Down-regulation of mitochondrial NADH and cytochrome b gene associated with high tumor stages in head and neck squamous cell carcinoma.

OBJECTIVE: This study aimed to determine mitochondrial mRNA expression levels and the relationships between these expression levels and various adverse clinicopathological characteristics. METHODS: The mRNA expression levels of all 12 genes encoded protein, located on the heavy-strand of mitochondrial DNA including cytochrome b, NADH1, NADH2, NADH3, NADH4, NADH4L, NADH5, ATPase6, ATPase8, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 2, cytochrome c oxidase subunit 3 were analyzed in 30 head and neck squamous cell carcinoma (HNSCC) and the corresponding normal tissues using reverse transcriptase quantitative real time PCR. Pearson Chi-square test was used to determine the relationships between these expression levels and categorical parameters. RESULTS: The expression levels of 12 mitochondrial mRNAs were observed in all 30 HNSCC patients with down-regulation, ranging from 43.3% to 76.7% and up-regulation, ranging from 10.0% to 36.7%. Furthermore, the number of cases with down-regulations in all 6 NADH and cytochrome b mRNA with TMN stages III and IV were significantly higher than that in stages I and II (p=0.049 and 0.007, respectively). CONCLUSION: Down-regulation of all mitochondrial NADH mRNA as well as mitochondrial cytochrome b mRNA was associated with high tumor stage among HNSCC patients.

Unveiling a novel biomarker panel for diagnosis and classification of well-differentiated thyroid carcinomas.

Thyroid cancer is the most common human endocrine malignancy with increasing global incidence. Papillary thyroid carcinomas (PTC) and follicular thyroid carcinomas (FTC) are well-differentiated thyroid cancers (WDTC) accounting for 95% of all thyroid cancer cases, with survival rates of almost 100% when diagnosed early. Since PTC and FTC have different modes of metastasis, they require different treatment strategies. Standard diagnosis by fine needle aspiration with cytopathological examination can be inaccurate in approximately 10-30% of all cases and difficult to definitively classify as WDTC. Currently, there is no single or panel of biomarkers available for thyroid cancer diagnosis and classification. This study identified novel biomarkers for thyroid cancer diagnosis and classification using proteomics, which may be translated into a biomarker panel for clinical application. Two-dimensional SDS-PAGE and mass spectrometry were used to identify potential biomarkers in papillary and follicular thyroid carcinoma cell lines, and the biomarkers were validated in five PTC and five FTC tissues, with their adjacent normal tissues from Thai patients. Eight biomarkers could distinguish PTC from normal tissues, namely enolase 1, triose phosphate isomerase, cathepsin D, annexin A2, cofilin 1, proliferating cell nuclear antigen (PCNA), copine 1 and heat shock protein 27 kDa (HSP27). These biomarkers can also discriminate FTC from normal tissues, except for annexin A2. On the contrary, annexin A2, cofilin 1, PCNA and HSP27 can be used to classify the types of WDTC. These findings have potential for use as a novel multi-marker panel for more accurate diagnosis and classification to better guide physicians on thyroid cancer treatment. Moreover, our results suggest the involvement of proteins in cell growth and proliferation, and the p53 pathway in the carcinogenesis of WDTC, which may lead to targeted therapy for thyroid cancer.

Expression of cdk6 in head and neck squamous cell carcinoma.

OBJECTIVE: Cdk6 is a key regulator during the G1/S cell cycle transition. Aberrant expression of cdk6 protein has been observed in many cancer types. However, little is known about the expression of cdk6 in head and neck squamous cell carcinoma (HNSCC) and its clinical significance. This study evaluated the expression of cdk6 in HNSCC and analyzed the relationship between cdk6 expression and clinicopathological parameters of HNSCC. MATERIALS AND METHODS: Expression of cdk6 was immunohistochemically investigated in 98 HNSCCs. Nuclear and cytoplasmic positive cells were counted separately. Data were presented as the percentage of positive cells. The correlation between the percentage of positive cells and clinicopathological factors was determined. RESULTS: Nuclear and cytoplasmic staining for cdk6 were detected in 91 cases and 97 cases, respectively. A significant correlation was found only between the percentage of nuclear positive cells and T classification (p value = 0.0410). Tumors with high nuclear cdk6-positive cells showed a linear trend toward advanced tumor status (p value = 0.0064). CONCLUSIONS: Cdk6 was highly expressed in HNSCC. Tumors with high nuclear cdk6 expression tended to have advanced tumor status. These results suggest that cdk6 plays a vital role in HNSCC and is involved in tumor progression of this cancer. CLINICAL RELEVANCE: An increased nuclear cdk6 expression is an unfavorable factor for HNSCC. Cdk6 may serve as a therapeutic target in this cancer.

Identification of novel allergen in edible insect, Gryllus bimaculatus and its cross-reactivity with Macrobrachium spp. allergens.

Edible insects have recently been promoted as a source of protein and have a high nutrition value. Identification of allergens and cross-reactivity between Macrobrachium spp. and the field cricket (Gryllus bimaculatus) is necessary for food safety control and to assist in the diagnosis and therapy of allergy symptoms. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins. Allergens were determined and identified by IgE-immunoblotting with pooled sera from prawn-allergic patients (n=16) and LC-MS/MS. Arginine kinase (AK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined as the important allergens in muscle of Macrobrachium rosenbergii whereas, hemocyanin (HC) was identified as an allergen in Macrobrachium spp. The allergens in Macrobrachium lanchesteri were identified as AK and HC. In addition, hexamerin1B (HEX1B) was identified as a novel and specific allergen in G. bimaculatus. The important allergen in G. bimaculatus and Macrobrachium spp. is AK and was found to cross-react between both species.

SLC35B2 expression is associated with a poor prognosis of invasive ductal breast carcinoma.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide, including Thailand, and is a major cause of mortality and morbidity, despite advances in diagnosis and treatment. Novel gene expression in breast cancer is a focus in searches for prognostic biomarkers and new therapeutic targets. MATERIALS AND METHODS: The mRNA expression of novel B4GALT4, SLC35B2, and WDHD1 genes in breast cancer were examined in invasive ductal breast carcinoma (IDC) patients using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Among these genes, increased expression of SLC35B2 mRNA was significantly associated with TNM stage III+IV of IDC (p<0.001). Hence, up-regulation of SLC35B2 may serve as a prognostic biomarker for poor prognosis, and is also a potential therapeutic target in breast cancer.

Identification of a novel allergen from muscle and various organs in banana shrimp (Fenneropenaeus merguiensis).

BACKGROUND: The increasing consumption of shellfish can cause an increase in allergic symptoms. Shrimp allergy can be species specific, but specific allergies in different organs have not been studied. Identification of allergens in muscle and others organs of banana shrimp is necessary for improved diagnostics of allergies for shrimp and food safety control. OBJECTIVE: To identify the IgE-binding proteins in various organs of Fenneropenaeus merguiensis by immunoblotting and tandem mass spectrometry. METHODS: Proteomic methods were used to investigate the allergenic proteins from banana shrimp. Proteins from muscle and various organs were separated by denaturing polyacrylamide gel electrophoresis. Allergens were analyzed by immunoblotting with pooled sera from shrimp allergic patients (n = 21) and tandem mass spectrometry. RESULTS: The important allergens in banana shrimp are arginine kinase, sarcoplasmic calcium-binding protein, myosin heavy chain, hemocyanin, enolase, and glyceraldehyde-3-phosphate dehydrogenase, which can be demonstrated by immunoblotting in muscle and shell. Moreover, vitellogenin, ovarian peritrophin 1 precursor, ß-actin, and 14-3-3 protein were suggested as allergens in the ovary at different stages of ovarian development. CONCLUSION: Ten allergens were identified as allergens in various organs, and they are suggested as novel allergens in banana shrimp. The major allergen in muscle and shell from this shrimp is arginine kinase, whereas the major allergen in the ovary is vitellogenin.

Aberrant O-GlcNAc-modified proteins expressed in primary colorectal cancer.

O-GlcNAcylation is a post-translational modification of serine and threonine residues which is dynamically regulated by 2 enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyze the addition and removal of a single N-acetylglucosamine (GlcNAc) molecule, respectively. This modification is thought to be a nutrient sensor in highly proliferating cells via the hexosamine biosynthesis pathway, a minor branch of glycolysis. Although emerging evidence suggests that O-GlcNAc modification is associated with many types of cancer, identification of O-GlcNAc-modified proteins and their role in cancer remain unexplored. In the present study, we demonstrated that O-GlcNAcylation is increased in primary colorectal cancer tissues, and that this augmentation is associated with an increased expression of OGT levels. Using 2-dimensional O-GlcNAc immunoblotting and LC-MS/MS analysis, 16 proteins were successfully identified and 8 proteins showed an increase in O-GlcNAcylation, including cytokeratin 18, heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), hnRNP H, annexin A2, annexin A7, laminin-binding protein, α-tubulin and protein DJ-1. Among these identified proteins, annexin A2 was further confirmed to show overexpression of O-GlcNAc in all cancer samples. The results, therefore, indicate that aberrant O-GlcNAcylation of proteins is associated with colorectal cancer and that identification of O-GlcNAc-modified proteins may provide novel biomarkers of cancer.

Determination of whole transcription profiles and specific pathways in invasive ductal breast carcinoma.

Breast cancer is the most common cancer affecting women worldwide including Thailand. Whole transcription profiles of invasive ductal breast carcinoma (IDC) obtained by oligonucleotide microarray should lead to a better understanding of the molecular basis of IDCs, allow for examination of specific markers for diagnosis, and provide novel targets for therapy. This study aimed to detect the whole transcript expression of approximately 35,000 target genes in Thai breast cancer patients, using Affymetrix GeneChip(®) Exon 1.0 Sense Target Arrays. Analysis revealed that the differential expression profiles of 928 genes (423 up-regulated and 505 down-regulated genes) were 2-fold or greater (unpaired t-test, p < 0.05) in invasive ductal breast cancer, compared with normal tissues. The Gene Ontology (GO) databases support important associations in 17 gene sets with p-value < 1E-10 and ≥ 4-fold changes, involving the tumorigenic pathways of cell cycles, extracellular regions, as well as cellular component organization. Likewise, the TGFBR and IL-6 pathways contain gene expression with statistically significant changes in IDC.

Proteomic analysis and abrogated expression of O-GlcNAcylated proteins associated with primary breast cancer.

O-GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc-modified proteins might be one of the vital malignant characteristics of cancer.

Quantitative real-time RT-PCR of ITGA7, SVEP1, TNS1, LPHN3, SEMA3G, KLB and MMP13 mRNA expression in breast cancer.

Breast cancer is the leading cause of cancer deaths among women worldwide, including Thailand. In the present study, the differential mRNA expression of SVEP1, LPHN3, KLB, ITGA7, SEMA3G, TNS1 and MMP13 genes was examined in breast cancer using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Among these genes, increased LPHN3 and MMP13 mRNA expression levels correlated with axillary-node metastasis (P=0.02). Multiple logistic regression analysis revealed that LPHN3 and MMP13 mRNA expression is significantly associated with axillary node status in breast cancer (P=0.04).

Ubiquitin-specific protease 14 expression associated with intrahepatic cholangiocarcinoma cell differentiation.

The purpose of this study was to identify the gene alterations amplified from AO16 primer and examine whether the expression patterns of USP14 in clinical specimens from patients with intrahepatic cholangiocarcinoma (ICC) is associated with cancer cells. DNA from tumor and corresponding normal tissues of 52 patients was amplified with 33 arbitrary primers. The DNA fragment that altered most frequently in ICC was cloned, sequenced, and identified by comparison with known nucleotide sequences in the genome database. The DNA copy numbers of the allelic alterations in cholangiocarcinoma were determined by quantitative real-time PCR and interpreted as allelic loss or DNA amplification by comparison with the reference gene. Associations between allelic imbalance and clinicopathological parameters of ICC patients were evaluated by X²-tests. The Kaplan-Meier method was used to analyze survival rates. Immunohistochemically, USP14 showed weak cytoplasmic staining in normal bile duct epithelial cells. It was strongly detected in 21 cancer patients (43.8%). There were correlations between USP14 expression level and the clinicopathological features of ICC, histological grade (P < 0.05). However, there were no significant differences in age, gender, tumor size, metastasis, lymph node metastasis, and staging. USP14 expression was related to cholangiocarcinoma cell differentiation. Due to their emerging role in control of multiple signaling pathways and oncoproteins, USP14 inhibitors may be useful for anticancer agents.

DNA amplification on chromosome 13q31.1 correlated with poor prognosis in colorectal cancer.

Colorectal cancer is a leading cause of cancer deaths worldwide. Genetic markers involved in prognosis of colorectal cancer are still being elucidated. In this study, genetic alterations associated with prognosis of colorectal cancer were determined using arbitrarily primed polymerase chain reaction (AP-PCR) and analyzed quantitatively by real-time PCR. Seven different DNA sequences, mapped on chromosomes 13q31.1, 9q31.1, 1q24, 4q31.3, 10q21, 11q13.4, and 13q13.3, were identified. Among these sequences, seven cases (23%) harbored DNA amplification in chromosome 13q31.1, and 9 (29%) and 7 (23%) presented genetic alterations in chromosome 1q24 and 11q13.4, respectively. Multivariate analysis showed that only DNA amplification in chromosome 13q31.1 was associated with poor survival among patients with colorectal cancer, with median survival time for chromosome 13q31.1 amplification versus no amplification of 64 versus 268 weeks (P = 0.001). This genetic alteration may have a prognostic role in colorectal cancer.

Genetic alteration in oral squamous cell carcinoma detected by arbitrarily primed polymerase chain reaction.

Oral cancer ranks as one of the top ten cancers in Thailand. Molecular carcinogenesis of this disease remains unknown. The purpose of this report was to identify the genetic alteration profile in Thai oral squamous cell carcinoma (OSCC) patients using arbitrarily primed PCR and to determine the association between genetic alterations and clinico-pathological characteristics. Band alteration profiles in the 32 OSCC tissues were compared with corresponding normal tissues amplified from 60 arbitrary primers using arbitrarily primed polymerase chain reaction (AP-PCR) were identified with 12 primers. Among these, 45 band patterns presented the alteration ranged from 36% to 88%. Primer AD15 at 750 base pairs (AD15-750 bp) was found to have both the highest band alteration (88%) and the highest band loss (37%). The highest DNA band amplification was found in primer AX11-1300 bp (56%). Primer AX-11 at 1300 base pairs at the altered frequency of 78% was significantly associated with smoking (p=0.007), and primer N20 at 800 base pairs showed association with low grade tumors (p=0.030). Our results indicate that AP-PCR is a useful technique for detect genetic alteration in oral squamous cell carcinoma and provide various genetic alternative data.

Effectiveness of inflammatory cytokines induced by sericin compared to sericin in combination with silver sulfadiazine cream on wound healing.

 Silk sericin (SS) has been shown to promote collagen synthesis during wound healing, but it lacks antimicrobial activity. We investigated the effectiveness and the induction of the inflammatory mediators IL-1bß and TNF-aα by SS, silver sulfadiazine (SSD) cream, and SS in combination with SSD cream on wound healing in rats. The results show that SS at 8% w/w partially inhibits SSD antibacterial activity. Treating wounds with a combination of SS and SSD did not induce significant wound size reduction when compared to other treatments. However, SS can promote collagen production in wounds even in the presence of SSD. Wounds treated with the combination of SS and SSD cream showed higher levels of IL-1ß and TNF-α when compared to wounds treated by SS alone, but the differences were not significant. Although SS may decrease the antimicrobial effect of SSD, SS in combination with SSD cream has the benefit of promoting collagen production without generating significant levels of inflammatory cytokines..

Identification of genetic alterations in thai breast cancer patients by arbitrarily primed polymerase chain reaction.

Breast cancer is the most common cancer among women worldwide. Genetic alterations prevalent in breast cancer are still being elucidated. In this report, changes in 30 breast cancer tissues, in comparison with normal tissues from Thai patients, were analyzed by arbitrarily primed polymerase chain reaction (AP-PCR). Genetic instability was detected by DNA fingerprinting obtained with 13 of 60 random primers. Of these, at least one amplification band, the incidence ranging from 27 to 80%, was observed in DNA amplified with 8 primers, whereas a band loss was exhibited with from 6 primers, the incidences ranging from 23 to 40%. Likewise, an amplification band amplified from primer D15 was observed in 80% of this patient group and a band loss produced from primer B12 presented in 40% of all cases. These results showed that AP-PCR is effective for the detection of genetic alterations in breast cancer tissues.

Genetic instability in cervical cancer detected by arbitrarily primed polymerase chain reaction.

The genetic instability in 54 Thai cervical cancer tissues were analyzed by Arbitrarily Primed Polymerase Chain Reaction (AP-PCR). The band alterations produced from 54 arbitrary primers were compared between the DNA finger printing from the patients and their corresponding normal cervical tissues. Results revealed 7 arbitrary primers provided DNA alteration patterns. Of these, an allelic loss in tumor DNA was found in DNA fingerprinting obtained from primers F-2 (64.8%), F-11 (68.5%), U-8 (51.9%), AE-3 (75.9%), AE-11 (53.7%), respectively. Moreover, DNA amplification was exhibited in patterns with primers B-12 (42.6%), J-16 (24.1%) and U-8 (70.4%). When genetic instability was investigated for associations with clinicopathological features, only the DNA amplified fragment with primer U-8 was significantly associated with stage II (P=0.030). Likewise, allelic loss amplified from arbitrary primer AE-3 showed significantly associate with age lower than 50 years old (P=0.003). Our findings suggest that the DNA alteration fragments produced from arbitrary primers of U-8 and AE-11 might be relevant to the pathogenesis of cervical cancer in Thai patients.

Polymorphism of glutathione S-transferase omega gene and risk of cancer.

Polymorphic glutathione S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to cancer. Though polymorphisms have been reported in GSTO1 and GSTO2, their predisposition to cancer risk has not yet been explored. In this case control study, 28 cases of hepatocellular carcinoma, 30 cases of cholangiocarcinoma, 31 cases of colorectal cancer, 30 cases of breast cancer and 98 controls were compared for frequencies of GSTO1 and GSTO2 genotypes. The statistical analysis provided the support for the difference in genotypic distribution for GSTO1*A140D between hepatocellular carcinoma (OR 23.83, CI 95%: 5.07-127), cholangiocarcinoma (OR 8.5, CI 95%: 2.07-37.85), breast cancer (OR 3.71, CI 95%: 1.09-13.02) and control. With regards to GSTO2*N140D polymorphism, there was no difference in genotypic distribution between all the types of cancer and control. The study suggests that GSTO1*A140D polymorphism could play an important role as a risk factor for the development of hepatocellular carcinoma, cholangiocarcinoma and breast cancer.

Proteomic Studies of Galectin-3 Expression in Human Thyroid Diseases by Immunodetection.

Galectin-3 expression in thyroid diseases was studied by 1-DE immunoblotting. Expression was markedly elevated in thyroid papillary carcinoma, compared to follicular adenoma, follicular carcinoma or non-neoplastic diseases. Galectin-3 expression was also elevated in malignant cancers of bone, breast, colon, esophagus, larynx, lung and ovary. Four cases of thyroid papillary carcinoma with metastasis gave 2-3 bands on 1-DE immunoblotting. 2-DE immunoblotting of gelectin-3 showed 3 dark spots with MW/pI 32.9/8.29, 31.0/8.40 and 30.0/8.40 and 2 light spots.

Cutaneous granuloma in systemic lymphoma: a case report in Thailand.

A 68-year-old woman presented with multiple erythematous infiltrative nodules and plaques on her face, trunk and extremities, 7 months after having complete remission from chemotherapy treatment of non-Hodgkin's lymphoma. Biopsy from the skin lesion showed tuberculoid granuloma without lymphoma. Special stains and culture were negative for micro-organism. Immunohistochemistry revealed polymorphic T and B cells infiltration without evidence of malignancy. The skin lesions subsided completely after corticosteroid treatment. Two months later, she developed brain involvement of lymphoma that responded well to radiation and chemotherapy.

OCT4 expression on a case of poorly differentiated (insular) carcinoma of the thyroid gland and minireview.

Poorly differentiated (insular) carcinoma of the thyroid gland is rare and defined as follicular-cell neoplasms that show limited evidence of structural follicular cell differentiation and occupy both morphologically and behaviourally an intermediate position between differentiated (follicular and papillary carcinomas) and undifferentiated (anaplastic) carcinomas. The authors report a case of a 37-year-old Thai woman who presented with a prolonged left thyroid nodule. Final pathological diagnoses of her mass were poorly differentiated (insular) carcinoma with lymphovascular invasion and nodular goiter. The tumor cell arrangements were nest (insular) and trabecular patterns with some follicular formations. Immunohistochemistry of the tumor cells revealed negative immunostaining for OCT4. Expression of OCT4 gene is involved in the regulation and maintenance of pluripotency of embryonic stem cells, germ cells, and in tumor cells. The authors believe that poorly differentiated (insular) carcinoma of the thyroid gland probably develops from the remnant of thyroid stem cells and is not associated with dedifferentiation (anaplasia or loss of cellular differentiation) from nodular goiter or cells of other thyroid carcinomas. Although there was negative immunostain for OCT4 in the presented case, the authors assumed that the tumor cells behave with an intermediate position between thyroid stem cells and prothyrocytes Also they do not behave with thyroblasts. Additionally, the tumor may be associated with new cellular dedifferentiation. However, there is only one case of immunohistochemistry of OCT4 in poorly differentiated (insular) carcinoma of the thyroid gland. Thus, prognosis of the presented still is mainly correlated with clinical and histological findings. Further research on expression of OCT4 gene on thyroid cancers and other malignant tumors relating to tumorigenic cancer cells (cancer stem cells) may be useful to prognostic evaluation and administration of a new chemotherapy and/or radiotherapy that is specific for tumor-initiating cells.