ΔNp63 overexpression promotes oral cancer cell migration through hyperactivated Activin A signaling.

Oral cancer is a common malignant tumor of the oral cavity that affects many countries with a prevalent distribution in the Indian subcontinent, with poor prognosis rate on account of locoregional metastases. Gain-of-function mutations in p53 and overexpression of its related transcription factor, p63 are both widely reported events in oral cancers. However, targeting these alterations remains a far-achieved aim due to lack of knowledge on their downstream signaling pathways. In the present study, we characterize the isoforms of p63 and using knockdown strategy, decipher the functions and oncogenic signaling of p63 in oral cancers. Using Microarray and Chromatin Immunoprecipitation experiments, we decipher a novel transcriptional regulatory axis between p63 and Activin A and establish its functional significance in migration of oral cancer cells. Using an orally bioavailable inhibitor of the Activin A pathway to attenuate oral cancer cell migration and invasion, we further demonstrate the targetability of this signaling axis. Our study highlights the oncogenic role of ΔNp63 - Activin A - SMAD2/3 signaling and provides a basis for targeting this oncogenic pathway in oral cancers.

Curcumin mediates selective aggregation of mutant p53 in cancer cells: A promising therapeutic strategy.

The increased stability of mutant p53 (Mutp53) plays a crucial role in its gain of function, making proteins involved in its stabilization promising targets for drug intervention. Although curcumin is known to exhibit anti-cancer effects, its role as a deubiquitinase (DUB) inhibitor in Mutp53 destabilization remains poorly explored. Our study demonstrates that curcumin treatment induced ubiquitination and destabilization of Mutp53 but not Wild-type p53 (WTp53) in cancer cells. Furthermore, proteasome and lysosome inhibitors failed to reverse the effect of curcumin, indicating Mutp53 destabilization is possibly via an alternate mechanism. Intriguingly, curcumin treatment also resulted in the nuclear aggregation of the Mutp53 protein, which was rescued by combined Dithiothreitol (DTT) treatment. Similar to curcumin, a broad-spectrum deubiquitinase inhibitor induced Mutp53 aggregation implying curcumin possibly acts by inhibiting deubiquitinases. Additionally, curcumin treatment inhibited colony-forming abilities, induced cytoplasmic vacuolation, and cell death selectively in Mutp53-expressing cells. Collectively, our study highlights the potential of curcumin as a promising therapeutic agent for targeting Mutp53-expressing cancer cells.

Elevated translationally controlled tumour protein promotes oral cancer progression and poor outcome.

BACKGROUND: Translationally controlled tumour protein (TCTP) is a multifunctional protein elevated in multiple cancers. However, studies on its role in oral carcinogenesis and prognosis are rare. We recently reported the role of its interacting partner, MCL1, in oral cancer progression and outcome. Hence, the present study aimed to assess TCTP expression in oral tumorigenesis and its association with patient outcomes alone and in combination with MCL1. METHODS: TCTP expression was assessed by immunohistochemistry and immunoblotting in oral tissues and cells, respectively. Cell viability post siRNA/dihydroartemisinin treatment was analysed by tetrazolium salt assay. Cell survival, invasion and tumorigenic potential post TCTP knockdown were assessed by clonogenic, Matrigel and soft-agar assays, respectively. The association of TCTP with patient outcome was analysed by Kaplan-Meier and Cox regression. RESULTS: TCTP was significantly overexpressed in oral premalignant lesions (p < 0.0001), oral tumours (p < 0.0001) and oral dysplastic and cancer cells versus normal oral mucosa and also in recurrent (p < 0.05) versus non-recurrent oral tumours. Further, elevated TCTP was significantly (p < 0.05) associated with poor recurrence free survival (RFS) and poor overall survival (OS; hazard ratio = 2.29; p < 0.05). Intriguingly, the high co-expression of TCTP and MCL1 further reduced the RFS (p < 0.05) and OS (p < 0.05; hazard-ratio = 3.49; p < 0.05). Additionally, TCTP knockdown decreased survival (p < 0.05), invasion (p < 0.01) and in vitro tumorigenic potential (p < 0.0001). Dihydroartemisinin treatment reduced TCTP levels and viability of oral cancer cells. CONCLUSION: Our studies demonstrate an oncogenic role of TCTP in oral cancer progression and poor outcome. Thus, TCTP may be a potential prognostic marker and therapeutic target in oral cancers.

Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

Elevated USP9X drives early-to-late-stage oral tumorigenesis via stabilisation of anti-apoptotic MCL-1 protein and impacts outcome in oral cancers.

BACKGROUND: Overexpression of anti-apoptotic MCL-1 protein in oral squamous cell carcinoma (OSCC) is linked to disease progression, therapy resistance and poor outcome. Despite its characteristic short half-life owing to ubiquitin-proteasome-dependent degradation, oral tumours frequently show elevated MCL-1 protein expression. Hence, we investigated the role of deubiquitinase USP9X in stabilising MCL-1 protein and its contribution to oral tumorigenesis. METHODS: Expression of MCL-1 and USP9X was assessed by immunoblotting and immunohistochemistry in oral cancer cell lines and tissues. The association between MCL-1 and USP9X was confirmed by coimmunoprecipitation and immunofluorescence. Cell death assessment was performed by MTT, flow cytometry and clonogenic assays. RESULTS: Both USP9X and MCL-1 are significantly elevated in oral premalignant lesions and oral tumours versus normal mucosa. USP9X interacts with and deubiquitinates MCL-1, thereby stabilising it. Pharmacological inhibition of USP9X potently induced cell death in OSCC cells in vitro and in vivo. The elevated expression of USP9X and MCL-1 correlated with poor prognosis in OSCC patients. CONCLUSION: We demonstrate the oncogenic role of USP9X in driving early-to-late stages of oral tumorigenesis via stabilisation of MCL-1, suggesting its potential as a prognostic biomarker and therapeutic target in oral cancers.

Novel nucleolar localization of clusterin and its associated functions in human oral cancers: An in vitro and in silico analysis.

Clusterin (CLU), a multifunctional chaperonic glycoprotein associated with diverse cellular functions has been shown to act as an oncogene or tumour suppressor gene in different cancers, implying a dual role in tumorigenesis. Here, we investigated the expression of CLU isoforms, their subcellular localization and functional significance in oral cancer cells. Significant downregulation of secretory CLU (sCLU) transcripts was observed in oral cancer cell lines and tumours versus normal cells while the nuclear CLU (nCLU) transcripts were undetectable. We demonstrated for the first time the nucleolar localization of sCLU, its response to different nucleolar stresses and association with cajal bodies post nucleolar stress. Functionally, knockdown of CLU revealed its negative association with ribosome biogenesis implying a possible tumour suppressor like role in oral cancers. Further, loss of sCLU in these cells also resulted in altered nuclear morphology and shrunken tubulin filaments. In addition, the levels of nucleolar Nucleophosmin 1(NPM1) and Fibrillarin, known to regulate nuclear morphology were downregulated indicating a possible role of sCLU in their stabilization. Further, an in silico docking approach to gain insights into the interaction of sCLU with nucleolar proteins NPM1, Fibrillarin, UBF and Nucleolin, revealed the involvement of a conserved region comprising of amino acid residues 140-155 of sCLU ß-chain, specifically via the Phe152 residue in hydrophobic interactions with these client nucleolar proteins indicating a possible stabilizing or regulatory role of sCLU. SIGNIFICANCE OF THE STUDY: This is the first study to demonstrate the nucleolar localization of sCLU and its associated functions in oral cancer cells. Downregulation of sCLU in oral cancer tissues and cell lines, and its negative association with ribogenesis suggest its tumour suppressor like role in oral cancers. The possible role of sCLU in stabilization or regulation of different nucleolar proteins thereby impacting their functions is also implicated.

Concomitant overexpression of Activin A and p63 is associated with poor outcome in oral cancer patients.

BACKGROUND: The present study aims to comprehensively analyze expression of Activin signaling components in oral cancer and to determine the predominant Activin expressed and its influence on prognosis. As our preliminary studies indicated regulation of Activin gene by p63, we also propose to assess its correlation with p63/p53 in oral tumors and its impact on outcome. METHODS: Expression of Activin subunits, receptors, and regulators was assessed by qRT-PCR and Western blotting. Correlation between Activin A and p63/p53 expression was evaluated in oral tumors by immunohistochemistry and their association with clinical outcome was determined by Kaplan-Meier curves and Cox regression. RESULTS: Activin ßA transcripts were upregulated (P = .013) in oral dysplastic and cancer cells compared with normal oral mucosa. Expression of Activin receptors and regulators was also altered. Activin ßA protein was significantly upregulated in oral tumors and adjacent normal tissues compared with normal oral mucosa (P < .0001). Expression of Activin ßA and p63 significantly correlated in oral tumors, correlation being stronger in tumors with high p53 (r = -.394, P = .005). Activin ßA overexpression was associated with advanced tumor stage (P = .021), positive nodes (P = .045), poor recurrence-free survival (P = .013), and overall survival (P = .024), while its concomitant overexpression with p63 was a better predictor of recurrence-free survival (HR = 10.66, CI: 1.41-80.19). CONCLUSIONS: Activin A overexpression is an early event in oral cancer pathogenesis and can independently predict survival. Moreover, in combination with p63 overexpression, it served as a better marker for poor prognosis. Activin A could thus be a promising target for improved outcome in oral cancer patients.

Impact of HPV 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy - A prospective observational study.

OBJECTIVE: With an aim to investigate the impact of Human Papilloma Virus (HPV) 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy, we undertook this prospective study. METHODS: Between May 2010 and April 2012, 150 histologically proven cervical cancer patients treated with radio (chemo) therapy were accrued. Cervical biopsies/brushings were collected at pre-treatment, end of treatment and at 3 monthly intervals up to 24months. Quantitative estimation of HPV 16/18 was done using real-time polymerase chain reaction (RT-PCR) and correlated with various clinical end-points. RESULTS: Out of 150 patients accrued, 135 patients were considered for final analysis. Pre-treatment HPV16/18 DNA was detected in 126 (93%) patients, with HPV-16 present in 91%. The mean log (±SD) HPV-16 and HPV-18 viral load at pre-treatment was 4.76 (±2.5) and 0.14 (±2.1) copies/10ng of DNA, respectively. Though significant decline in viral load was observed on follow-ups (p<0.0001); by 9-month follow-up, 89 (66%) patients had persistence of HPV infection. Patients with persistent HPV 16/18 infection had a significantly higher overall and loco-regional relapses [44/89 (49%) and 29/89 (32%)] as compared to HPV clearance by 9months [12/43 (28%) and 5/43 (11%)] with p=0.024 and p=0.02, respectively. Also, persistent HPV infection by 24-month showed a significant impact on loco-regional control (LRC) and recurrence-free survival (RFS). CONCLUSION: In locally advanced cervical cancers treated with radical radio (chemo) therapy, persistent HPV 16/18 infection is significantly high in immediate post-treatment period and correlated with higher loco-regional, overall relapses and was also associated with early relapses.

Comparative evaluation of the clonogenic capacity of periodontal ligament fibroblasts in Hank's balanced salt solution and egg albumen: An in vitro study.

BACKGROUND/AIM: The type of storage media for short-term storage of an avulsed tooth is a critical determinant for the success of tooth replantation. If immediate replantation of an avulsed tooth is not possible, it is advised to store the tooth in a suitable storage medium. The viability and clonogenicity of periodontal ligament fibroblasts (PDLF) determines the success of replantation of an avulsed tooth. The aim of this study was to evaluate the effect on the clonogenic capacity of PDLF's upon storage in Hank's balanced salt solution (HBSS) and egg albumen. METHODS: Fibroblast cell culture was established from a human premolar tooth extracted for orthodontic purposes. The PDLF cells thus obtained were treated with either Dulbecco's modified Eagle's medium (DMEM; as a positive control), HBSS, or egg albumen for different durations at room temperature and then allowed to grow in DMEM medium until visible colonies appeared which were then fixed, stained, and scored manually. RESULTS: With increase in the duration of storage in both egg albumen as well as HBSS, there was a reduction in the clonogenic capacity of the PDLF's as compared to DMEM. However, storage in egg albumen led to a significant reduction in the clonogenic capacity of PDLF's (8%-16% for egg albumen) compared to HBSS (80%-90%). CONCLUSION: Due to its limited ability to support the clonogenicity of PDLF's, egg albumen is a poor storage medium for an avulsed tooth compared to either DMEM or HBSS.

A simple cost-effective modification improves the quality of immunocytochemical staining in cervical scrape samples characterized by presence of excess mucus.

Immunocytochemistry (ICC) is a very important tool in a diverse range of biomedical research as well as in diagnostic cytopathology. Smears prepared from cervical scrapes contain a large amount of overlying mucus that interferes with the standard immunocytochemical staining protocol. A modified ICC protocol is described, which involves pretreatment of these smears with 1 mg/ml solution of Ambroxol hydrochloride in methanol for 1 hour. Source of Ambroxol hydrochloride was a 30 mg Mucolite™ tablet, at a cost of 1.70 rupees (∼3·5 US cents) per tablet. This mucolytic solution effectively clears the mucus, facilitating the accessibility of the antibody to the antigenic determinants. This pretreatment resulted in the increased percentage of positively stained cells as well as staining intensity, leading to improved overall ICC staining and score. This is a novel modification that can be cost-effectively applied in ICC staining protocols for cytology samples characterized by the presence of excess mucus.

Myeloid cell leukemia-1: a formidable barrier to anticancer therapeutics and the quest of targeting it.

The antiapoptotic B cell lymphoma-2 (Bcl-2) family members are apical regulators of the intrinsic pathway of apoptosis that orchestrate mitochondrial outer membrane permeabilization (MOMP) through interactions with their proapoptotic counterparts. Overexpression of antiapoptotic Bcl-2 family proteins has been linked to therapy resistance and poor prognosis in diverse cancers. Among the antiapoptotic Bcl-2 family members, predominant overexpression of the prosurvival myeloid cell leukemia-1 (Mcl-1) has been reported in a myriad of hematological malignancies and solid tumors, contributing to therapy resistance and poor outcomes, thus making it a potential druggable target. The unique structure of Mcl-1 and its complex regulatory mechanism makes it an adaptive prosurvival switch that ensures tumor cell survival despite therapeutic intervention. This review focusses on diverse mechanisms adopted by tumor cells to maintain sustained elevated levels of Mcl-1 and how high Mcl-1 levels contribute to resistance in conventional as well as targeted therapies. Moreover, recent developments in the Mcl-1-targeted therapeutics and the underlying challenges and considerations in designing novel Mcl-1 inhibitors are also discussed.

Human Papillomavirus-Related Multiphenotypic Sinonasal Carcinoma with Unique HPV type 52 Association: A Case Report with Review of Literature.

Human papillomavirus (HPV)-related multiphenotypic sinonasal carcinoma (HMSC) is a recently described distinctive clinicopathologic entity defined by association to high risk HPV, localization to sinonasal tract and close histologic resemblance to salivary gland tumors. Lack of awareness of its pathologic features and biology among pathologists and oncologists make this entity susceptible to misdiagnosis and erroneous management. Herein, we illustrate a case of HMSC of the nasal cavity associated with heretofore unreported subtype HPV-52 and discuss the challenges associated with diagnosis and management of this rare tumor. A 48-year-old woman with intermittent epistaxis for 6 months presented with a nasal mass and underwent middle turbinectomy. Histology showed a tumor with features typical of adenoid cystic carcinoma (ACC) in the form of basaloid cells and cribriform architecture. However, careful inspection revealed findings uncommon in ACC; such as surface pagetoid tumor spread, areas of solid sheets of myoepithelial cells accompanied by increased mitotic figures which prompted immunohistochemistry. Multidirectional differentiation into ductal (CK7, AE1/AE3) and myoepithelial (p63, p40, S100, calponin) lineage together with strong and diffuse immunopositivity for p16 distinguished this tumor from ACC. HPV genotyping was positive for high risk HPV subtype HPV52, which confirmed the diagnosis of HMSC. HPV-related multiphenotypic sinonasal carcinoma is an under-recognized unique clinicopathologic entity that needs awareness to avoid mistaking it for commoner salivary gland tumors. Making accurate diagnosis of this newly-described tumor is imperative in order to understand its biology and to develop optimal therapeutic strategies.

BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells.

We have previously reported overexpression of antiapoptotic MCL-1 protein in human oral cancers and its association with therapy resistance and poor prognosis, implying it to be a potential therapeutic target. Hence, we investigated the efficacy and mechanism of action of Obatoclax, a BH3 mimetic pan BCL-2 inhibitor in human oral cancer cell lines. All cell lines exhibited high sensitivity to Obatoclax with complete clonogenic inhibition at 200-400 nM concentration which correlated with their MCL-1 expression. Mechanistic insights revealed that Obatoclax induced a caspase-independent cell death primarily by induction of a defective autophagy. Suppression of autophagy by ATG5 downregulation significantly blocked Obatoclax-induced cell death. Further, Obatoclax induced interaction of p62 with key components of the necrosome RIP1K and RIP3K. Necrostatin-1 mediated inhibition of RIP1K significantly protected the cells from Obatoclax induced cell death. Moreover, Obatoclax caused extensive mitochondrial stress leading to their dysfunction. Interestingly, MCL-1 downregulation alone caused mitochondrial stress, highlighting its importance for mitochondrial homeostasis. We also demonstrated in vivo efficacy of Obatoclax against oral cancer xenografts and its synergism with ionizing radiation in vitro. Our studies thus suggest that Obatoclax induces autophagy-dependent necroptosis in oral cancer cells and holds a great promise in the improved management of oral cancer patients.

Human papillomavirus in head and neck cancer in India: Current status and consensus recommendations.

Human papillomavirus (HPV) associated head and neck squamous cell cancers (HNSCC) have become increasingly common in the West, but the same cannot be said about India. These cancers have a different biology and confer a better prognosis, however, its current role in the management of patients in India is not clearly defined. At the 35th Indian Cooperative Oncology Network conference held in September 2016, a panel of radiation, surgical and medical oncologists, pathologists, and basic scientists from across the country having experience in clinical research with respect to HPV in HNSCC reviewed the available literature from India. All the ideas and facts were thereafter collated in this report. Various topics of controversy in dealing with the diagnosis and management of HPV-associated HNSCC have been highlighted in this report in context to the Indian scenario. Furthermore, the prevalence of the same and its association with tobacco and high-risk sexual behavior has been touched on. Conclusively, a set of recommendations has been proposed by the panel to guide the practicing oncologists of the country while dealing with HPV-associated HNSCC.

High expression of survivin and its splice variants survivin ΔEx3 and survivin 2 B in oral cancers.

OBJECTIVES: We have previously reported inactivation of p53 in 46% of Indian patients with oral cancer. Survivin, a p53 target gene and an inhibitor of apoptosis protein (IAP), is overexpressed in several cancers, including oral cancers. Studies assessing the role of survivin and its splice variants in oral cancers are, however, rare. MATERIALS AND METHODS: The expression of 6 survivin isoforms in 4 oral cancer cell lines (AW8507, AW13516, UPCI-SCC040, UPCI-SCC029 B), a dysplastic oral cell line (DOK), 75 paired oral tumor and adjacent normal tissues, and 12 normal oral tissue samples from healthy individuals was analyzed by real-time PCR. The expression was correlated with clinicopathologic parameters, which included age, sex, tumor-node-metastasis (TNM) staging, tobacco and/or alcohol consumption, site, and differentiation status of tumor. RESULTS: This is the first study to find overexpression of the 6 characterized survivin isoforms in oral cancers compared with normal tissues (P < .05). Additionally, a significant (P < .05) correlation among the fold changes of all 6 survivin isoforms was observed. Survivin wild type (wt) was the predominantly expressed isoform in oral cell lines and tumor tissues versus normal tissues (P < .05). Among the minor isoforms, survivin ΔEx3 and survivin 2 B were dominantly expressed, whereas survivin 2 α and survivin 3 α overexpression was found for the first time. Further high survivin 3 B expression exhibited a significant association (P < .05) with poorly differentiated tumors. Interestingly the combined expression of the antiapoptotic survivin isoforms, survivin wt, survivin ΔEx3, and survivin 3 B, exhibited a significant association with TNM staging of the tumor. CONCLUSIONS: Our studies thus indicate that oral cancers overexpress the antiapoptotic survivin variants, which exhibit an association with advanced tumor stage, implying a role for these variants in oral tumorigenesis.

Expression of bcl-2 and bax in chewing tobacco-induced oral cancers and oral lesions from India.

Deregulation of oncogenes and tumor suppressor genes involved in apoptosis has been associated with tumor development and progression. To investigate the involvement of apoptosis regulating proteins in oral cancer in Indian patients, primarily associated with chewing tobacco habits, immunohistochemical expression of bcl-2 and bax was examined in 63 oral squamous cell carcinomas, and 31 putative premalignant lesions. Our studies revealed overexpression of tumor specific cytoplasmic bcl-2 in 56% and bax in 43% oral cancers. The oral cancers in the Indian patients are preceded by premalignant oral lesions; hence oral lesions were examined for bcl-2 and bax expression. We observed aberrant expression of bcl-2 in 16% oral lesions comprising leukoplakias and SMF and bax in 55% oral lesions. We have already reported, p53 expression in these oral cancers and lesions. It was noteworthy that 30% oral cancers demonstrated a p53+bcl2+ pattern, and 14% samples exhibited p53+bcl2+bax+ pattern. However, none of the oral lesions showed concurrent deregulation of p53 and bcl-2 or all the three genes. Interestingly 45% oral lesions were p53-bax+ as compared to 18% oral cancers; while 39% oral lesions were bcl2-bax+ as compared to 14% oral cancers, indicating overexpression of bax in oral lesions, in the absence of p53 and bcl-2 proteins. Significant correlation was observed between positive nodal status and bcl2+ (p=0.047) and p53+bcl-2+ (p=0.01) in oral cancers. Kaplan Meier survival analysis showed significantly (p=0.059) higher survival in patients with p53- oral tumors than with p53+ tumors. Our studies thus indicate frequent overexpression of apoptosis regulators bcl-2, bax and p53 proteins in oral cancers, and a subset of oral lesions, representing early events in oral car-cinogenesis. The aberrant bcl-2 expression and loss of p53 function observed, may play an important role in the tumorigenesis of oral cancers by allowing escape from apoptosis and enabling additional genetic alterations to accrue.

Overexpression of Mcl-1L splice variant is associated with poor prognosis and chemoresistance in oral cancers.

BACKGROUND: Altered expression of Mcl-1, an anti-apoptotic member of the Bcl-2 family, has been linked to the progression and outcome of a variety of malignancies. We have previously reported the overexpression of Mcl-1 protein in human oral cancers. The present study aimed to evaluate the clinicopathological significance of the expression of three known Mcl-1 isoforms in oral tumors and the effect of targeting Mcl-1L isoform on chemosensitivity of oral cancer cells. METHODS: The expression of Mcl-1 isoforms- Mcl-1L, Mcl-1S & Mcl-1ES was analyzed in 130 paired oral tumors and 9 oral cell lines using quantitative real-time PCR & protein by western blotting. The Mcl-1 mRNA levels were correlated with clinicopathological parameters and outcome of oral cancer patients. The effect of Mcl-1L shRNA or Obatoclax (a small molecule Mcl-1 inhibitor), in combination with Cisplatin on chemosensitivity of oral cancer cells was also assessed. RESULTS: Anti-apoptotic Mcl-1L was predominantly expressed, over low or undetectable pro-apoptotic Mcl-1S and Mcl-1ES isoforms. The Mcl-1L transcripts were significantly overexpressed in all cancer cell lines and in 64% oral tumors versus adjacent normals (P<0.02). In oral cancer patients, high Mcl-1L expression was significantly associated with node positivity (P = 0.021), advanced tumor size (P = 0.013) and poor overall survival (P = 0.002). Multivariate analysis indicated Mcl-1L to be an independent prognostic factor for oral cancers (P = 0.037). Mcl-1L shRNA knockdown or its inhibition by Obatoclax in combination with Cisplatin synergistically reduced viability and growth of oral cancer cells than either treatment alone. CONCLUSION: Our studies suggest that overexpression of Mcl-1L is associated with poor prognosis and chemoresistance in oral cancers. Mcl-1L is an independent prognostic factor and a potential therapeutic target in oral cancers.

Raman spectroscopic study of radioresistant oral cancer sublines established by fractionated ionizing radiation.

Radiotherapy is an important treatment modality for oral cancer. However, development of radioresistance is a major hurdle in the efficacy of radiotherapy in oral cancer patients. Identifying predictors of radioresistance is a challenging task and has met with little success. The aim of the present study was to explore the differential spectral profiles of the established radioresistant sublines and parental oral cancer cell lines by Raman spectroscopy. We have established radioresistant sublines namely, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B from its parental UPCI:SCC029B cell line, by using clinically admissible 2Gy fractionated ionizing radiation (FIR). The developed radioresistant character was validated by clonogenic cell survival assay and known radioresistance-related protein markers like Mcl-1, Bcl-2, Cox-2 and Survivin. Altered cellular morphology with significant increase (p<0.001) in the number of filopodia in radioresistant cells with respect to parental cells was observed. The Raman spectra of parental UPCI:SCC029B, 50Gy-UPCI:SCC029B and 70Gy-UPCI:SCC029B cells were acquired and spectral features indicate possible differences in biomolecules like proteins, lipids and nucleic acids. Principal component analysis (PCA) provided three clusters corresponding to radioresistant 50Gy, 70Gy-UPCI:SCC029B sublines and parental UPCI:SCC029B cell line with minor overlap, which suggest altered molecular profile acquired by the radioresistant cells due to multiple doses of irradiation. The findings of this study support the potential of Raman spectroscopy in prediction of radioresistance and possibly contribute to better prognosis of oral cancer.

Bcl-xL protein: predictor of complete tumor response in patients with oral cancer treated with curative radiotherapy.

BACKGROUND: We earlier observed altered expression of p53 and Bcl-xL in oral cancer cell lines/tissues and wanted to evaluate these proteins for prediction of radiotherapy response and outcome. METHODS: Thirty-nine paraffin-embedded, pretreatment oral cancer biopsies were analyzed for protein expression using immunohistochemistry and correlated with tumor response to radiotherapy and disease-free survival (DFS). RESULT: High p53 (p = .040) was observed in female versus male patients. Increased p53 intensity (p = .063) was observed in tobacco habitués (chewers ± smokers) versus patients with no habits. In univariate analysis, nodal positivity (p = .044) and favorable/complete tumor response (p = .002) exhibited a significant correlation with DFS, whereas tumor response emerged as an independent predictor of DFS in multivariate analysis. Significantly high Bcl-xL (p = .048) was observed in the unfavorable versus favorable responders. CONCLUSION: Our study suggests that Bcl-xL expression along with clinical parameters may be useful for identifying patients with oral cancer likely to draw maximum benefit from curative radiotherapy.

Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells.

BACKGROUND: Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy. METHODS: The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR). RESULTS: Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells, suggesting a synergistic effect of the Mcl-1L siRNA with IR on radiosensitivity. Interestingly, during the development of radioresistant sublines using FIR, high expression of Mcl-1L was observed. CONCLUSION: Our studies suggest that Mcl-1L isoform has an important role in the survival and radioresistance of OSCC and may be a promising therapeutic target in oral cancers.