Guggulsterone Targets Smokeless Tobacco Induced PI3K/Akt Pathway in Head and Neck Cancer Cells

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

Nuclear and Cytoplasmic Accumulation of Ep-ICD Is Frequently Detected in Human Epithelial Cancers

BACKGROUND: We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. METHODOLOGY AND RESULTS: Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. CONCLUSIONS: Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers

Overexpression of Prothymosin Alpha Predicts Poor Disease Outcome in Head and Neck Cancer

In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard's ratio, HR = 5.2, 95% CI = 2.3–11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease

Nuclear S100A7 Is Associated with Poor Prognosis in Head and Neck Cancer

Tissue proteomic analysis of head and neck squamous cell carcinoma (HNSCC) and normal oral mucosa using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and liquid chromatography-mass spectrometry, led to the identification of a panel of biomarkers including S100A7. In the multi-step process of head and neck tumorigenesis, the presence of dysplastic areas in the epithelium is proposed to be associated with a likely progression to cancer; however there are no established biomarkers to predict their potential of malignant transformation. This study aimed to determine the clinical significance of S100A7 overexpression in HNSCC.Immunohistochemical analysis of S100A7 expression in HNSCC (100 cases), oral lesions (166 cases) and 100 histologically normal tissues was carried out and correlated with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients. Overexpression of S100A7 protein was significant in oral lesions (squamous cell hyperplasia/dysplasia) and sustained in HNSCC in comparison with oral normal mucosa (p(trend)<0.001). Significant increase in nuclear S100A7 was observed in HNSCC as compared to dysplastic lesions (p = 0.005) and associated with well differentiated squamous cell carcinoma (p = 0.031). Notably, nuclear accumulation of S100A7 also emerged as an independent predictor of reduced disease free survival (p = 0.006, Hazard ratio (HR = 7.6), 95% CI = 1.3-5.1) in multivariate analysis underscoring its relevance as a poor prognosticator of HNSCC patients.Our study demonstrated nuclear accumulation of S100A7 may serve as predictor of poor prognosis in HNSCC patients. Further, increased nuclear accumulation of S100A7 in HNSCC as compared to dysplastic lesions warrants a large-scale longitudinal study of patients with dysplasia to evaluate its potential as a determinant of increased risk of transformation of oral premalignant lesions

Slug is a predictor of poor prognosis in esophageal squamous cell carcinoma patients.

BACKGROUND:Slug, a regulator of epithelial mesenchymal transition, was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. This study aimed to determine the clinical significance of Slug overexpression in ESCC and determine its correlation with clinicopathological parameters and disease prognosis for ESCC patients. METHODS:Immunohistochemical analysis of Slug expression was carried out in archived tissue sections from 91 ESCCs, 61 dysplastic and 47 histologically normal esophageal tissues. Slug immunopositivity in epithelial cells was correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. RESULTS:Increased expression of Slug was observed in esophageal dysplasia [cytoplasmic, 24/61 (39.3%) cases, p = 0.001, odd's ratio (OR) = 4.7; nuclear, 11/61 (18%) cases, p < 0.001, OR = 1.36] in comparison with normal esophageal tissues. The Slug expression was further increased in ESCCs [cytoplasmic, 64/91 (70.3%) p < 0.001, OR = 10.0; nuclear, 27/91 (29.7%) p < 0.001, OR = 1.42]. Kaplan Meier survival analysis showed significant association of nuclear Slug accumulation with reduced disease free survival of ESCC patients (median disease free survival (DFS) = 6 months, as compared to those that did not show overexpression, DFS = 18 months; p = 0.006). In multivariate Cox regression analysis nuclear Slug expression [p= 0.005, Hazard's ratio (HR) = 2.269, 95% CI = 1.289 - 3.996] emerged as the most significant independent predictor of poor prognosis for ESCC patients. CONCLUSIONS:Alterations in Slug expression occur in early stages of development of ESCC and are sustained during disease progression. Slug may serve as a diagnostic biomarker and as a predictor of poor disease prognosis to identify ESCC patients that are likely to show recurrence of the disease

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

Abstract Background Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. Methods Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. Results MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (ptrend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. Conclusions Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC

ST and nicotine - induced Akt pathway activation.

<p>SCC4 cells (2–3×10⁶) were treated with (<b>A</b>) ST (20 µg/ml) or (<b>B</b>) nicotine (10 µM), for the indicated time intervals and whole-cell extracts were prepared. Whole-cell extracts (60 µg protein) were resolved on 10% SDS-PAGE, electrotransferred to a PVDF membrane and non-specific binding was blocked with 5% non-fat milk overnight. Protein expression was determined by probing with phospho-specific antibodies for pAkt (thr-309), pAkt (ser-473). pGSKβ3, pRaf, pPDK1 and Akt using enhanced chemiluminescence method. Western blotting for α-tubulin was done to show equal protein loading.</p

ST and nicotine induces Bad phosphorylation enhances interaction with 14-3-3ζ.

<p>SCC4 were treated with (<b>A</b>) ST (20 µg/ml) or (<b>B</b>) with nicotine (10 µM) for different time intervals and immunoprecipitation assays were carried out using whole cell lysates and analyzed by western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014728#s4" target="_blank">Materials and Methods</a>. 14-3-3ζ was immunoprecipitated using specific antibody and the bound pBad protein co-precipitated with 14-3-3ζ was determined by western blot analysis. Reverse immunoprecipitation assays were carried out in which pBad was immunoprecipitated, followed by western blotting analysis of 14-3-3ζ using a specific antibody against this protein. (<b>C</b>) Figure shows the hypothetical model for inhibition of ST / nicotine induced activation of PI3K/Akt pathway by GS. Our results showed treatment with ST and /or nicotine activates Akt (phosphorylated at Ser-473 and Ser-309) resulting in phosphorylation of its downstream targets Raf, GSK3β, and pS6, whereas GS treatment inhibits activation of Akt pathway. Interestingly, pre-treatment of head and neck cancer cells with GS inhibits activation of Akt and its downstream targets (Raf, GSK3β, and pS6) on exposure to ST / nicotine. GS pre-treatment also releases Bad from inhibitory action of 14-3-3ζ, thereby activating intrinsic pathway of apoptosis. Thus, our results demonstrated GS as a potential therapeutic agent for ST-induced head and neck carcinogenesis.</p

(A) AKT is co-localized with Bax in cytoplasm.

<p>SCC4 cells (5×10³) were plated on coverslips and incubated with a mouse antibody against human Bax and a rabbit antibody against human AKT antibodies. Alexa flour®594 -conjugated anti-rabbit (red) and Fluorescein isothiocyanate-conjugated (green) anti-mouse secondary antibodies were used to visualize Akt (red) and Bax (green) localization patterns using a fluorescent microscope. Panel (i) DAPI stained nuclei in blue color; (ii) cytoplasmic expression of Bax; (iii) cytoplasmic expression of Akt protein; and (iv) merged photomicrograph (ii) and (iii) showing co-localization of Bax and Akt. <b>(B)</b> Phosphorylation of Bax at (Ser-184) and Bad (Ser-136) results in retention of Bax and Bad in cytosol. SCC4 cells were kept untreated or treated with 50 µM GS for 4 h, ST (20 µg/ml) for 6 h, 10 µM nicotine for 4 h. SCC4 cells were pre-treated with 50 µM GS for 4 h, followed by ST for 6 h or with nicotine for 4 h. Cytoplasmic (C) and Mitochondrial (M) extracts were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014728#s4" target="_blank">materials and methods</a> and separated on 10% SDS-PAGE. Proteins were then electro-transferred on PVDF membrane followed by blocking with 5% non-fat milk overnight. Blots were incubated with specific antibodies against Bax and Bad. Protein expression was determined using enhanced chemiluminescence method. Purity of the subcellular fractions obtained was determined using western blot for mitochondrial protein, succinate dehydrogenase. β-actin was used as a loading control.</p