Epoxyazadiradione exhibit activities in head and neck squamous cell carcinoma by targeting multiple pathways.

The head and neck squamous cell carcinoma (HNSCC) constitute about 90% of all head and neck cancers. HNSCC falls in the top 10 cancers in men globally. Epoxyazadiradione (EPA) and Azadiradione (AZA) are the limonoids derived from the medicinal plant Azadirachta indica (popularly known as Neem). Whether or not the limonoids exhibit activities against HNSCC and the associated mechanism remains elusive. Herein, we demonstrate that EPA exhibits stronger activity in HNSCC in comparison to AZA. The limonoids obeyed the Lipinski's rule of 5. EPA exhibited activities in a variety of HNSCC lines like suppression of the proliferation and the induction of apoptosis. The limonoid suppressed the level of proteins associated with anti-apoptosis (survivin, Bcl-2, Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9). Further, the expression of proapoptotic Bax and caspase-9 cleavage was induced by the limonoid. Exposure of EPA induced reactive oxygen species (ROS) generation in the FaDu cells. N-acetyl-L-cysteine (ROS scavenger) abrogated the down-regulation of tumorigenic proteins caused by EPA exposure. EPA induced NOX-5 while suppressing the expression of programmed death-ligand 1 (PD-L1). Further, hydrogen peroxide induced NF-κB-p65 nuclear translocation and EPA inhibited the translocation. Finally, EPA modulated the expression of lncRNAs in HNSCC lines. Overall, these results have shown that EPA exhibit activities against HNSCC by targeting multiple cancer related signalling molecules. Currently, we are evaluating the efficacy of this molecule in mice models.

Phenotypic and Functional Characteristics of Th17 (CD4+IL17A+) Cells in Human Oral Squamous Cell Carcinoma and Its Clinical Relevance.

Recent studies have suggested an important role of T helper 17 (Th17) cells in tumor biology however, their phenotypic and functional aspects are poorly understood in context with oral cancer. We therefore, investigated the various phenotypic and functional markers of Th17 cells elucidating their relevance in oral squamous cell carcinoma (OSCC). Multi-color flow cytometry (FACs) was used to analyze the frequency and different markers of circulating Th17 cells ex vivo in peripheral blood mono-nuclear cells (PBMCs) from 69 OSCC patients and 35 healthy controls. Percent Mean ± SEM of different types of cells were compared between the two groups using Mann-Whitney U test. We found significantly (p < 0.0001) increased frequency of Th17 cells in patients as compared to controls. These cells were found to express CCR6 profoundly but not CXCR4, CD62L, and CCR7 as chemokine receptors. Additionally, it expressed HLA-DR, CD69, and CD25 moderately but CD28 and CD161 highly. The cytokine profiling revealed 3 subsets namely Th17/1 (IL17A+IFNγ+), Th17/inflammatory (IL17A+IL8+), and Th17/2 (IL17A+IL4+) which were found to be elevated in patients as compared to controls. The early stage patients had a shift toward Th17/1 type and vice versa. Our results suggest that Th17 cells may have effector immune functions in oral cancer immunity through CCR6, CD161, HLA-DR, CD69, CD28 receptors and inducing Th17/1 type of cells expressing polyfunctional antitumor IFNγ cytokine. Thus, novel immune-boosting regimens based on enhancement of Th17 cells in oral cancer patients may provide therapeutic benefits in them.

Garcinol inhibits tumour cell proliferation, angiogenesis, cell cycle progression and induces apoptosis via NF-κB inhibition in oral cancer.

Garcinol, a polyisoprenylated benzophenone is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Its ability to inhibit tumour growth has been demonstrated in certain cancers. In this study, we evaluated the potential anti-tumour effects of garcinol on oral squamous cell carcinoma (OSCC) cells. Three OSCC cell lines (SCC-4, SCC-9 and SCC-25) were treated with garcinol for 48 h and its effect on growth and proliferation, clonogenic survival, cell cycle and apoptosis was studied by MTT, clonogenic assay, propidium iodide (PI) staining and annexin-V binding assay, respectively. The alteration in expression of NF-κB and COX-2 was studied by western blot analysis and that of VEGF by ELISA. Garcinol treatment significantly (p < 0.001) inhibited the growth and proliferation and colony formation of OSCC cells with a concomitant induction of apoptosis and cell cycle arrest. It did not show toxic effect on normal cells. It significantly (p < 0.05) reduced the expression of NK-κB and COX-2 expression in treated cells as compared to untreated controls besides inhibiting VEGF expression. It appears that garcinol exerts anti-proliferative, pro-apoptotic, cell-cycle regulatory and anti-angiogenic effects on oral cancer cells through inhibition of NF-κB and COX-2. Thus, garcinol may be developed as a potential chemopreventive and/or chemotherapeutic agent for treatment of oral squamous cell carcinoma.

Prognostic significance of cytoplasmic p27 in oral squamous cell carcinoma.

OBJECTIVE: p27 is a cell cycle-dependent kinase inhibitor whose presence in nucleus is associated with good prognosis. Recent studies propose that when localized to cytoplasm, it functions as an oncogene and confers a poorer prognosis. This study aimed at analysing the subcellular localization of p27 and its prognostic significance in oral squamous cell carcinomas (OSCCs). METHODS: Immunohistochemistry for p27 was carried out on 60 cases of OSCC (30 cases each of those with lymph node metastasis [LN+ve SCC] and without lymph node metastasis [LN-ve SCC]) and 30 normal mucosa. The relationship between p27 localization and prognosis was analysed statistically. RESULTS: Nuclear immunopositivity was seen in 15%, 23%, 7% and 60%, while cytoplasmic immunopositivity was seen in 80%, 63%, 97% and 43% of all SCC, LN+ve OSCC, LN-ve SCC cases and normal mucosa, respectively. There was a significant inverse correlation between nuclear and cytoplasmic p27 immunopositivity (P = 0.001). Nodal status and tumour stage were the only two parameters that correlated with disease-free survival (DFS) in OSCC cases. However, in LN+ve SCC, a significantly shortened DFS was seen in cases with cytoplasmic p27 expression compared to those without (P = 0.02). Conversely, LN+ve SCC with nuclear p27 had longer DFS on comparison with those without (P = 0.04). CONCLUSIONS: To the best of our knowledge, this is the first study to analyse cytoplasmic localization of p27 in OSCC and correlate with prognosis. Cytoplasmic localization is associated with poor prognosis in OSCC with lymph node metastasis allowing the consideration of cytoplasmic p27 in predicting prognosis and targeted therapeutic approaches.

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells.

Development of peptide inhibitor as a therapeutic agent against head and neck squamous cell carcinoma (HNSCC) targeting p38alpha MAP kinase.

BACKGROUND: The p38alpha MAP kinase pathway is involved in inflammation, cell differentiation, growth, apoptosis and production of pro-inflammatory cytokines TNF-alpha and IL-1beta. The overproduction of these cytokines plays an important role in cancer. The aim of this work was to design a peptide inhibitor on the basis of structural information of the active site of p38alpha. METHODS: A tetrapeptide, VWCS as p38alpha inhibitor was designed on the basis of structural information of the ATP binding site by molecular modeling. The inhibition study of peptide with p38alpha was performed by ELISA, binding study by Surface Plasmon Resonance and anti-proliferative assays by MTT and flow cytometry. RESULTS: The percentage inhibition of designed VWCS against pure p38alpha protein and serum of HNSCC patients was 70.30 and 71.5%, respectively. The biochemical assay demonstrated the K(D) and IC50 of the selective peptide as 7.22 x 10(-9) M and 20.08 nM, respectively. The VWCS as inhibitor significantly reduced viability of oral cancer KB cell line with an IC50 value of 10 microM and induced apoptosis by activating Caspase 3 and 7. CONCLUSIONS: VWCS efficiently interacted at the ATP binding pocket of p38alpha with high potency and can be used as a potent inhibitor in case of HNSCC. GENERAL SIGNIFICANCE: VWCS can act as an anticancer agent as it potentially inhibits the cell growth and induces apoptosis in oral cancer cell-line in a dose as well as time dependent manner. Hence, p38alpha MAP kinase inhibitor can be a potential therapeutic agent for human oral cancer.

Over Expression of Cancer Stem Cell Marker CD44 and Its Clinical Significance in Patients with Oral Squamous Cell Carcinoma.

Cancer stem cell marker CD44 is a cell-surface glycoprotein which is involved in various cellular functions such as cell-cell interactions, cell adhesion, haematopoiesis and tumour metastasis. The CD44 gene transcription is partly activated by beta-catenin and Wnt signalling pathway, the later pathway being linked to tumour development. However, the role of CD44 in oral squamous cell carcinoma (OSCC) is not well understood. We investigated the expression of CD44 in peripheral circulation, tumour tissues of oral cancer patients and oral squamous cell carcinoma cell lines by ELISA and quantitative (q)-RTPCR. Relative CD44s mRNA expression was significantly higher in peripheral circulation (p = 0.04), tumour tissues (p = 0.049) and in oral cancer cell lines (SCC4, SCC25 p = 0.02, SCC9 p = 0.03). Circulating CD44total protein levels were also significantly (p < 0.001) higher in OSCC patients that positively correlated with increasing tumour load and loco-regional spread of the tumour. The circulating tumour stem cell marker CD44 appears to be a potent indicator of tumour progression and may be useful for developing suitable therapeutics strategies for patients with oral squamous cell carcinoma.

Circulating Vimentin Over-Expression in Patients with Oral Sub Mucosal Fibrosis and Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common (90%) types of oral carcinomas in the world. It is the 2nd most common and 3rd deadliest cancer in India. The lack of early detection marker is one of the major causes of worst prognosis. The vimentin belongs to intermediate filament family proteins which plays significant role in maintaining cellular integrity. Over-expression of vimentin has been widely reported in many epithelial cancers however the information regarding its prevalence in the oral cancers still needs further scientific intervention. The expression level of circulating vimentin protein in serum samples (n = 30) of oral submucous fibrosis (OSMF), OSCC patients and healthy controls were measured by performing ELISA. The serum level of vimentin was significantly higher in OSMF (p < 0.01) and OSCC (p < 0.003) patients as compared to healthy subjects. The circulating vimentin levels showed a gradual increase with increasing disease status (normal < OSMF < OSCC). Circulatory levels of vimentin may ba useful indicator of disease progression and as a suitable target for therapeutic intervention of oral submucous fibrosis and oral carcinoma.

Serum Survivin in Oral Submucosal Fibrosis and Squamous Cell Carcinoma.

Survivin, an inhibitor of apoptosis protein is a biomarker of significance in prognostication of many malignancies. In the current study we investigated the serum survivin levels in patients with oral submucosal fibrosis (OSMF) and squamous cell carcinoma (OSCC). Serum was isolated from, peripheral blood collected of clinically and histopathologically confirmed OSMF and OSCC patients. Circulating level of survivin was measured in patients and control subjects by ELISA and analyzed further using Kruskal-Wallis test and two-sample Wilcoxon rank-sum (Mann-Whitney) test. Serum Survivin levels were significantly reduced in the OSCC group as compared to the control group. No significant correlation was noted between the serum survivin level and various clinicopathological characteristics of OSCC and OSMF patients. Our study suggests that free, wild form of circulating survivin probably has no role in predicting the prognosis of oral cancer or the malignant transformation potential of oral submucosal fibrosis.

Cinnamomum zeylanicum Extract and its Bioactive Component Cinnamaldehyde Show Anti-Tumor Effects via Inhibition of Multiple Cellular Pathways.

Cinnamomum zeylanicum is a tropical plant with traditional medicinal significance that possesses antimicrobial, antifungal, anti-parasitic, and anti-tumor properties. Here, we have elucidated the anti-tumor effects of Cinnamomum zeylanicum extract (CZE) and its bioactive compound cinnamaldehyde (CIN) on oral cancer and elucidated underlying molecular mechanisms. Anti-tumor activities of CZE and CIN were demonstrated by various in vitro experiments on oral cancer cells (SCC-4, SCC-9, SCC-25). The cell proliferation, growth, cell cycle arrest, apoptosis, and autophagy were analyzed by MTT, clonogenic assay, propidium iodide, annexin-V-PI, DAPI, and acridine orange staining, respectively. The binding affinity of CIN towards dihydrofolate reductase and p38-MAP kinase alpha was analyzed by molecular docking. Western blot assay was performed to assess the alteration in the expression of various proteins. CZE and CIN treatment significantly inhibited the growth and proliferation of oral cancer cells in a dose-dependent manner. These treatments further induced apoptosis, cell cycle arrest, and autophagy. CZE and CIN inhibited the invasion and cytoplasmic translocation of NF-κB in these cell lines. CIN showed a high affinity to MAP kinase P38 alpha and dihydrofolate reductase with binding affinities of -6.8 and -5.9 kcal/mol, respectively. The cancer cells showed a decreased expression of various PI3k-AKT-mTOR pathways related to VEGF, COX-2, Bcl-2, NF-κB, and proteins post-treatment.

Correlation of cyclin D1 expression with aggressive DNA pattern in patients with tobacco-related intraoral squamous cell carcinoma.

BACKGROUND & OBJECTIVES: Cyclin D1 has been strongly implicated in cell proliferation particularly in the G1/S checkpoint of the cell cycle, and prognoses in human malignancies. We investigated the correlation between cyclin D1 overexpression and clinicopathological features as well as cell cycle parameters to understand its clinical significance in patients with tobacco-related oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemistry for cyclin D1 and DNA flowcytometry for cell cycle parameters was done on paraffin embedded tumour samples from 45 patients with OSCC RESULTS: Higher expression of cyclin D1 was observed only in 30 (66.6%) of 45 cases that correlated with advanced age (P <0.02), higher tumour stage (P<0.01), histological differentiation and lymph node metastasis (P <0.01). Analysis of nuclear DNA pattern revealed cyclin D1 immunoreactivity in tumours with aggressive DNA pattern such as aneuploidy (P<0.05) and higher S phase fraction (P<0.04). INTERPRETATION & CONCLUSIONS: Higher expression of cyclin D1 in oral cancer appears to be closely linked to cell proliferation, differentiation and lymph node invasion. Pre-operative evaluation of cyclin D1 in biopsy specimen may be useful in planning the most appropriate treatment strategies in patients with tobacco-related OSCC.

Fas receptor (CD95) & Fas ligand (CD178) expression in patients with tobacco-related intraoral squamous cell carcinoma.

BACKGROUND & OBJECTIVES: Fas receptor and Fas Ligand (FasL) system has been implicated in the resistance to apoptosis, insensitivity to chemotherapy and in providing immune privileged status to most of the tumours. However, no reports are available on Fas and FasL expression in patients with tobacco-related oral carcinoma. Therefore, the present study was undertaken to observe Fas and FasL expression and their correlation with clinicopathological features as well as cell cycle parameters. METHODS: Immunohistochemistry for Fas, FasL and DNA flow cytometry for cell cycle parameters was successfully done on 41 paraffin embedded tumour and 10 normal samples. The results were evaluated for possible association of Fas and FasL with clinicopathological features and cell cycle parameters. RESULTS: Weak Fas expression was observed on the cell membrane only in 2 of 41 (5%) oral tumours while FasL immunoreactivity was seen in 26 of 41 (63.4%) tumours. In contrast, all ten normal oral tissues exhibited strong cytoplasmic and membrane Fas receptor immunoreactivity but absence of FasL staining. Older patients, greater tumour size and lymph node positivity were found to be associated with high expression of FasL. Significantly higher (P<0.01) expression of FasL was observed in oral tumours with aggressive DNA pattern like aneuploidy and high S-phase fraction. INTERPRETATION & CONCLUSIONS: Downregulation of Fas receptor and up-regulation of Fas ligand appear to be an important feature of tobacco-related intraoral carcinoma. Association of FasL expression with advanced clinical stage and aggressive DNA pattern suggests that the Fas and FasL system may be used as an important prognostic variable in patients with tobacco-related intraoral squamous cell carcinoma.

Single nucleotide polymorphisms in TNF-alpha, TNFR2 gene and TNF-alpha production in Asian Indians.

Polymorphisms in the TNF-alpha and TNF receptor type 2 (TNFR2) genes in Asian Indians are not well understood. We investigated single nucleotide polymorphisms in the TNF-alpha 5'-flanking promoter/enhancer region and in exons 6, 9 and 10 of TNFR2 gene by PCR-restriction length polymorphism (PCR-RFLP) and PCR-sequence specific primer (SSP) techniques, respectively. The results showed single bi-allelic polymorphism in TNF-alpha (-308G > A) and in TNFR2 exons 6 (676 T > G) and 9 (1176 G > A). Additionally, three bi-allelic polymorphisms (1663 G > A, 1668G > T and 1690C > T) were observed in the exon 10 of TNFR2 gene. The distribution of polymorphic alleles distinctly differed in Aryan and Dravidian Indian communities. The TNF-alpha and TNFR2 genotype and allele frequencies of Asian Indians stand closer to other Asian populations but distant from Caucasians. Furthermore, TNF-alpha -308 GA genotype was associated with significantly higher production of TNF-alpha as compared to GG genotype. These polymorphisms may be related to variation in TNF-alpha and TNFR2 expression during immune response to various stimuli in Asian Indians.

Targeted disruption of PI3K/Akt/mTOR signaling pathway, via PI3K inhibitors, promotes growth inhibitory effects in oral cancer cells.

PURPOSE: The phosphoinositide-3-kinase (PI3K) pathway is the frequently altered in human cancer. This has led to the development and study of novel PI3K inhibitors for targeted therapy and also to overcome resistance to radiotherapy. METHOD: The anti-tumour effects of PI3K inhibitors (PI-828, PI-103 and PX-866) in terms of cell proliferation, colony formation, induction of apoptosis, cell cycle arrest, invasion, autophagy, and pNF-κB/p65 translocation in SCC-4, SCC-9 and SCC-25 cells were studied by performing MTT, clonogenic, DAPI staining, propidium iodide staining, annexin-V binding, matrigel invasion, acridine orange staining and immuno-fluorescence assay. Western blot assay was performed to assess the alteration in the expression of various proteins. RESULT: PI-828 and PI-103 treatment exhibited dose-dependent inhibition of growth and proliferation of OSCC cells with a concomitant induction of apoptosis, altered cell cycle regulation and decreased invasiveness (p < 0.01). PX-866 induced apoptosis, cell cycle arrest, autophagy and a significant decrease in the invasiveness of oral cancer cells as compared to untreated cells (p < 0.01). These compounds significantly reduced expression of COX-2, cyclin-D1 and VEGF in the treated cells besides cytoplasmic accumulation of pNF-κB/p65 protein. In addition to PI3Kα, inactivation of downstream components, i.e. Akt and mTOR was seen. CONCLUSION: PI3K inhibitors such as PI-103, PI-828 and PX-866 may be developed as potential therapeutic agents for effective treatment of oral squamous cell carcinoma (OSCC) patients, associated with activated PI3K/Akt pathway.

Association of TNF-alpha and TNFR1 promoters and 3' UTR region of TNFR2 gene polymorphisms with genetic susceptibility to tobacco-related oral carcinoma in Asian Indians.

Tobacco-related oral squamous cell carcinoma is a common malignancy in Asian people. It accounts for almost 40% of cancers among Indian men and 3% in the Western world. Smokeless tobacco has been shown to induce tumor necrosis factor-alpha (TNF-alpha), which, along with its receptors, is over-expressed in people with oral carcinoma. Single nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor genes may affect their expression and may be a potential determinant of susceptibility to tobacco-related oral carcinomas. We assessed SNPs in TNF-alpha(-308, -238) and TNF receptor 1 (TNFR1; -609) promoters by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and at four sites of TNF receptor 2 gene (TNFR2; exon 9 site 1176; exon 10 sites 1663, 1668 and 1690) by PCR-sequence-specific primers (PCR-SSP) techniques, respectively, in 94 patients and 130 healthy controls. TNF-alpha-308 G allele was significantly lower (Pc=0.004; OR=3.85), whereas A allele was significantly higher (Pc=0.004; OR=0.25) in patients compared with controls. No significant change was observed at -238 promoter site between the two groups. In the case of TNF receptors, both TNFR1 -609 TT (Pc=0.006; OR=15.3) and TNFR2 1690 CT (Pc=0.018; OR=5.6) genotypes were significantly lower in patients compared with controls. It seems that TNF-alpha-308 G/A may be related to susceptibility, whereas -609 TT TNFR1 and 1690 C/T TNFR2 SNPs may be protective to tobacco-related oral squamous cell carcinoma. These SNPs may be useful as a marker for high-risk groups among Asian Indians.

Tinospora Cordifolia Induces Cell Cycle Arrest in Human Oral Squamous Cell Carcinoma Cells.

Natural products with medicinal value are gradually gaining importance in clinical research due to their well-known property of no side effects as compared to drugs. Tinospora cordifolia (Guduchi) has been used for centuries in Ayurvedic system of medicine for treating various ailments including cancer. In present study, we found that the Tinospora cordifolia extracts (TCE) induced inhibition of proliferation of KB cells was associated with arrest of G0/G1-phase of cell cycle. The effectiveness of TCE in checking the growth of KB cells without altering the growth of normal peripheral blood mononuclear cells (PBMC) indicates that Tinospora cordifolia has differential effect on normal and malignant cells hence, it may have therapeutic potential in cancer.

Operculina turpethum extract inhibits growth and proliferation by inhibiting NF-κB, COX-2 and cyclin D1 and induces apoptosis by up regulating P53 in oral cancer cells.

OBJECTIVES: Herbal drugs are popularly emerging as complementary and alternative medicines in cancer patients because of their cost effectiveness and minimal side-effects. The extract of Operculina turpethum (OT) is known to have antipyretic, anti-inflammatory and purgative properties. Since it is popularly known have antiinflammatory activity, we investigated its anti-tumor activity on four oral squamous cell carcinoma cell lines (OSCC) namely, (SCC-4, KB, SCC-9 and SCC-25). DESIGN: Antitumor activities of Operculina turpathum extract (OTE) was investigated by MTT and clonogenic assay, effect on cell cycle and apoptosis induction by Annexin-V/propidium iodide (PI) staining and flow cytometry and invasive potential of the tumor was determined by matrigel assay. The expression of various proteins involved in these mechanisms was analysed by western blotting. RESULTS: OTE specifically inhibited the growth and colony formation of OSCC cells in a dose-dependent manner via inhibiting NF-κB and its downstream target COX-2. It further arrested cell cycle at G0/G1 phase by inhibiting cyclin-D1 and induced early apoptosis by up-regulating P53 in OSCC cells. It also limits the invasion capacity of OSCC cells by up to 55-60%. CONCLUSIONS: OTE shows antitumor activities in OSCC cells by inhibiting NF-κB, COX-2 and cyclin D1 and upregulation of p53 expression. It may be developed as a safe and promising alternative chemopreventive/chemotherapeutic agent for oral cancer.

Age related changes in Fas (CD95) and Fas ligand gene expression and cytokine profiles in healthy Indians.

Ageing in human and animal models show changes in many aspects of protective immunity, particularly lymphopoenia and progressive decline in immune functions leading to increased frequency of infection and neoplasia. However, the exact mechanism of these defects is still unclear. In this study, elderly subjects showed a decline in CD3+ and CD4+ T-cell subsets as well as serum IL-2 levels. Serum IL-6 was significantly raised while expression of its signaling receptor gp130 was significantly impaired in elderly as compared to the younger ones. Additionally, all the elderly individuals showed constitutive expression of Fas and FasL mRNA; however, none of the younger individuals expressed mRNA transcripts constitutively although induced expression was seen in both the groups. Similarly, frequency of Fas and FasL expressing CD4+ and CD8+ T-cell subsets were significantly (p < 0.001) higher in elderly subjects as compared to the younger ones. Elderly individuals also showed a significantly (p < 0.001) higher frequency of activation induced cell death (AICD). Since interaction of Fas with its cognate ligand (FasL) activates death inducing caspases leading to apoptosis, and gp130 induces anti-apoptotic signal through STAT-3 pathway, these results suggest that the decline in protective immune functions in aged individuals may be related to Fas and FasL mediated apoptosis of peripheral T-cell subsets.

Expression of Cell Cycle-associated Proteins p53, pRb, p16, p27, and Correlation With Survival: A Comparative Study on Oral Squamous Cell Carcinoma and Verrucous Carcinoma.

Verrucous carcinoma (VC) is a well-differentiated form of squamous cell carcinoma (SCC) with better prognosis. Differences in molecular pathogenesis between the 2 have not been well-characterized. We conducted this study to evaluate immunohistochemical expression of cell-cycle regulatory proteins p53, pRb, p16, and p27 in SCC and VC, compare the expression in these 2 neoplasms, and assess if these markers have any diagnostic or prognostic value. Sixty cases of SCC with and without lymph node metastasis and 31 cases of VC were studied. Immunohistochemical analysis for p53, pRb, p16, and p27 was performed and the results were analyzed. SCC was most frequent in tongue (52%), whereas VC in buccal mucosa (81%). Mean age of SCC patients was significantly lower than in VC. Majority of SCCs were in stage III and IV (63%), whereas VCs were in stage I and II (84%). p53 immunopositivity was more frequent in SCC (65%) than in VC (23%) (P≤0.001). VC had lower p53 as compared with well-differentiated SCC and SCC without lymph node metastasis. No significant difference was seen in pRb, p16, and p27 expression. Disease-free survival (DFS) at 1 year for SCC was 57% whereas it was 80% for VC (P=0.02). DFS and overall survival of SCC correlated with nodal status and stage; cell-cycle-associated protein expression had no association with DFS. To conclude, p53 immunoexpression differs in SCC and VC, suggesting different pathogenesis, and it may have some utility as an adjunct to morphology to differentiate between the 2. Expression of cell-cycle-associated proteins does not influence survival in SCC.

Effect of rhythmic breathing (Sudarshan Kriya and Pranayam) on immune functions and tobacco addiction.

Stress, a psychophysiological process, acts through the immune-neuroendocrine axis and affects cellular processes of body and immune functions, leading to disease states including cancer. Stress is also linked to the habit of tobacco consumption and substance abuse, which in turn also leads to diseases. Sudarshan Kriya (SK) and Pranayam (P), rhythmic breathing processes, are known to reduce stress and improve immune functions. Cancer patients who had completed their standard therapy were studied. SK and P increased natural killer (NK) cells significantly (P <0.001) at 12 and 24 weeks of the practice compared to baseline. Increase in NK cells at 24 weeks was significant (P <0.05) compared to controls. There was no effect on T-cell subsets after SK and P either in the study group or among controls. SK and P helped to control the tobacco habit in 21% of individuals who were followed up to 6 months of practice. We conclude that the inexpensive and easy to learn and practice breathing processes (SK and P) in this study demonstrated an increase in NK cells and a reduction in tobacco consumption. When confirmed in large and randomized studies, this result could mean that the regular practice of SK and P might reduce the incidence and progression of cancer.