"Aberrant sialylation plays a significant role in oral squamous cell carcinoma progression".

OBJECTIVE: Aberrant glycosylation, mainly sialylation and fucosylation, is recently considered as a major hallmark of cancer. Aberrant sialylation has long been associated with various neoplastic diseases. However, role of aberrant sialylation in oral cancer is still in its infancy. The present study aimed to examine mRNA expressions of α-2, 3, α-2, 6 sialyltransferase (ST) families and sialidase in 160 human oral cancer tissues. METHODS: mRNA expression of ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, ST6GAL1, and neuraminidase 3 (NEU3) was analyzed by RT-qPCR in 80 paired malignant and adjacent normal tissues from oral cancer patients. RESULTS: The results indicated significant (P ≤ .05) down-regulation of various STs (ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, and ST6GAL1) and sialidases (NEU3) in malignant tissues as compared to adjacent normal tissues. Higher mRNA levels of ST3GAL2 and ST3GAL3 were significantly associated with advanced stage of the disease, lymph node involvement, and perineural invasion, which denote their role in progression and metastasis of oral cancer. Present study also revealed altered sialylation patterns according to anatomical site of the disease and tobacco habit. CONCLUSION: The study demonstrated significant role of elevated mRNA levels of ST3GAL2 and ST3GAL3 in disease progression and metastasis of oral carcinoma.

Overexpression of VEGF165 is associated with poor prognosis of cervical cancer.

BACKGROUND: Cervical cancer is a major health hazard to Indian women. Human papillomavirus (HPV) infection is an established risk factor for cervical carcinogenesis. However, understanding the cervical cancer biology beyond HPV infection is very crucial to predict aggressive behavior, prognosis, treatment response and survival. In the present study, we explored the role of vascular endothelial growth factor A (VEGFA) isoforms, VEGFC and VEGFD in cervical cancer progression and its association with HPV 16 and 18 infections. MATERIAL AND METHODS: A total of 110 cervical cancer tissues and 50 normal cervical tissues were collected for the study. Reverse transcription-polymerase chain reaction was employed to analyze tissue VEGFA isoforms, VEGFC and VEGFD expression. RESULTS: VEGF165 was significantly higher, whereas VEGFC and VEGFD were significantly lower in malignant cervical carcinoma tissues as compared to normal cervix tissues. Expression levels of VEGF121 and VEGFC were significantly associated with type of tumor growth while VEGF165 was significantly associated with lymph node metastasis. VEGF165 transcript levels were significantly higher in patients with squamous cell carcinoma (SCC) and developed recurrence. Most strikingly, higher VEGF165 expression was significantly associated with worst disease-free survival (DFS) specifically in patients with SCC. CONCLUSION: Association of VEGF165 with lymph node metastasis, disease recurrence and worst DFS indicated that VEGF165 is an important prognostic factor in cervical carcinogenesis.

Role of aberrant glycosylation enzymes in oral cancer progression.

BACKGROUND: Carcinogenesis, a multistep process involves sequential changes during neoplastic transformation. The various hallmarks of cancer aid in cell survival, proliferation, and dissemination. Aberrant glycosylation, a recently defined hallmark of cancer, is influenced by glycosylation enzymes during carcinogenesis. Therefore, the present study measured α-2,3 and α-2,6 sialyltransferase (ST), sialidase, and α-L-fucosidase activity in patients with oral precancerous conditions (OPC) and oral cancer patients. SUBJECTS: The study enrolled 100 oral cancer patients, 50 patients with OPC, 100 healthy controls, and 46 posttreatment follow-ups of oral cancer patients. Blood and saliva were collected from all the participants. MATERIALS AND METHODS: Sialidase activity was measured by spectrofluorimetric method, α-2,3 and α-2,6 ST by ELISA using biotinylated lectins, and α-L-fucosidase by spectrophotometric method. RESULTS: The results depicted increased levels of sialidase, α-2,3 and α-2,6 ST, α-L-fucosidase in patients with OPC and oral cancer patients. Receiver operating characteristic curve indicated significant discriminatory efficacy in distinguishing controls and oral cancer patients for serum and salivary sialidase and α-L-fucosidase activity, and serum α-2,6 ST. Furthermore, serum and salivary α-L-fucosidase activity and serum sialidase activity significantly distinguished controls and patients with OPC. Serum and salivary sialidase, α-L-fucosidase, and serum α-2,3 ST activity were higher in patients with metastasis as compared to nonmetastatic patients. Higher values of serum α-L-fucosidase activity were significantly associated with low-overall survival. CONCLUSION: The increased levels of enzymes correlated with tumor initiation, progression, and metastasis in oral cancer patients. The alterations in glycosyltransferases/glycosidases thus support the view of glycosylation as a hallmark of cancer.

Inhibition of Aggregation of Mutant Huntingtin by Nucleic Acid Aptamers In Vitro and in a Yeast Model of Huntington's Disease.

Elongated polyglutamine stretch in mutant huntingtin (mhtt) correlates well with the pathology of Huntington's disease (HD). Inhibition of aggregation of mhtt is a promising strategy to arrest disease progression. In this work, specific, high-affinity RNA aptamers were selected against monomeric mhtt (51Q-htt). Some of them inhibited its aggregation in vitro by stabilizing the monomer. They also recognized 103Q-htt but not 20Q-htt (nonpathogenic length). Inhibition of aggregation corresponded with reduced leakage of a fluorescent probe from liposomes and diminished oxidative stress in RBCs. The presence of aptamers was able to rescue the sequestration of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by aggregated mhtt. Some of the aptamers were able to enhance the partitioning of mhtt in the soluble fraction in a yeast model of HD. They were also able to rescue endocytotic defect due to aggregation of mhtt. The beneficial effect of a combination of aptamers was enhanced with improvement in cell survival. Since HD is a monogenic autosomal dominant disorder, aptamers may be developed as a viable strategy to slow down the progress of the disease. Since they are nonimmunogenic and nontoxic, aptamers may emerge as strong candidates to reduce protein-protein interaction and hence protein aggregation in protein misfolding disorders in general.

Melatonin: Emerging Player in the Management of Oral Cancer.

Oral cancer (OC) has emerged as a major medical and social issue in many industrialized nations due to the high death rate. It is becoming increasingly common in people under the age of 45, although the underlying causes and mechanisms of this increase remain unclear. Melatonin, as a pleiotropic hormone, plays a pivotal role in a wide variety of cellular and physiological functions. Mounting evidence supports melatonin's ability to modify/influence oral carcinogenesis, help in the reduction of the incidence of OC, and increase chemo- and radiosensitivity. Despite its potential anti-carcinogenic effects, the precise function of melatonin in the management of OC is not well understood. This review summarizes the current knowledge regarding melatonin function in anti-carcinogenesis mechanisms for OC. In addition, clinical assessment and the potential therapeutic utility of melatonin in OC are discussed. This review will provide a basis for researchers to create new melatonin-based personalized medicines for treating and preventing OC.

Association of Interactions between Metabolic 'Caretaker' Genes, p53, MDM2, and Tobacco Use with the Risk of Oral Cancer: A Multifactor Dimensionality Reduction Approach.

BACKGROUND: The present study investigated the association of interactions between gene polymorphisms in metabolic 'caretaker' genes (Phase I: CYP1A1, CYP2E1; Phase II: GSTM1, GSTT1), the cell cycle regulatory gene, p53, along with its negative controller, MDM-2, and the environment variable (tobacco). A nonparametric model, multifactor dimensionality reduction (MDR), was applied to analyse these interactions. MATERIALS AND METHODS: This case-control study was carried out on 242 subjects. Genomic DNA was extracted from peripheral blood lymphocytes.11 gene variants with an exposure variable (tobacco use) were analysed using MDR to identify the best locus model for gene-gene and gene-environment interactions. Statistical significance was evaluated using a 1000-fold permutation test using MDR permutation testing software (version 1.0 beta 2). The value of p<0.05 was considered statistically significant. RESULTS: The best three-locus model for gene-gene interaction included two of the p53 gene polymorphisms; rs17878362 (intron 3) and rs1042522 (exon 4) and rs6413432 in the Phase I gene, CYP2E1(DraI). The three-locus model to evaluate the gene-environment interaction included two intronic polymorphisms of the p53 gene, that is, rs17878362 (intron 3) and rs1625895 (intron 6), and rs4646903 in the Phase I gene CYP1A1*2C. The interaction graphs revealed independent main effects of the tobacco and p53 polymorphism, rs1042522 (exon 4), and a significant additive interaction effect between rs17878362 (intron 3) and rs1042522 (exon 4). CONCLUSIONS: The nonparametric approach highlighted the potential role of tobacco use and variations in the p53 gene as significant contributors to oral cancer risk. The findings of the present study will help implement preventive strategies in both tobacco use and screening using a molecular pathology approach.

Alterations in p53 Influence hTERT, VEGF and MMPs Expression in Oral Cancer Patients.

BACKGROUND: Mutant p53 is the crucial molecule in the etiopathogenesis of oral cancer. Therefore, we aimed to evaluate the impact of alterations of the p53 gene and its negative feedback regulator, MDM2, on the expression of hTERT, VEGF, and MMPs; the critical genes involved in oral cancer progression. MATERIAL AND METHODS: p53 and MDM2 genotyping were done by PCR-RFLP. p53 mutation analysis was performed using PCR-SSCP and sequencing. hTERT, VEGFA isoforms, MMP2, and MMP9 mRNA levels were analyzed by semi-quantitative Reverse Transcriptase PCR. RESULTS: Arg allele at p53 exon 4 was significantly associated with overexpression of hTERT, MMP2, and MMP9 individually. Expression of hTERT, VEGF A isoforms, MMP2 and MMP9 were significantly altered in the presence of p53 and MDM2 polymorphisms and p53 mutations in a specific combination. Mutant p53, Arg allele at p53 exon 4 locus, and G/G/or T/T genotype at MDM2revealed increased expression of hTERT, VEGF A isoforms, and MMP2/9. CONCLUSION: This study provides evidence that apart from mutant p53, naturally occurring sequence variants in p53codon 72 (Arg72Pro) (rs1042522) and MDM2 (rs2279744) significantly alter the expression of hTERT, VEGF-A isoforms, and MMP2/9 in a specific combination. The differential interaction of codon 72 variants with MDM2, hTERT, VEGF-A isoforms and MMP2/9 play a role in the aggressiveness of oral cancer. The results have important implications for oral cancer progression and should be explored for innovative treatment options.

Curbing the Deregulation of Glycosylation in Tongue Carcinoma Cells with Natural Compounds.

BACKGROUND: Aberrant glycosylation has been recently considered as a major hallmark of cancer. Furthermore, we have reported that aberrant glycosylation, mainly sialylation and fucosylation, plays a major role in oral cancer progression and metastasis. OBJECTIVE: In the present study, we evaluated the role of tobacco compounds (4-NQO, NNK, Benzopyrene), natural compounds (Curcumin, Butein and Piceatannol) and commonly used chemotherapeutic compound (Cisplatin) on sialylation and fucosylation transcript levels in the tongue cancer cell line (SAS). METHODS: The SAS cells were treated with the tobacco compounds, natural compounds and Cisplatin after obtaining their IC50 values using MTT assay. After 24 hr treatment of the compounds, RNA was isolated from the cells and converted to cDNA. RT-qPCR was performed for mRNA expression of glycosylation transcripts. RESULTS: The treatment of tobacco compounds on the SAS cells resulted in increased mRNA levels of ST3GAL1, NEU3, FUT5 and FUT6 in a dose-dependent manner. The treatment of Curcumin and Butein resulted in lower mRNA levels of FUT8, whereas dose-dependent higher mRNA levels of FUT3 were also observed after the treatment of Curcumin. SAS cells exhibited a dose-dependent decrease in ST3GAL2, FUT5 and FUT8 mRNA after Piceatannol treatment. Furthermore, Cisplatin treatment on the SAS cells resulted in increased mRNA levels of FUT3 as the concentration increased from 100 µM to 200 µM. While, treatment of Cisplatin resulted in decreased mRNA levels of ST3GAL2, ST3GAL3, FUT5 and FUT8 in a dose-dependent manner. All together, the data revealed Piceatannol as a potent synergistic for Cisplatin to target the altered glycosylation for better treatment management of tongue carcinoma. CONCLUSION: The study provides a normal approach of targeting aberrant glycosylation with natural compounds, which may open the possibility of newer therapeutic strategies using natural compounds alone or in combination with other conventional therapies.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.

RNA Aptamers Rescue Mitochondrial Dysfunction in a Yeast Model of Huntington's Disease.

Huntington's disease (HD) is associated with the misfolding and aggregation of mutant huntingtin harboring an elongated polyglutamine stretch at its N terminus. A distinguishing pathological hallmark of HD is mitochondrial dysfunction. Any strategy that can restore the integrity of the mitochondrial environment should have beneficial consequences for the disease. Specific RNA aptamers were selected that were able to inhibit aggregation of elongated polyglutamine stretch containing mutant huntingtin fragment (103Q-htt). They were successful in reducing the calcium overload, which leads to mitochondrial membrane depolarization in case of HD. In one case, the level of Ca2+ was restored to the level of cells not expressing 103Q-htt, suggesting complete recovery. The presence of aptamers was able to increase mitochondrial mass in cells expressing 103Q-htt, along with rescuing loss of mitochondrial genome. The oxidative damage to the proteome was prevented, which led to increased viability of cells, as monitored by flow cytometry. Thus, the presence of aptamers was able to inhibit aggregation of mutant huntingtin fragment and restore mitochondrial dysfunction in the HD cell model, confirming the advantage of the strategy in a disease-relevant parameter.

Designing aptamers which respond to intracellular oxidative stress and inhibit aggregation of mutant huntingtin.

Targeted expression of a therapeutic agent is a major bottleneck in designing a drug delivery system. Protein aggregation and elevated oxidative stress are associated with the onset of many neurodegenerative disorders, including Huntington's disease (HD). An oxidative stress-inducible promoter, i.e. Thioredoxin 2, was employed to design a sensor for protein aggregation. RNA aptamers specific for mutant huntingtin were expressed only in cells where aggregation of mutant huntingtin occurred. A nine-fold increase in RNA expression was seen when aptamer sequences were cloned under the Trx2 promoter. Expression of aptamer resulted in reduced protein aggregation and decreased oxidative stress, which, in turn, reduced the expression of aptamers by two-fold. Reduction in aggregation led to increased cell survival. The aptamers were not expressed in cells expressing wild-type huntingtin in the soluble form. This rational and simple design will allow the use of this construct for the targeted expression of other therapeutic nucleic acid molecules as well.

Challenges with osmolytes as inhibitors of protein aggregation: Can nucleic acid aptamers provide an answer?

Protein aggregation follows some common motifs. Whether in the formation of inclusion bodies in heterologous overexpression systems or inclusions in protein conformational diseases, or aggregation during storage or transport of protein formulations, aggregates form cross beta-sheet structures and stain with amyloidophilic dyes like Thioflavin T and Congo Red, irrespective of the concerned protein. Traditionally, osmolytes are used to stabilize proteins against stress conditions. They are employed right from protein expression, through production and purification, to formulation and administration. As osmolytes interact with the solvent, the differential effect of the stress condition on the solvent mostly determines the effect of the osmolyte on protein stability. Nucleic acid aptamers, on the other hand, are highly specific for their targets. When selected against monomeric, natively folded proteins, they bind to them with very high affinity. This binding inhibits the unfolding of the protein and/or monomer-monomer interaction which are the initial common steps of protein aggregation. Thus, by changing the approach to a protein-centric model, aptamers are able to function as universal stabilizers of proteins. The review discusses cases where osmolytes were unable to provide stabilization to proteins against different stress conditions, a gap which the aptamers seem to be able to fill.

Specific detection of tetanus toxoid using an aptamer-based matrix.

Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.