail vein injection of liposomal curcumin, with intraperitoneal cisplatin, into nude mice growing xenograft HNSCC tumors. Introduction of curcumin and suboptimal concentrations of cisplatin showed a significant suppressive effect compared with treatment with either agent alone. Reduced expression of cyclin D1, IκBα, phospho-IκBα, and IKKβ occurred in cisplatin- and curcumin-treated cell lines. Confocal microscopy showed expression of IKKβ in the nucleus of the cell lines. Chromatin immunoprecipitation assay on DNA isolated from IKKβ immunoprecipitated samples showed PCR amplification of interleukin-8 promoter sequences, a binding site of NFκB, indicating an interaction between IKKβ and NFκB. Curcumin inhibited IKKβ in the cytoplasm and nucleus, leading to reduced NFκB activity, with no effect on phospho-AKT. In vivo studies showed significant growth inhibition of xenograft tumors treated with a combination of liposomal curcumin and cisplatin. The suppressive effect of curcumin was mediated through inhibition of cytoplasmic and nuclear IKKβ, resulting in inhibition of NFκB activity. Cisplatin treatment led to cellular senescence, indicating an effect mediated by p53 activation. The mechanisms of the two agents through different growth signaling pathways suggest potential for the clinical use of subtherapeutic doses of cisplatin in combination with curcumin, which will allow effective suppression of tumor growth while minimizing the toxic side effects of cisplatin. Mol Cancer Ther; 9(10); 2665–75. ©2010 AACR.
Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a poor prognosis for advanced tumors. There were 40,490 new cases of HNSCC in the United States in 2006, accounting for ∼3% of adult malignancies (1). The worldwide incidence exceeds half a million cases annually. The significant morbidity associated with current treatment modalities, which include disfiguring surgery, chemotherapy, and radiation, has led to continuing investigation of potential alternative and less toxic therapies.
Current treatment regimens for HNSCC include use of platinum-based chemotherapy such as cisplatin (2). Cisplatin is not effective when used as a single agent, but its efficacy is significantly enhanced when used with radiation therapy and/or in combination with other chemotherapeutic drugs. Thus, cisplatin/radiation or combination chemotherapy is the cornerstone of treatment of many cancers, including head and neck cancer. An example of this is the combination of cisplatin and cetuximab in the treatment of metastatic/recurrent head and neck cancer (3). Initial platinum responsiveness is high, but the majority of cancer patients eventually relapse with cisplatin-resistant disease. Many mechanisms of cisplatin resistance have been proposed, including changes in cellular uptake and efflux of the drug, inhibition of apoptosis, and increased DNA repair. We have hypothesized that cisplatin induces cell cycle arrest through a p16/p53-dependent pathway (4). Cisplatin has several dose-dependent side effects, including renal, otologic, and bone marrow suppression. Thus, a suboptimal level of cisplatin in combination with nontoxic agents would be useful for effective treatment of HNSCC.
Curcumin (diferuloylmethane) is the major component of the spice turmeric and is derived from the rhizome of the East Indian plant Curcuma longa (Fig. 1A). It has been consumed as a dietary supplement for centuries and has also been shown to prevent tumor initiation, proliferation, and metastasis in breast, colon, oral, and other human cancers (5). Curcumin is soluble only in organic solvents, but liposomal formulations have been studied in the treatment of pancreatic cancer (6).
Delivery of curcumin is limited by its poor bioavailability and insolubility in saline. Previous studies from our laboratory have shown that curcumin treatment suppresses growth of HNSCC cell lines in vitro and reduces tumor volume in vivo via topical application (7). More recently, our laboratory has shown the use of liposome-encapsulated curcumin for intravenous administration of curcumin, and bioavailability studies confirmed the presence of curcumin in circulating blood and liver (8). Its growth-inhibitory effect was mediated through the inhibition of transcription factor NFκB, resulting in the downregulation of IκBα, cyclin D1, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and interleukin-8 (IL-8) genes.
In the present study, we investigated whether curcumin would enhance the suppressive effect of cisplatin in HNSCC. Two HNSCC cell lines, CAL27 and UM-SCC1, were treated with curcumin and cisplatin individually or in combination. Nude mice with subcutaneous xenograft CAL27 tumors were administered with cisplatin alone or a combination of cisplatin and liposomal curcumin. In addition, the mechanism of action of curcumin, inhibiting transactivation of NFκB, mediated through cytoplasmic and nuclear IKKβ, was studied.
Materials and Methods
HNSCC cell lines CAL27 and UM-SCC1, representing oral cancers, were used. CAL27 was obtained from the American Type Culture Collection, and UM-SCC1 was obtained from Dr. Thomas E. Carey (University of Michigan, Ann Arbor, MI). CAL27 cells were characterized by the American Type Culture Collection using polyphasic (genotypic and phenotypic) testing methods. UM-SCC1 cells were not characterized by our laboratory after receiving from the University of Michigan. Cell lines were grown in DMEM containing high glucose (4,500 μg/mL) and 1 mmol/L glutamine, 100 IU/mL penicillin, and 10% fetal bovine serum (Sigma).
Liposomal curcumin preparation
A 9:1 ratio of lipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (Sigma-Aldrich) was dissolved in tert-butanol at a concentration of 10 mg/mL. Sterile water (1/20 volume) was added and 1 part curcumin (purity, 97%; Cayman Chemical) was added for a final lipid/curcumin ratio of 10:1. The solution was sterile filtered, frozen in dry ice and acetone, and lyophilized overnight. Liposomal curcumin was suspended in sterile 0.9% NaCl at 65°C to yield a 100 mmol/L stock solution.
Curcumin and cisplatin treatment of HNSCC cell lines
Cells were plated in 24-well plates (30,000 per well) and allowed to grow for 24 hours. After serum starvation for 24 hours, cells were treated with cisplatin [10 μmol/L (i.e., 3 μg/mL) or 20 μmol/L (i.e., 6 μg/mL)] for 5 hours or with liposomal curcumin (100 μmol/L) for 8 hours. Combination treatment consisted of addition of cisplatin 3 hours after the addition of liposomal curcumin. Untreated cells or those treated with empty liposomes alone (100 μmol/L) were used as controls. Cells were then allowed to incubate in serum-containing medium at 37°C, and cell viability was measured at 24 hours. Growth assays were done at least twice and each time in triplicates.
Cell viability assay
Growth medium was aspirated out of wells and 1.0 mL of MTT solution (1 mg/mL in complete medium; Sigma-Aldrich) was added to each well. Cells were then incubated at 37°C for 4 hours. The MTT solution was aspirated out of the wells, air dried for 5 minutes, and dissolved in isopropanol, and absorbance values were read in an ELISA microplate reader at 570 nm as described (4).
Western blot analysis
Proteins were extracted from cells or xenograft tumors using radioimmunoprecipitation assay lysis buffer containing a complete protease inhibitor cocktail (Roche Diagnostics). Western blotting was done using 20 to 30 μg of denatured proteins on 4% to 20% SDS acrylamide gels (Invitrogen, Inc.). Proteins transferred onto nitrocellulose were hybridized following the established protocol (7) to the antibodies (cyclin D1, phospho-IκBα, IκBα, histone H1, and histone H3; Santa Cruz Biotechnology). All Western blot analyses were done at least twice.
Cells were grown overnight on coverslips to semiconfluence (70–80%) and treated with liposomal curcumin for 30 minutes, 1 hour, or 4 hours. Cells were then treated with tumor necrosis factor α (TNFα) for 15 minutes. Untreated cells and cells treated with empty liposomes for 4 hours were included as controls. Immunofluorescence was done using the IKKβ, NFκB, phospho-AKT, and phospho-IκBα antibodies (Santa Cruz Biotechnology) following the established protocol (7). Confocal microscopy was done following established protocol using a Zeiss microscope.
Chromatin immunoprecipitation assay
UM-SCC1 cells grown to 80% confluency were treated with TNFα for 1 hour and cross-linked with 37% formaldehyde for 10 minutes at 37°C. Chromatin immunoprecipitation (ChIP) assays were done using ChIP Assay kit (Millipore) following the manufacturer's protocol. Immunoprecipitations were carried out using antibodies to IgG (Santa Cruz Biotechnology), IKKβ (Cell Signaling), and NFκB (Calbiochem). DNA isolated from the input as well as the immunoprecipitated samples were amplified using IL-8 and β-actin primers (9). The following primers were used in the PCR: IL-8, 5′-GGGCCATCAGTTGCAAATC-3′ (forward) and 5′-TTCCTTCCGGTGGTTTCTTC-3′ (reverse); β-actin, 5′-CAACGCCAAAACTCTCCCTC-3′ (forward) and 5′-ATCGGCAAAGGCGAGGCTCTG-3′ (reverse). DNAs were denatured at 95°C for 1 minute, annealed at 55°C for 1 minute, and extended at 72°C for 1 minute for 35 cycles. The PCR products were separated on 10% polyacrylamide gels and stained with ethidium bromide, and images were captured using the Kodak Gel documentation system.
CAL27 cells grown to semiconfluence in six-well plates were treated with liposomal curcumin (100 μmol/L) for 8 hours or cisplatin (20 μmol/L) for 5 hours. Growth medium was replaced and cells were grown for 4 days. Cells were then stained with β-galactosidase, and blue senescence colonies were visualized under the microscope. Photographs were taken using a Nikon camera.
Curcumin binding studies
CAL27 cells grown to semiconfluence in 100-mm dishes were treated with liposomal curcumin (100 μmol/L) for 8 hours. After medium replacement, cells were grown for 24 hours and proteins were extracted from total cell lysates. Immunoprecipitation was done with IKKβ or phospho-S6K antibody, and the precipitate was washed five times with the cell lysate buffer. Curcumin was extracted using ethyl acetate/methanol mixture and vacuum dried, and tandem mass spectrometry (MS/MS) analysis was carried out on a triple-quadrupole spectrophotometer as described (8). Curcumin peak identification and intensity measurement was determined using a standard curcumin run as a control. Untreated cells and cells treated with liposome alone were used as negative controls.
HNSCC xenograft tumors in mice
Five-week-old female athymic nude mice (nu/nu; Harlan) were used for in vivo experiments. Animals were injected with 2 × 106 cells in the right flank to form xenograft tumors. Animals were housed in sterile rodent microisolator caging, with filtered cage top. Two to four animals were housed in each cage, with animals resting directly on bedding. They were given free access to sterile water and food. All cages, covers, and bedding were sterilized weekly. All animal procedures were approved by the Institutional Animal Care and Use Committee of the West Los Angeles Veterans Affairs Medical Center, in accordance with the USPHS Policy on Humane Care and Use of Laboratory Animals.
Treatment of nude mice with liposomal curcumin and cisplatin
Once subcutaneous nodules were visible at the inoculation site (days 7–10 after inoculation), liposomal curcumin (50 mg/kg, 1 mg for the 20-g mouse in a maximal volume of 100 μL) or equal amount of empty liposomes was administered via tail vein injection three times a week for 4 weeks. In the fourth week, cisplatin (100 μL of 7.5 μg/mL) was administered through intraperitoneal injection. There were four groups in the study: mice with no treatment (nine), mice treated with cisplatin (seven), mice treated with empty liposomes and cisplatin (five), and mice treated with liposomal curcumin and cisplatin (six). Tumor size was measured weekly using Vernier calipers, and tumor volume was calculated using the formula V = 4/3pW2L, where W is half of the shorter axis diameter and L is half of the longer axis diameter as described (10). At the end of the fourth week, mice were sacrificed and tumors were removed.
For the MTT absorbance, cell viability was set to be 100% at baseline, and the percent of viable cells [(viable cell count in the experiment group/mean viable cell count) × 100] per replicate was calculated at each of the posttreatment days. To examine the differences in the reduction of cell viability over time among groups, ANOVA modeling was used with the following covariates: group, time (measured in days), and a group-by-time interaction term. The model also allows heterogeneous variance across groups.
Time trends in tumor volume among experimental groups were examined using the piecewise regression model, which allows for the trend after the injection of cisplatin at week 3 to be different from the trend before injection of cisplatin. Although mice were followed over time, their IDs were not recorded due to technical difficulties. Thus, the common repeated-measures model cannot be used in this situation. Instead, the piecewise regression model was used, with the assumption of heterogeneous variance across groups to analyze tumor volume growth. All statistical analyses were carried out with the SAS System for Windows (version 9.2)
Growth inhibition of HNSCC cell lines in vitro with liposomal curcumin and cisplatin
Two HNSCC cell lines, CAL27 and UM-SCC1, representing aggressive oral cancers were tested for growth inhibition with cisplatin or liposomal curcumin alone or in combination. All experiments were done in triplicate in 24-well plates, and viable cell counts were measured at 24, 48, 72, and 96 hours after treatment. Empty liposomes, curcumin alone, and cisplatin alone had a significantly greater growth inhibition in CAL27 cells over time (i.e., slope of growth inhibition) compared with the untreated control (P = 0.0015, <0.0001, and <0.0001, respectively; Fig. 1B and C). There was significantly greater effect in liposomal curcumin–treated cells in combination with cisplatin compared with cisplatin alone–treated cells (P = 0.0152). Statistical significance was not observed for combination treatment of liposomal curcumin and cisplatin versus liposomes and cisplatin. In UM-SCC1 cells, liposomes alone did not show an inhibitory effect. Significant growth inhibition over time was seen in the presence of cisplatin (P = 0.0486 and 0.0011, for 10 and 20 μmol/L, respectively), curcumin alone (P = 0.0064), curcumin in combination with 10 μmol/L cisplatin (P = 0.0016), and liposomes in combination with 10 μmol/L cisplatin (P = 0.0247) compared with control. We have also observed a significantly greater effect over time in curcumin-treated cells compared with cells treated with liposomes (P = 0.0011). The combination treatment, liposomal curcumin and 10 μmol/L cisplatin, showed an effect similar to that seen in cells treated with 20 μmol/L cisplatin.
Reduced expression of NFκB-activated genes in curcumin and cisplatin-treated cells
We have previously shown that curcumin and cisplatin downregulate the expression of genes involved in cell cycle and apoptosis and also that cisplatin induces cellular senescence through the upregulation of p16 and p53 tumor suppressor genes (4, 7). Our studies have also shown that the effect of curcumin is mediated by the inhibition of IKKβ kinase, resulting in reduced phosphorylation of IκBα (inhibitor of NFκB) and retention of NFκB in the cytoplasm (11). Thus, cisplatin and curcumin seem to suppress cell growth through independent mechanisms.
To verify that the two agents act via different mechanisms, we did expression analysis of proteins collected from the drug-treated cell lines. Both cisplatin and curcumin showed reduced expression of cyclin D1 and phospho-IκBα in CAL27 and UM-SCC1 cell lines (Fig. 2A). Because our previous studies have shown lower-level expression of these two proteins in CAL27 cells, in the present experiments, the cells were pretreated with TNFα for 15 minutes before the addition of drugs. A higher concentration of cisplatin (20 μmol/L) showed greater reduction in the expression of cyclin D1 and phospho-IκBα in comparison with treatment with 10 μmol/L cisplatin. In addition, combined treatment with 10 μmol/L cisplatin and liposomal curcumin resulted in greater inhibition, equaling that of treatment with 20 μmol/L cisplatin. Empty liposomes did not affect the expression of these two proteins. Western blot analysis showing reduced expression of IκBα and IKKβ in cisplatin (20 μmol/L)– and liposomal curcumin (100 μmol/L)–treated CAL27 cells (Fig. 2B) further indicated a possible direct effect on the expression of these two proteins by cisplatin and curcumin.
To confirm further that curcumin inhibited phospho-IκBα expression, immunofluorescence was done on CAL27 cells treated with liposomes or liposomal curcumin. Phospho-IκBα expression was mostly seen in the cytoplasm, and the expression level increased with the addition of TNFα (Fig. 2C). Treatment with empty liposomes resulted in increased localization of the protein to the perinuclear region. However, curcumin treatment led to reduced protein expression in the cytoplasm and in the perinucleus of the cells, confirming the inhibitory activity of curcumin on IKK kinase, resulting in decreased phosphorylation of IκBα.
To show that cisplatin inhibits cell growth through a senescence pathway, CAL27 cells treated with liposomal curcumin or cisplatin were stained for the expression of β-galactosidase, a marker for cellular senescence. Untreated cells contained a low-level background blue staining due to cell death from confluent cell culture in 4 days of study protocol (Fig. 2D). Although there was cell killing in curcumin-treated cells, there was no observable blue stain, indicating a senescence-independent cell death by liposomal curcumin. As expected, cisplatin-treated cells showed a high-level expression of β-galactosidase, correlating to senescence-mediated cell death of HNSCC cells.
Curcumin binds to IKKβ protein subunit of the IKK complex
Because we have previously shown that curcumin inhibited IKK kinase activity (11), we did MS/MS analysis to determine whether there was a direct binding of curcumin to IKKβ protein. Cell lysates of CAL27 treated with curcumin were immunoprecipitated with IKKβ or a control phospho-S6K antibody, and the antibody-bound products were extracted with organic solvents. Analysis by MS showed the presence of curcumin peak in the cell lysates. Intensity measurements using a curcumin standard showed a 5-fold increased binding of curcumin to IKKβ in comparison with phospho-S6K (Supplementary Table S1), indicating an interaction between curcumin and IKKβ.
Nuclear expression of IKKβ protein in HNSCC cell lines
To verify further whether curcumin binding resulted in the inhibition of IKKβ expression as was seen in Western blotting, confocal microscopy was done. We observed expression of IKKβ both in the cytoplasm and in the nucleus of CAL27 and UM-SCC1 cell lines (Fig. 3A). Although the nuclear presence of the IKKα protein has been shown before (12), there is only one recent report indicating the expression of IKKβ protein in the nucleus of normal and neoplastic B lymphocytes (9). Thus, for the first time, we have seen IKKβ expression in the nucleus of HNSCC cell lines. To confirm this finding, we used two different IKKβ antibodies and HeLa cells, a cervical cancer cell line shown before to contain cytoplasmic IKKβ expression. Expression of IKKβ in HeLa cells was seen in the cytoplasm as well as in the nucleus of the cells (Fig. 3A). Costaining with 4′,6-diamidino-2-phenylindole (DAPI; left column), a nuclear-specific stain, showed magenta staining of the nucleus (last column in Fig. 3A), confirming nuclear expression of IKKβ in HeLa and HNSCC cell lines. Whereas treatment with liposomes did not have an effect on the expression of IKKβ, liposomal curcumin treatment resulted in an inhibitory effect both in the cytoplasmic and in the nuclear expression of this protein (last row in Fig. 3A). As a confirmation to cytoplasmic and nuclear localization of IKKβ, we did confocal microscopy for nuclear-specific proteins histones H1 and H3. The results showed complete localization of histone H1 to the nucleus (Fig. 3B). Expression of histone H3 was seen as speckles inside the nucleus as wells in the perinuclear/cytoplasmic region (Fig. 3B). Western blot hybridization studies of the cytoplasmic and nuclear extracts of CAL27 and HeLa cells further confirmed the presence of IKKβ in the nucleus of these two cell lines (Fig. 3C). Immunoprecipitation with nuclear IKKβ from CAL27 cells and hybridization to histone H3 antibody showed binding of histone H3, indicating a role for nuclear IKKβ in chromatin remodeling (Fig. 3C). There was also hybridization to NFκB in these immunoblots, indicating an interaction between NFκB and IKKβ proteins in the cytoplasm as well as in the nucleus. These interactions were confirmed by the hybridization of IKKβ antibody to the immunoprecipitates of cytoplasmic and nuclear NFκB (Fig. 3C). Finally, ChIP assay showed amplification of IL-8 promoter sequences (NFκB binding site) in the DNA isolated from the IKKβ antibody immunoprecipitated samples, indicating an interaction between IKKβ and NFκB (Fig. 3D). IL-8 promoter amplification was not seen with the IgG antibody precipitated samples used as a control. There was no amplification of β-actin promoter sequences in any of the immunoprecipitated samples again used as a control in the ChIP assay.
To verify the effect of curcumin on nuclear IKKβ and on NFκB, liposome- and liposomal curcumin–treated CAL27 cells were studied by immunofluorescence. Expression was also measured with the addition of TNFα, a NFκB upregulating molecule. IKKβ expression was seen in the nucleus, and there was a minimal effect with the addition of TNFα (Fig. 4A). Treatment with liposome did not alter the expression. However, reduced expression was observed in as little as 30 minutes after curcumin treatment. The presence of IKKβ in nucleosome-like particles again showed a possible role of this protein in chromatin remodeling. Expression of NFκB was seen in the cytoplasm, and increased nuclear transport occurred with the addition of TNFα (Fig. 4B). NFκB expression in liposome-treated cells resembled that of control cells. Reduction in the expression of NFκB could be seen in curcumin-treated cells. However, the inhibitory effect was pronounced with the blockage of NFκB transport in the presence of TNFα, confirming the inhibitory effect of curcumin on the transcriptional activation of NFκB. Here, the results clearly show the effect of curcumin on cytoplasmic IKKβ, leading to the inhibition of IKKβ kinase activity. Finally, we did not see an inhibitory effect on phospho-AKT expression in the presence or absence of TNFα (Fig. 4C), confirming our previous finding that curcumin inhibited NFκB by an AKT-independent mechanism in HNSCC cells.
Enhanced growth inhibition of in vivo xenograft tumors with combination (liposomal curcumin and cisplatin) therapy
CAL27 xenograft tumors were grown in nude mice, and tumor dimensions were measured weekly with calipers. Once it was evident that xenograft tumors were forming (7 days), liposomes and liposomal curcumin in saline were injected via the tail vein three times a week for 4 weeks. In the fourth week, mice also received cisplatin by intraperitoneal injection. A slower tumor growth rate of liposomal curcumin–treated mice for the first 3 weeks confirmed our earlier findings (8). Results from the piecewise regression model indicated that in comparison with the control, a greater inhibitory effect over time (i.e., estimated slope of growth) was seen with the curcumin-cisplatin combination treatment before and after receiving cisplatin. However, the estimated difference in slopes of growth did not reach statistical significance (P = 0.1098). A box plot is a convenient way of graphically depicting groups of numerical data through their five-number summaries (minimum, lower quartile, median, upper quartile, and maximum). The tumor volume over the first 3 weeks for cisplatin alone (Fig. 5B, top right) and curcumin-cisplatin (Fig. 5B, bottom right) did not go beyond 100 mm (Fig. 5B, dashed lines), except one observation at week 3 in the cisplatin alone group. In addition, a smaller variation in tumor volume is seen 1 week after the injection of cisplatin (week 4) for these two groups. This analysis again showed growth inhibition of xenograft tumors in the combination treatment in comparison with cisplatin alone or the controls (Fig. 5B). The tumor size shown in Fig. 5C showed growth inhibition with cisplatin alone and an enhanced growth reduction with the inclusion of curcumin in the combination treatment. Western blot analysis showed a marginal inhibitory effect on the expression of cyclin D1 in cisplatin-treated tumors (Fig. 5D). However, liposomal curcumin treatment in combination with cisplatin resulted in a marked decrease in cyclin D1 expression correlating to the inhibitory effect on tumor growth.
The mechanism of action of cisplatin includes cell cycle arrest and initiation of apoptosis (13). We and others have shown that cisplatin induces cellular senescence through activation of p53 and p16 proteins, and there is strong evidence that p53 plays a role in cisplatin sensitivity. It also seems that cisplatin-induced growth arrest in human cancer cells has characteristics of senescence in addition to apoptosis (14). In the present investigation, we used HNSCC cell lines containing mutated p53 and lacking p16 expression. The senescence-associated β-galactosidase activity was seen in the CAL27 cells following cisplatin exposure, suggesting that growth of cancer cells containing mutated p53 function can also be inhibited by cisplatin via nuclear transportation of p53 protein (4). Previous results further suggest that functional expression of p16 augments the effect of cisplatin in cell killing. Because p16 and p53 are functionally active in the nucleus, it is likely that cisplatin plays an essential role in the nuclear transport and stabilization of these proteins for cell cycle arrest and apoptosis. We have shown here that in the presence of cisplatin, there is reduced expression of NFκB-regulated proteins cyclin D1 and phospho-IκBα, thereby indicating control of NFκB transactivation by cisplatin through the nuclear p53 protein.
Tumor cells evade apoptosis by downregulation of apoptotic proteins and upregulation of antiapoptotic proteins. Expression of growth signaling pathways gives them survival advantage and allows them to resist therapy-induced apoptosis (15). We and others have shown that curcumin-mediated head and neck cancer cell growth inhibition occurs through the inhibition of NFκB activity (8, 16–18). Recently, we have reported that the inhibitory effect of curcumin on NFκB is by the inhibition of IKK kinase (11). In the present investigation, we have shown that this inhibition could be due to direct interaction between curcumin and IKKβ. Further, for the first time, we have shown the presence of IKKβ in the nucleus whose function seems to be related to transcriptional activation of NFκB by chromatin remodeling. Additional studies including ChIP assay will be required to determine the proteins recruited by IKKβ for this NFκB transactivation.
Transcription factor NFκB and the serine/threonine kinase AKT play critical roles in cancer cell survival and have been shown to be activated in various malignancies (19, 20). Some have shown that curcumin acts via an AKT-dependent pathway in cell growth regulation (21, 22). Previous studies in our laboratory revealed that liposomal curcumin treatment suppressed the activation of NFκB without affecting the expression of phospho-AKT or its downstream target phospho-S6 kinase (8). Here, we have confirmed using immunofluorescence that curcumin affects NFκB expression via an AKT-independent mechanism.
Recent investigative trials include gene-targeted therapies in HNSCC, including cetuximab, bevacizumab, and erlotinib, targeted against epidermal growth factor receptor (EGFR; refs. 23–27). Although anti-EGFR therapies are active in some patients, eventually disease in nearly all patients will become refractory to therapy (28). Mutations in the EGFR receptor seem to play a significant role in the resistance to EGFR therapy. Thus, efforts are under way to identify alternate therapies, including the use of curcumin in combination with radiation and/or chemotherapeutic drugs in hepatic carcinoma, ovarian carcinoma, and HNSCC (29–31).
In conclusion, we have used the combination of curcumin and cisplatin to show enhanced growth suppression in HNSCC. We believe that this occurs through two different molecular pathways. Curcumin affects NFκB transactivation by inhibiting IKK kinase activity both in the cytoplasm and in the nucleus, possibly through an interaction with IKKβ (Fig. 6). We hypothesize that nuclear IKKβ in combination with histone H3 is involved in chromatin remodeling of the NFκB transcription binding sites. Inhibition of IKKβ by curcumin therefore results in reduction in NFκB-occupied sites in chromatin, leading to a decrease in NFκB-mediated transcription. Cisplatin, in contrast, downregulates NFκB through a p53-mediated pathway. We therefore believe that the therapeutic potential of cisplatin will be enhanced with the addition of curcumin, with lower, less toxic doses of cisplatin required for its cytotoxic effect.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
We thank Dr. Yasutada Akiba for confocal microscopy and Dr. Ke Wei Zhao for help in acquiring photographic images.
Grant Support: VA Greater Los Angeles Healthcare, West Los Angeles Surgical Education Research Center, and UCLA Academic Senate (M.B. Wang); NIH grant R21 CA116826-01 (M.B. Wang); and Veterans Administration (Washington, DC) Merit grant (E.S. Srivatsan).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.