Salah O. Ibrahim

Intercountry prevalences and practices of betel-quid use in south, southeast and eastern asia regions and associated oral preneoplastic disorders: An international collaborative study by asian betel-quid consortium of south and east Asia

Health risks stemming from betel‐quid (BQ) chewing are frequently overlooked by people. Updated epidemiological data on the increased BQ use among Asian populations using comparable data collection methods have not been widely available. To investigate the prevalence, patterns of practice and associated types of oral preneoplastic disorders, an intercountry Asian Betel‐quid Consortium study (the ABC study) was conducted for Taiwan, Mainland China, Malaysia, Indonesia, Nepal and Sri Lanka. A random sample of 8,922 subjects was recruited, and the data were analyzed using survey‐data modules adjusted for the complex survey design. Chewing rates among men (10.7–43.6%) were significantly higher than women (1.8–34.9%) in Taiwan, Mainland China, Nepal and Sri Lanka, while womens rates (29.5–46.8%) were higher than that for men (9.8–12.0%) in Malaysia and Indonesia. An emerging, higher proportion of new‐users were identified for Hunan in Mainland China (11.1–24.7%), where Hunan chewers have the unique practice of using the dried husk of areca fruit rather than the solid nut universally used by others. Men in the Eastern and South Asian study communities were deemed likely to combine chewing with smoking and drinking (5.6–13.6%). Indonesian women who chewed BQ exhibited the highest prevalence of oral lichen planus, oral submucous fibrosis and oral leukoplakia (9.1–17.3%). Lower schooling, alcohol drinking and tobacco smoking were identified as being associated with BQ chewing. In conclusion, the ABC study reveals the significant cultural and demographic differences contributing to practice patterns of BQ usage and the great health risks that such practices pose in the Asian region.

Prevalence of viral (HPV, EBV, HSV) infections in oral submucous fibrosis and oral cancer from India

Abstract Conclusion: There is a high prevalence of human papilloma viruses (HPV) in oral submucous fibrosis (OSMF) and the etiologic implication of this finding warrants further studies. Objective: The prevalence of oral squamous cell carcinoma (OSCC) and OSMF is high in India, and the diseases are partly attributed to high consumption of betel quid containing areca nut and tobacco. This study investigated the prevalence of HPV, herpes simplex virus (HSV), and Epstein–Barr virus (EBV) DNA in two groups of patients using betel quid with tobacco, those with OSMF (n = 12) and those with OSCC (n = 62). Methods: DNA was extracted from all the samples and viral genome was examined by PCR/DNA sequencing. HPV-positive samples were analyzed separately for the high-risk types HPV 16 and 18. Results: HPV DNA, HSV DNA, and EBV DNA were detected in 11 (91%), 1 (8%), and 3 (25%) of the 12 samples from patients with OSMF compared with 15 (24%), 3 (5%), and 18 (29%), respectively, from 62 patients with OSCC. HPV 16 and 18 DNA was detected in 8/12 (67%) in the OSMF group and 10/62 (16%) in the OSCC group. The difference between presence of HPV DNA in OSMF and OSCC groups was statistically significant, while the difference between HSV and EBV DNA content in OSMF and OSCC groups was insignificant.

Presence of human papilloma virus, herpes simplex virus and Epstein-Barr virus DNA in oral biopsies from Sudanese patients with regard to toombak use

Using PCR/DNA sequencing, we investigated the prevalence of human papillomavirus (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA in brush biopsies obtained from 150 users of Sudanese snuff (toombak) and 25 non-users of toombak in formalin-fixed paraffin-embedded tissue samples obtained from 31 patients with oral dysplasias (25 toombak users and 6 non-users), and from 217 patients with oral cancers (145 toombak users and 72 non-users). In the brush tissue samples from toombak users, HPV was detected in 60 (40%), HSV in 44 (29%) and EBV in 97 (65%) of the samples. The corresponding figures for the 25 samples from non-users were 17 (68%) positive for HPV, 6 (24%) positive for HSV and 21 (84%) for EBV. The formalin-fixed samples with oral dysplasias were all negative for HPV. In the 145 oral cancer samples from toombak users, HPV was detected in 39 (27%), HSV in 15 (10%) and EBV in 53 (37%) of the samples. The corresponding figures for the samples from non-users were 15 (21%) positive for HPV, 5 (7%) for HSV and 16 (22%) for EBV. These findings illustrate that prevalence of HSV, HPV and EBV infections are common and may influence oral health and cancer development. It is not obvious that cancer risk is increased in infected toombak users. These observations warrant further studies involving toombak-associated oral lesions, to uncover the possible mechanisms of these viral infections in the development of oral cancer, and the influence of toombak on these viruses.

Population Burden of Betel Quid Abuse and Its Relation to Oral Premalignant Disorders in South, Southeast, and East Asia: An Asian Betel-Quid Consortium Study

OBJECTIVES We investigated the population burden of betel quid abuse and its related impact on oral premalignant disorders (OPDs) in South, Southeast, and East Asia. METHODS The Asian Betel-Quid Consortium conducted a multistage sampling of 8922 representative participants from Taiwan, Mainland China, Malaysia, Indonesia, Nepal, and Sri Lanka. Participants received an interviewer-administered survey and were examined for oral mucosal disorders. RESULTS The prevalence of betel quid abuse was 0.8% to 46.3% across 6 Asian populations. The abuse frequency was over 40.5% for current chewers, with the highest proportion in Nepalese and Southeast Asian chewers (76.9%-99.6%). Tobacco-added betel quid conferred higher abuse rates (74.4%-99.6%) among Malaysian, Indonesian, and Sri Lankan men than did tobacco-free betel quid (21.8%-89.1%). Gender, lower education level, younger age at chewing initiation, and clustering of familial betel quid use significantly contributed to higher abuse rates. Indonesian betel quid abusers showed the highest prevalence of OPDs and had a greater risk of OPDs than did nonabusers. CONCLUSIONS Betel quid abuse is high in regions of Asia where it is customarily practiced, and such abuse correlates highly with OPDs. By recognizing abuse-associated factors, health policies and preventive frameworks can be effectively constructed to combat these oral preneoplasms.

S100A14 regulates the invasive potential of oral squamous cell carcinoma derived cell-lines in vitro by modulating expression of matrix metalloproteinases, MMP1 and MMP9

Despite the differential expression of S100A14 (a newly identified S100 member) in various human cancers including oral squamous cell carcinomas (OSCCs), its biological role in tumour invasion has not been characterised. The aim of this study was thus to investigate the possible role of S100A14 in OSCC cell invasion. Using immunohistochemistry in normal (n=13), dysplastic (n=10) and OSCC (n=16) archival tissues, S100A14 protein was found to be down-regulated/lost with concomitant membrane to cytoplasmic translocation in OSCCs, especially in the invading tumour islands. These expression data were corroborated by profiling S100A14 mRNA expression using quantitative RT-PCR (qRT-PCR) in an in vitro human OSCC progression model consisting of cell-lines derived from normal (n=3), dysplastic (n=3) and OSCC (n=8) tissues. Employing in vitro Matrigel invasion assay, we demonstrated that retroviral vector mediated over-expression of S100A14 resulted in significant decrease in the invasive potential of OSCC derived CaLH3 and H357 cell-lines whereas siRNA mediated knockdown resulted in significant increase in the invasive potential of CaLH3 cell-line. Pathway focused PCR array and validation using qRT-PCR revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines. Further, S100A14 over-expression was found to be associated with suppression of MMP9 gelatinolytic activity in CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in 19 cases of OSCCs. Collectively, these data provide the first evidence for a role of S100A14 protein in regulation of OSCC cell invasion by modulating expression of MMP1 and MMP9.

Expression profile of the S100 gene family members in oral squamous cell carcinomas

BACKGROUND Several of the S100 gene members have been reported to be differentially expressed in many human pathological conditions, in particular, the malignancies. Identification and quantification of the differentially expressed S100 gene members in oral squamous cell carcinoma (OSCC) might facilitate their use as potential diagnostic and/or prognostic markers or targets for therapy. METHODS we examined the expression profile of 16 members of the S100 gene family at the mRNA level by semiquantitative reverse transcription-polymerase chain reaction (sRT-PCR) in 27 cases of OSCCs/their pair-wised normal controls obtained from Sudanese patients, and confirmed the sRT-PCR results by performing quantitative real time-polymerase chain reaction (qRT-PCR) for 6 of the 16 genes examined. RESULTS With sRT-PCR, 4 (25%; S100A4, S100A6, S100A8, S100A14) out of the 16 S100 gene members examined were found to be significantly down-regulated (P < 0.05) in the tumors compared to the normal controls. None of the S100 gene members examined were found to be significantly up-regulated in the tumors. qRT-PCR results confirmed the significant down-regulation of the S100A4, S100A6, and S100A14 genes in the tumors examined. CONCLUSION S100 gene family members might play an important role in the pathogenesis of the OSCCs examined. Findings of the present work warrant in-depth studies of the S100 gene family members, in particular, the S100A4, S100A6, S100A8, and S100A14 to further understand their possible role(s) in OSCC tumorigenesis.

Mutations of the p53 gene in oral squamous-cell carcinomas from sudanese dippers of nitrosamine-rich toombak and non-snuff-dippers from the Sudan and Scandinavia

Using PCR‐SSCP/DNA sequencing methods, we analyzed 14 oral squamous‐cell carcinomas (OSCCs) and 8 pre‐malignant oral lesions from different Sudanese patients for prevalence of mutations in exons 5 to 9 of the p53 gene in relation to toombak‐dipping status. OSCCs (14 from Sudan, 28 from Scandinavia), and 3 pre‐malignant oral lesions from Sudanese non‐dippers were used as controls. A statistically significant increased incidence in mutations of the p53 gene was found in OSCCs from toombak dippers (93%; 13/14), as compared with those from non‐dippers in Sudan (57%; 8/14) and in Scandinavia (61%; 17/28) respectively. In OSCCs from dippers, mutations were found in exons 5 to 9, while in those from non‐dippers they were found in exons 5, 7, 8, 9, and no mutations were found in exon 8 in any of the OSCCs from Sudan. Certain types of mutations, however, were similar with respect to exposure to toombak. OSCCs from dippers showed 15 transversions, 9 transitions, 3 insertions and one deletion, compared with 7 transversions, 2 transitions and one deletion found in OSCCs from Sudanese non‐dippers, and 9 transversions, 17 transitions and 2 insertions found in those from non‐dippers in Scandinavia. No mutations were found in any of the non‐malignant oral lesions in relation to dipping or non‐dipping status. These findings suggest that (i) the use of toombak plays a significant role in induction of increased p53 gene mutations, (ii) mutations observed were similar to those induced by tobacco‐specific N‐nitrosamines (TSNAs) in experimental animal models and those already reported in toombak dippers, (iii) types of mutations associated with TSNAs were similar in the exposed and the control groups, (iv) a novel mutation in exon 6 was found in the OSCCs from toombak dippers, (v) the p53 exons 5 (codon 130), 6 (codons 190, 216) and 7 (codons 229, 249, 252) mutations are probable hot spots for toombak‐related OSCCs. Further studies are necessary to validate the increased incidence and exon locations of the p53‐gene mutations as a biomarker of malignant transformation in populations in which the oral use of tobacco is habitual. Int. J. Cancer 81:527–534, 1999.

Gene expression profile in oral squamous cell carcinomas and matching normal oral mucosal tissues from black Africans and white Caucasians: the case of the Sudan vs. Norway.

Expression profile of 588 known genes relating to tumour biology, was examined between oral squamous cell carcinomas (OSCCs) and matching normal oral mucosal tissues (NOMTs) obtained from Sudanese (n=11) and Norwegian (n=11) patients. cDNA probes were synthesised from total RNA and hybridised with the Atlas human cancer cDNA expression array membranes. RT-PCR and immunohistochemistry were applied to confirm the expression pattern of a subset of the 588 genes. Differences in expression of the genes examined were found between the OSCCs and the NOMTs on the Atlas membranes. Several of these genes were either up- or down-regulated 1.6-fold or higher in the OSCCs compared to the NOMTs in the cases from the two populations. We found that 181 (31%) and 195 (33%) genes were either up-regulated or down-regulated in the OSCCs from the Sudan and Norway, respectively. From the total number of genes (n=376) found expressed in the OSCCs investigated from the two countries, 53 genes (14%) showed common expression profile [35 (66%) were up-regulated and 18 (34%) were down-regulated] and 70 genes (19%) showed opposite regulation status. Results of the RT-PCR and immunohistochemistry confirmed the hybridisation data. These findings may provide an OSCCs-specific gene expression profile in patients from the two countries, suggesting that alterations of 123 genes are common in these OSCCs regardless of ethnic differences or other socio-cultural risk factors between the patients from the two countries. The findings might further suggest that specific genes are frequently involved in these OSCCs, which may provide novel clues as diagnostic, prognostic biomarkers and/or targets for therapy. The Atlas human cancer cDNA expression array technique can be useful to examine and describe the expression profile of known genes frequently involved in OSCCs from different populations.

Polyester copolymer scaffolds enhance expression of bone markers in osteoblast‐like cells

In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering.

S100A14 inhibits proliferation of oral carcinoma derived cells through G1-arrest.

Altered expression of S100A14 has been reported in various human cancers including oral squamous cell carcinomas (OSCCs). Its biological functions in carcinogenesis, however, are largely unknown. This study aimed to investigate the functional role of S100A14 in tumor cell proliferation and its possible functional association with p53. S100A14 protein was found to be gradually down-regulated during the transition from normal to dysplastic and carcinoma cells in an in vitro human OSCC progression model. When over-expressed by employing retroviral expression vector, S100A14 inhibited proliferation of CaLH3 and OSCC1, OSCC cell-lines harboring wild type (wt) p53, by inducing G1-arrest. This G1-arrest correlated with up-regulation of p21 both in the CaLH3 and OSCC1 cell-lines. shRNA mediated silencing of p53 led to partial suppression of p21 in S100A14 over-expressing CaLH3 cells, indicating that p21 up-regulation was, at least, partly dependent on p53. We further demonstrated that nuclear accumulation of p53 occurred with over-expression of S100A14 in CaLH3 cells. Our data suggest a novel role of S100A14 in OSCC cell proliferation by inducing G1-arrest and also indicate a functional link between S100A14 and the tumor suppressor protein p53.