Mysore S. Veena

Curcumin: A review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma

Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. Curcumin has been used extensively in Ayurvedic medicine for centuries, as it is nontoxic and has a variety of therapeutic properties including anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer activities via its effect on a variety of biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigenesis and metastasis. Curcumin has shown anti-proliferative effect in multiple cancers, and is an inhibitor of the transcription factor NF-κB and downstream gene products (including c-myc, Bcl-2, COX-2, NOS, Cyclin D1, TNF-α, interleukins and MMP-9). In addition, curcumin affects a variety of growth factor receptors and cell adhesion molecules involved in tumor growth, angiogenesis and metastasis. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and treatment protocols include disfiguring surgery, platinum-based chemotherapy and radiation, all of which may result in tremendous patient morbidity. As a result, there is significant interest in developing adjuvant chemotherapies to augment currently available treatment protocols, which may allow decreased side effects and toxicity without compromising therapeutic efficacy. Curcumin is one such potential candidate, and this review presents an overview of the current in vitro and in vivo data supporting its therapeutic activity in head and neck cancer as well as some of the challenges concerning its development as an adjuvant chemotherapeutic agent.

Curcumin Suppresses Growth of Head and Neck Squamous Cell Carcinoma

Purpose: The purpose of this study was to determine whether curcumin would trigger cell death in the head and neck squamous cell carcinoma (HNSCC) cell lines CCL 23, CAL 27, and UM-SCC1 in a dose-dependent fashion. Experimental Design: HNSCC cells were treated with curcumin and assayed for in vitro growth suppression using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide and fluorescence-activated cell sorting analyses. Expression of p16, cyclin D1, phospho-Iκβ, and nuclear factor-κβ (NF-κβ) were measured by Western blotting, gel shift, and immunofluorescence. Results: Addition of curcumin resulted in a dose-dependent growth inhibition of all three cell lines. Curcumin treatment resulted in reduced nuclear expression of NF-κβ. This effect on NF-κβ was further reflected in the decreased expression of phospho-Iκβ-α. Whereas the expression of cyclin D1, an NF-κβ–activated protein, was also reduced, there was no difference in the expression of p16 at the initial times after curcumin treatment. In vivo growth studies were done using nude mice xenograft tumors. Curcumin was applied as a noninvasive topical paste to the tumors and inhibition of tumor growth was observed in xenografts from the CAL27 cell line. Conclusions: Curcumin treatment resulted in suppression of HNSCC growth both in vitro and in vivo. Our data support further investigation into the potential use for curcumin as an adjuvant or chemopreventive agent in head and neck cancer.

Liposome-Encapsulated Curcumin Suppresses Growth of Head and Neck Squamous Cell Carcinoma In vitro and in Xenografts through the Inhibition of Nuclear Factor κB by an AKT-Independent Pathway

Purpose: The purpose of this study was to determine whether a liposomal formulation of curcumin would suppress the growth of head and neck squamous cell carcinoma (HNSCC) cell lines CAL27 and UM-SCC1 in vitro and in vivo. Experimental Design: HNSCC cell lines were treated with liposomal curcumin at different doses and assayed for in vitro growth suppression using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A reporter gene assay was done on cell lines to study the effect of liposomal curcumin on nuclear factor κB (NFκB) activation. Western blot analysis was done to determine the effect of curcumin on the expression of NFκB, phospho-IκBα, phospho-AKT (pAKT), phospho-S6 kinase, cyclin D1, cyclooxygenase-2, matrix metalloproteinase-9, Bcl-2, Bcl-xL, Mcl-1L, and Mcl-1S. Xenograft mouse tumors were grown and treated with intravenous liposomal curcumin. After 5 weeks, tumors were harvested and weighed. Immunohistochemistry and Western blot analyses were used to study the effect of liposomal curcumin on the expression of NFκB and pAKT. Results: The addition of liposomal curcumin resulted in a dose-dependent growth suppression of both cell lines. Liposomal curcumin treatment suppressed the activation of NFκB without affecting the expression of pAKT or its downstream target phospho-S6 kinase. Expression of cyclin D1, cyclooxygenase-2, matrix metalloproteinase-9, Bcl-2, Bcl-xL, Mcl-1L, and Mcl-1S were reduced, indicating the effect of curcumin on the NFκB pathway. Nude mice xenograft tumors were suppressed after 3.5 weeks of treatment with i.v. liposomal curcumin, and there was no demonstrable toxicity of liposomal curcumin upon autopsy. Immunohistochemistry and Western blot analysis on xenograft tumors showed the inhibition of NFκB without affecting the expression of pAKT. Conclusions: Liposomal curcumin suppresses HNSCC growth in vitro and in vivo. The results suggest that liposomal curcumin is a viable nontoxic therapeutic agent for HNSCC that may work via an AKT-independent pathway.

Curcumin Enhances the Effect of Cisplatin in Suppression of Head and Neck Squamous Cell Carcinoma via Inhibition of IKKβ Protein of the NFκB Pathway

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.

The Malignant Pleural Effusion as a Model to Investigate Intratumoral Heterogeneity in Lung Cancer

Malignant Pleural Effusions (MPE) may be useful as a model to study hierarchical progression of cancer and/or intratumoral heterogeneity. To strengthen the rationale for developing the MPE-model for these purposes, we set out to find evidence for the presence of cancer stem cells (CSC) in MPE and demonstrate an ability to sustain intratumoral heterogeneity in MPE-primary cultures. Our studies show that candidate lung CSC-expression signatures (PTEN, OCT4, hTERT, Bmi1, EZH2 and SUZ12) are evident in cell pellets isolated from MPE, and MPE-cytopathology also labels candidate-CSC (CD44, cMET, MDR-1, ALDH) subpopulations. Moreover, in primary cultures that use MPE as the source of both tumor cells and the tumor microenvironment (TME), candidate CSC are maintained over time. This allows us to live-sort candidate CSC-fractions from the MPE-tumor mix on the basis of surface markers (CD44, c-MET, uPAR, MDR-1) or differences in xenobiotic metabolism (ALDH). Thus, MPE-primary cultures provide an avenue to extract candidate CSC populations from individual (isogenic) MPE-tumors. This will allow us to test whether these cells can be discriminated in functional bioassays. Tumor heterogeneity in MPE-primary cultures is evidenced by variable immunolabeling, differences in colony-morphology, and differences in proliferation rates of cell subpopulations. Collectively, these data justify the ongoing development of the MPE-model for the investigation of intratumoral heterogeneity, tumor-TME interactions, and phenotypic validation of candidate lung CSC, in addition to providing direction for the pre-clinical development of rational therapeutics.

Curcumin treatment suppresses IKKβ kinase activity of salivary cells of patients with head and neck cancer: a pilot study.

Purpose: To determine whether curcumin would inhibit IκB kinase β (IKKβ) kinase activity and suppress expression of proinflammatory cytokines in head and neck squamous cell carcinoma cancer (HNSCC) patients. Experimental Design: Saliva was collected before and after subjects chewed curcumin tablets. Protein was extracted and IKKβ kinase activity measured. Interleukin (IL)-6 and IL-8 levels in the salivary supernatants were measured by ELISA. IL-6, IL-8, and other interleukin were also measured independently with ELISA to confirm the inhibitory effect of curcumin on expression and secretion of salivary cytokines. Results: Curcumin treatment led to a reduction in IKKβ kinase activity in the salivary cells of HNSCC patients (P < 0.05). Treatment of UM-SCC1 cells with curcumin as well as with post-curcumin salivary supernatant showed a reduction of IKKβ kinase activity. Significant reduction of IL-8 levels (P < 0.05) was seen in post-curcumin samples from patients with dental caries. Although there was reduced IL-8 expression in 8 of 21 post-curcumin samples of HNSCC patients, the data did not reach statistical significance. Saliva samples from HNSCC patients were also analyzed in a blinded fashion for expression of cytokines. IL-10, IFN-γ, IL-12p70, and IL-2 clustered together, and granulocyte macrophage colony stimulating factor and TNF-α clustered together. Log10 ratio analysis showed decrease in expression of all nine cytokines in both the salivary supernatant and salivary cells of curcumin-treated samples. Conclusions: Curcumin inhibited IKKβ kinase activity in the saliva of HNSCC patients, and this inhibition correlated with reduced expression of a number of cytokines. IKKβ kinase could be a useful biomarker for detecting the effect of curcumin in head and neck cancer. Clin Cancer Res; 17(18); 5953–61. ©2011 AACR.

Cancer stem cells, microRNAs, and therapeutic strategies including natural products

Embryonic stem cells divide continuously and differentiate into organs through the expression of specific transcription factors at specific time periods. Differentiated adult stem cells on the other hand remain in quiescent state and divide by receiving cues from the environment (extracellular matrix or niche), as in the case of wound healing from tissue injury or inflammation. Similarly, it is believed that cancer stem cells (CSCs), forming a smaller fraction of the tumor bulk, also remain in a quiescent state. These cells are capable of initiating and propagating neoplastic growth upon receiving environmental cues, such as overexpression of growth factors, cytokines, and chemokines. Candidate CSCs express distinct biomarkers that can be utilized for their identification and isolation. This review focuses on the known and candidate cancer stem cell markers identified in various solid tumors and the promising future of disease management and therapy targeted at these markers. The review also provides details on the differential expression of microRNAs (miRNAs), and the miRNA- and natural product-based therapies that could be applied for the treatment of cancer stem cells.

Suppression of Interleukin 6 and 8 Production in Head and Neck Cancer Cells With Curcumin via Inhibition of Iκβ Kinase

OBJECTIVES To evaluate the effect of curcumin on production of interleukin 6 (IL-6) and 8 (IL-8) in head and neck squamous cell carcinoma (HNSCC) cell lines and to determine the mechanism by which these effects are modulated. Curcumin suppression of HNSCC is believed to be partly due to inhibition of the transcription factor nuclear factor-kappa beta (NF-kappa beta). Interleukin 6 and IL-8 are cytokines induced by NF-kappa beta activation with elevated levels in the serum of patients with HNSCC. DESIGN We treated HNSCC cell lines CCL23, CAL27, UM-SCC1, and UM-SCC14A with increasing doses of curcumin and measured IL-6 and IL-8 levels using an enzyme-linked immunosorbent assay. SETTING Levels of NF-kappa beta, Ikappa beta kinase (IKK), and phosphorylated Ikappa beta were analyzed by means of Western blot. The IKK activity was measured in UM-SCC14A cells using an IKK-specific Ikappa beta alpha substrate after treatment with curcumin. MAIN OUTCOME MEASURES Reverse transcription-polymerase chain reaction was performed to determine the effect of curcumin on the expression of IL-6 and IL-8. RESULTS Curcumin treatment resulted in dose-dependent inhibition of IL-6 and IL-8 in all cell lines. All cell lines had similar NF-kappa beta levels; however, UM-SCC1 and UM-SCC14A had significantly higher Ikappa beta kinase levels and required considerably higher doses of curcumin before inhibition of IL-6 and IL-8 occurred. Curcumin treatment resulted in inhibition of IKK activity and inhibition of IL-6 and IL-8 expression. CONCLUSIONS Curcumin significantly reduces IL-6 and IL-8 levels in HNSCC cell lines. This mechanism appears to be mediated via inhibition of Ikappa beta-kinase activity in the NF-kappa beta pathway. Interleukins 6 and 8 have potential use as biomarkers to measure the efficacy of treatment with curcumin.

Hyperactivated JNK Is a Therapeutic Target in pVHL-Deficient Renal Cell Carcinoma

Clear cell renal cell carcinomas (RCC), the major histologic subtype of RCC accounting for more than 80% of cases, are typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Although accumulation of hypoxia-inducible factor alpha (HIF-α) is the most well-studied effect of VHL inactivation, direct inhibition of HIFα or restoration of wild-type pVHL protein expression has not proved readily feasible, given the limitations associated with pharmacologic targeting of transcription factors (i.e., HIF-α) and gene replacement therapy of tumor suppressor genes (i.e., VHL). Here, we have established that phosphorylated c-Jun, a substrate of the c-Jun-NH(2)-kinase (JNK), is selectively activated in clear cell RCC patient specimens. Using multiple isogenic cell lines, we show that HIF-α-independent JNK hyperactivation is unique to the pVHL-deficient state. Importantly, pVHL-deficient RCCs are dependent upon JNK activity for in vitro and in vivo growth. A multistep signaling pathway that links pVHL loss to JNK activation involves the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal to sequentially stimulate TAK1 (MAPKKK), MKK4 (MAPKK), and JNK (MAPK). JNK stimulates c-Jun phosphorylation, activation, and dimerization with c-Fos to form a transcriptionally competent AP1 complex that drives transcription of the Twist gene and induces epithelial-mesenchymal transition. Thus, JNK represents a novel molecular target that is selectively activated in and drives the growth of pVHL-deficient clear cell RCCs. These findings can serve as the preclinical foundation for directed efforts to characterize potent pharmacologic inhibitors of the JNK pathway for clinical translation.

Inactivation of the Cystatin E/M Tumor Suppressor Gene in Cervical Cancer

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5′aza 2‐deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.