pregna-4-17-diene-3-16-dione and Carcinoma--Squamous-Cell

Understanding the molecular pathways perturbed in smokeless tobacco- (ST) associated head and neck squamous cell carcinoma (HNSCC) is critical for identifying novel complementary agents for effective disease management. Activation of nuclear factor-kappaB (NF-κB) and cyclooxygenase-2 (COX-2) was reported in ST-associated HNSCC by us [Sawhney,M. et al. (2007) Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco. Int. J. Cancer, 120, 2545-2556]. In search of novel agents for treatment of HNSCC, we investigated the potential of guggulsterone (GS), (4,17(20)-pregnadiene-3,16-dione), a biosafe nutraceutical, in inhibiting ST- and nicotine-induced activation of NF-κB and signal transducer and activator of transcription (STAT) 3 pathways in HNSCC cells. GS inhibited the activation of NF-κB and STAT3 proteins in head and neck cancer cells. This inhibition of NF-κB by GS resulted from decreased phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha the inhibitory subunit of NF-κB. Importantly, treatment of HNSCC cells with GS abrogated both ST- and nicotine-induced nuclear activation of NF-κB and pSTAT3 proteins and their downstream targets COX-2 and vascular endothelial growth factor. Furthermore, GS treatment decreased the levels of ST- and nicotine-induced secreted interleukin-6 in culture media of HNSCC cells. In conclusion, our findings demonstrated that GS treatment abrogates the effects of ST and nicotine on activation of NF-κB and STAT3 pathways in HNSCC cells that contribute to inflammatory and angiogenic responses as well as its progression and metastasis. These findings provide a biologic rationale for further clinical investigation of GS as an effective complementary agent for inhibiting ST-induced head and neck cancer.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Nucleus; Chromatin Immunoprecipitation; Commiphora; Cyclooxygenase 2; Ganglionic Stimulants; Head and Neck Neoplasms; Humans; NF-kappa B; Nicotine; Pregnenediones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Tobacco, Smokeless; Tumor Cells, Cultured

Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad.. Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad.. In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

Topics: bcl-2-Associated X Protein; bcl-Associated Death Protein; Carcinoma, Squamous Cell; Chromones; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Head and Neck Neoplasms; Humans; Models, Biological; Morpholines; Nicotine; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pregnenediones; Signal Transduction; Tobacco, Smokeless; Tumor Cells, Cultured; Up-Regulation

The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells.. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies.. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP).. GS targets 14-3-3 zeta associated cellular pathways for reducing proliferation and inducing apoptosis in head and neck cancer cells, warranting its investigation for use in treatment of head and neck cancer.

Topics: 14-3-3 Proteins; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Head and Neck Neoplasms; Humans; Immunoprecipitation; Mitochondria; Phosphorylation; Pregnenediones; Receptors, Cytoplasmic and Nuclear; Time Factors

Treatment of human head and neck squamous cell carcinoma (HNSCC) cell lines with guggulsterone, a widely available, well-tolerated nutraceutical, demonstrated dose-dependent decreases in cell viability with EC(50)s ranging from 5 to 8 microM. Guggulsterone induced apoptosis and cell cycle arrest, inhibited invasion and enhanced the efficacy of erlotinib, cetuximab and cisplatin in HNSCC cell lines. Guggulsterone induced decreased expression of both phosphotyrosine and total signal transducer and activator of transcription (STAT)-3, which contributed to guggulsterone's growth inhibitory effect. Hypoxia-inducible factor (HIF)-1alpha was also decreased in response to guggulsterone treatment. In a xenograft model of HNSCC, guggulsterone treatment resulted in increased apoptosis and decreased expression of STAT3. In vivo treatment with a guggulsterone-containing natural product, Guggulipid, resulted in decreased rates of tumor growth and enhancement of cetuximab's activity. Our results suggest that guggulsterone-mediated inhibition of STAT3 and HIF-1alpha provide a biologic rationale for further clinical investigation of this compound in the treatment of HNSCC.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Cetuximab; Cisplatin; Commiphora; Drug Synergism; Erlotinib Hydrochloride; Female; Head and Neck Neoplasms; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Nude; Neoplasm Transplantation; Phytotherapy; Plant Preparations; Pregnenediones; Quinazolines; STAT3 Transcription Factor