Chamsai Pientong

Bioactive constituents from the leaves of Clinacanthus nutans Lindau

Three chlorophyll derivatives (phaeophytins) were isolated from the chloroform extract of Clinacanthus nutans Lindau leaves by means of chromatographic techniques and bioactivity-guided fractionation to give three pure compounds. Structure elucidation of the isolated compounds was carried out on the basis of spectral analyses. Three of these were known compounds with structures related to chlorophyll a and chlorophyll b namely 13(2)-hydroxy-(13(2)-R)-phaeophytin b, 13(2)-hydroxy-(13(2)-S)-phaeophytin a and 13(2)-hydroxy-(13(2)-R)-phaeophytin a. These compounds, which have not previously been reported in this plant, were shown to have anti-herpes simplex activity. They exhibited anti-HSV-1F activity at subtoxic concentrations. Their inhibitory activity affected the virus before viral entry to the host cells. This effect might be virucidal or interference with viral adsorption or penetration.

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

Enhanced expression of the HPV 16 E6‐E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6‐E7 genes. It binds to four E2 binding sites (E2BSs 1–4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6‐E7 oncogenes. In the majority of HPV‐transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra‐chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1–4 of the HPV 16 URR in a series of 18 HPV16‐positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.

E2 proteins of high risk human papillomaviruses down-modulate STING and IFN-κ transcription in keratinocytes.

In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.

Risk factors for cholangiocarcinoma in high-risk area of Thailand: Role of lifestyle, diet and methylenetetrahydrofolate reductase polymorphisms

BACKGROUND AND AIM Cholangiocarcinoma (CCA) is the most common cancer in Northeast Thailand. Endemicity of Opisthorchis viverrini (OV) - a known carcinogen - is responsible, but although infection is very common, the lifetime risk of CCA is only 5%. Other co-factors must exist, including aspects of lifestyle or diet along with variations in genetic susceptibility to them. Change in methylenetetrahydrofolate reductase (MTHFR) activity may influence both DNA methylation and synthesis. This study investigates risk factors for CCA with a focus on lifestyle, diet and MTHFR polymorphisms. METHODS Nested case-control study within cohort study was conducted. 219 subjects with primary CCA were each matched with two non-cancer controls from the same cohort on sex, age at recruitment and presence/absence of OV eggs in stool. Lifestyle and dietary data were obtained at recruitment. MTHFR polymorphisms were analyzed using PCR with high resolution melting analysis. The associations were assessed using conditional logistic regression. RESULTS Consumption of alcohol, raw freshwater fish and beef sausage increased the risk of CCA, while fruit and/or vegetables consumption reduced risk. There were interactions between MTHFR and preserved freshwater fish and beef. These dietary items are either a source of OV or of pre-formed nitrosamine, folate and antioxidants that are of possible relevance in OV carcinogenesis. CONCLUSIONS Primary prevention of CCA in high-risk population is based upon efforts to reduce OV infection. Reduced consumption of alcohol and preserved meats, and increased consumption of dietary folate, actions with a wider preventive potential, may also help in the reduction of CCA burden.

Association of human papillomavirus type 16 long control region mutation and cervical cancer

BackgroundThe variation of human papillomavirus (HPV) genes or HPV variants demonstrates different risks of cervical cancer. Mutation in the long control region (LCR) at YY1-motifs is one of the mechanisms for enhancing viral oncogene expression during the course of cancer cell progression. In Thai women, cervical cancers are almost always associated with HPV16 variant sub-lineage Asian (HPV16As); however, the mechanism involved remains elusive. The aim of this study was to understand further the oncogenic potential of HPV16As.MethodsA total of 82 HPV16-positive specimens from Thai women were selected from formalin-fixed paraffin-embedded cervical tissues, and the full length E6 gene of each specimen was amplified and sequenced. LCRs of the HPV16As-positive cases were amplified and sequenced to analyze their polymorphisms. Transcriptional activities of the HPV16As LCRs were then compared with sub-lineage European (EUR), sub-lineage Asian-American 1 (AA1) and HPV16 prototype by insertion of the LCRs into the pGL3-Basic vector.ResultsThe HPV16 DNA sequences were classified as HPV16 prototype (18.3%), Asian (As, 61%), Asian American-1 (AA1, 8.5%), European (EUR, 7.3%), Asian African-2 (AFR2, 3.7%) and Java-135C (J135C, 1.2%). The prevalence of HPV16As was 30% in low-grade squamous intraepithelial lesion (LSIL), while that in high-grade squamous intraepithelial lesion (HSIL) and squamous cell cervical carcinoma (SCC) were 63.9% and 66.7%, respectively, which demonstrates a significant association of HPV16As with the disease severity. LCR polymorphisms from 43 HPV16As positive cases were analyzed by PCR-sequencing. Thirty-eight nucleotide variation positions spanned nucleotide positions 7157–82. Ten new mutations found in the HPV16As LCRs were located predominantly at the enhancer and proximal to the 3’-end of the early promoter. The LCRs of the common HPV16As, EUR and AA1 showed 5, 13 and 23-fold higher activity than the HPV16 prototype LCR, while those of the new nucleotide variations of As showed 19 (As-sv1) and 30 (As-sv14) -fold higher activity than the HPV16 prototype.ConclusionsHPV16As DNA sequence variation, especially at the proximal to early promoter in the LCR, enhances transcriptional activity. This could be one of the possible mechanisms for HPV16As-associated cervical cancer development.

Human Papillomavirus Genotypes and Cervical Cancer in Northeast Thailand

Human papillomavirus (HPV) is a major cause of cervical cancer. More than 100 HPV genotypes have been identified; however the distribution varies geographically and according to ethnicity. The purpose of this study was to investigate the prevalence and distribution of HPV subtypes among Northeast Thai women. Subjects included 198 cases of SCCA and 198 age-matched, healthy controls. HPV-DNA was amplified by PCR using the consensus primers GP5+/6+ system followed by reverse line blot hybridization genotyping. The prevalence of high-risk HPV infection was 21 (10.1%) and 152 (76.8%) in the controls and in the cases, respectively. High-risk HPV significantly increased the risk for cervical cancer with an OR of 42.4 (95%CI: 22.4-81.4, p<0.001) and an adjusted OR of 40.7-fold (95%CI: 21.5-76.8, p <0.001). HPV-16 was the most prevalent HPV type in the SCCA (56.2%) followed by HPV-58 (17.8%) and HPV-18 (13.6%); whereas HPV-58 (46.4%) was a prominent genotype in the controls followed by HPV-16 (39.3%) and unidentified HPV types (25.0%). These findings indicate that HPV infection remains a critical risk factor for SCCA; particularly, HPV-16, HPV-58 and HPV-18. In order to eradicate cervical cancer, sustained health education, promoted use of prophylactics and a HPV-58 vaccine should be introduced in this region.

Risk Factors for Colon Cancer in Northeastern Thailand : Interaction of MTHFR Codon 677 and 1298 Genotypes with Environmental Factors

Background Polymorphisms in methylenetetrahydrofolate reductase (MTHFR), such as MTHFR C677T and A1298C, are associated with several cancers. This study aimed to evaluate the effects of MTHFR polymorphisms on colon cancer risk and possible interactions with environmental factors in a population from northeastern Thailand. Methods This hospital-based case–control study was conducted during 2002–2006; 130 colon cancer cases and 130 age- and sex-matched controls were enrolled. Information was collected and blood samples were obtained for assay of MTHFR C677T and A1298C polymorphisms by polymerase chain reaction with restriction fragment length polymorphism techniques. Associations between variables of interest and colon cancer were assessed using conditional logistic regression. Results Increased risk of colon cancer was associated with alcohol consumption and bowel habits. Alcohol drinkers who consumed ≤0.50 or >0.50 units of alcohol per day had elevated risks (ORadj = 3.5; 95% CI: 1.19–10.25 and ORadj = 1.71; 95% CI: 0.74–3.96, respectively). The risk was also higher in subjects with frequent constipation (11.69; 2.18–62.79) and occasional constipation (3.43; 1.72–6.82). An interaction was observed between the MTHFR C677T polymorphism and freshwater fish consumption on colon cancer risk (P value for interaction = 0.031). Interactions were observed between the MTHFR A1298C polymorphism and bowel habits, family history of cancer, alcohol consumption, and beef consumption on colon cancer risk (P-value for interaction = 0.0005, 0.007, 0.067, 0.003, respectively). Conclusions In a Thai population, colon cancer risk was associated with alcohol and beef consumption, bowel habits, and family history of cancer. Interactions between MTHFR polymorphisms and environmental factors were also observed.

The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection.

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.

Risk factors for cervical cancer in northeastern Thailand: detailed analyses of sexual and smoking behavior.

Cervical cancer is a serious public health problem in Thailand. We investigated possible risk factors for cervical cancer including HPV infection, p53 polymorphism, smoking and reproductive history among women in Northeast Thailand using a case control study with 177 cases and age-matched controls. Among the HPV carriers, a significantly increased risk for cervical cancer with an OR of 36.97 (p<0.001) and an adjusted OR of 38.07 (p<0.001) were observed. Early age at first sexual exposure, and multiple sexual partners increased the risk of cervical cancer with ORs ranging between 1.73-2.78 (p<0.05). The interval between menarche and first sexual intercourse<6 years resulted in a significant increase in the risk for cervical cancer with ORs ranging between 3.32-4.09 and the respective adjusted OR range for the 4-5 and 2-3 year-old groups were 4.09 and 2.92. A higher risk was observed among subjects whose partner had smoking habits, whether currently or formerly; with respective ORs of 3.36 (p<0.001) and 2.17 (p<0.05); and respective adjusted ORs of 2.90 (p<0.05) and 3.55 (p<0.05). Other smoking characteristics of the partners including smoking duration≥20 years, number of cigarettes smokes≥20 pack-years and exposure time of the subject to passive smoking≥5 hrs per day were found to be statistically significant risks for cervical cancer with adjusted ORs of 3.75, 4.04 and 11.8, respectively. Our data suggest that the risk of cervical cancer in Thai women is substantially associated with smoking characteristics of the partner(s), the interval between menarche and first sexual intercourse as well as some other aspects of sexual behavior.

Prevalence of human papillomavirus type 16 and its variants in abnormal squamous cervical cells in Northeast Thailand.

OBJECTIVES To investigate the prevalence of HPV, HPV16, and HPV16 variants in scraped cervical cells cytologically diagnosed as normal cervical cell and in formalin-fixed, paraffin-embedded tissues of cervical intraepithelial neoplasia II-III and squamous cervical carcinoma in Northeast Thailand. METHODS All samples were subjected to PCR using consensus GP5+/GP6+ primers. HPV16 was genotyped by Southern blot hybridization and reverse line blot hybridization. The HPV16 E6 gene was amplified and sequenced. RESULTS HPV infections were found in 33.8% of normal cervical cells, 97.3% of cervical intraepithelial neoplasia II-III, and 100% of squamous cervical carcinomas. The prevalence of HPV16 increased significantly with histological grade (normal cervical cell, 16.7%; cervical intraepithelial neoplasia II-III, 38.9%; squamous cervical carcinoma, 75%). The most common variant found was the Asian (As) (58.7%) followed by the European (E) lineage (41.3%). The HPV16 As lineages showed a risk association in 73.9% of squamous cervical cancer and 57.1% of cervical intraepithelial neoplasia II-III, while no increased risk was observed in the E lineages. CONCLUSION Our study demonstrates that HPV16, in particular the As variant, was the major causative agent associated with cervical cancer in Northeast Thailand, and our study suggests that some mutations of the E6 gene in this variant, which leads to amino acid changes, may be more carcinogenic.