Search

Background and Objectives : Cervical cancer is one of the most common types of frequently found cancers in Thailand. One of the causative agents is the infection of the high-risk human papillomavirus (HPV) type 16 and 18. Traditional medicines are rich sources of bioactive compounds which are a valuable source for the development of novel cancer therapies. In this study, the therapeutic effects of 3 traditional medicines, Kerra TM , KS TM , and Minoza TM , were studied on HeLa and CaSki cells. Materials and Methods : The effects of Kerra TM , KS TM , and Minoza TM on cancer cells were evaluated through cytotoxicity and cell death assays. The infection assay using HPV-16 pseudovirus was also carried out. Results : All traditional medicines efficiently suppressed cell growths of HeLa and CaSki, with Kerra TM being the most potent anticancer agent followed by KS TM and Minoza TM . Kerra TM at 158 µg/mL and 261 µg/mL significantly increases the percentage inhibition of the HPV-16 pseudovirus infection in a pre-attachment step in a dose-dependent manner, while KS TM at 261 µg/mL efficiently inhibited viral infection in both pre-attachment and adsorption steps. However, Kerra TM , KS TM , and Minoza TM at subtoxic concentrations could not reduce the viral E6 mRNA expressions of HPV-16 and HPV-18. Cell death assay by acridine orange/ethidium bromide showed that Kerra TM increased population of dead cells in dose-dependent manner in both CaSki and HeLa. The percentage of secondary necrosis in Kerra TM -treated CaSki was higher than that of HeLa cells, while the percentage of late apoptotic cells in HeLa was higher than that of CaSki, indicating that HeLa was more susceptible to Kerra TM than CaSki. For KS TM and Minoza TM , these extracts at 250 µg/mL promoted autophagy over cell death. At 500 µg/mL, the percentage of dead cells in Kerra TM was higher than that of KS TM and Minoza TM . Conclusions : Kerra TM is a potent traditional medicine for promoting cancer cell death. Kerra TM is possibly useful in the prevention and treatment of cervical cancer. Further investigation will be carried out to gain a better understanding of the biochemical mechanism and the pharmacological activity underlying this effect.

Deregulated expression of viral E6 and E7 genes often caused by viral genome integration of high-risk human papillomaviruses (HR-HPVs) into host DNA and additional host genetic alterations are thought to be required for the development of cervical cancer. However, approximately 15% of invasive cervical cancer specimens contain only episomal HPV genomes. In this study, we investigated the tumorigenic potential of human cervical keratinocytes harboring only the episomal form of HPV16 (HCK1T/16epi). We found that the HPV16 episomal form is sufficient for promoting cell proliferation and colony formation of parental HCK1T cells. Ectopic expression of host oncogenes, MYC and PIK3CAE545K, enhanced clonogenic growth of both early- and late-passage HCK1T/16epi cells, but conferred tumor-initiating ability only to late-passage HCK1T/16epi cells. Interestingly, the expression levels of E6 and E7 were rather lower in late-passage than in early-passage cells. Moreover, additional introduction of a constitutively active MEK1 (MEK1DD) and/or KRASG12V into HCK1T/16epi cells resulted in generation of highly potent tumor-initiating cells. Thus an in vitro model for progression of cervical neoplasia with episomal HPV16 was established. In the model, constitutively active mutation of PIK3CA, PIK3CAE545K, and overexpression of MYC, in the cells with episomal HPV16 genome were not sufficient, but an additional event such as activation of the RAS-MEK pathway was required for progression to tumorigenicity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wongjampa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Cervical cancer screening can effectively reduce new cervical cancer cases, including in Thailand. The abnormal results are subsequently referred for colposcopy. To avoid unnecessary colposcopy, an efficient triage is still needed for validation. This study aimed to investigate the overall positivity of cytology-based screening, HPV detection, and p16/Ki-67 dual staining and evaluate different triage strategies for predictive diagnosis of abnormal cervical lesions in northeastern Thailand., Methods: Cervical cells were collected from 191 women who came for cervical screening in the gynecological outpatient department during March 2019-February 2020. Pap smear samples were classified into 6 groups including 17 atypical glandular cells (AGC), 21 atypical squamous cells of undetermined significance (ASC-US), 7 atypical squamous cells - cannot exclude HSIL (ASC-H), 26 low-grade squamous intraepithelial lesions (LSILs), 19 high-grade SILs (HSILs) and 101 no squamous intraepithelial lesion (noSIL). Polymerase chain reaction (PCR) was performed for HPV DNA detection. HPV genotyping was determined by reverse line blot hybridization. P16/Ki-67 dual staining was performed by using CINtec PLUS Cytology kit. Biopsies from abnormal screening were collected for surgical pathology classification., Results: High-risk HPV (HR-HPV) infection was 2.97%, 29.41%, 38.10%, 57.14%, 46.15% and 84.21% in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL cytology respectively. P16/ Ki-67 in noSIL, AGC, ASC-US, ASC-H, LSIL and HSIL was 0.99%, 5.88%, 9.52%, 42.86%, 26.92% and 63.16%, respectively (P-value < 0.001). Among p16/Ki-67 positive cases, 96.15% (25/26) were infected with HPV and 84.62% (22/26) were HR-HPV. The overall positivity of each and co-testing between cytology or HPV DNA testing or p16/Ki-67 dual staining was evaluated. In each cervical lesion, primary HPV DNA testing showed the highest sensitivity, but low specificity. The combined all HPV/HR-HPV with p16/Ki-67 detection increased the specificity of abnormal cervical lesions., Conclusion: P16/Ki-67 dual stain cytology in HPV-positive women performs well for diagnosis of abnormal cervical lesions and should be considered for management of HPV-positive women to avoid unnecessary colposcopy referrals.

Objective: We investigated human papillomavirus (HPV) infection and detected anal squamous intraepithelial lesions by modified liquid-based cytology (LBC) and p16/Ki67 dual-staining., Methods: Anal swabs (n=393) were collected from patients with HIV infection. Anal cells were kept in 95% ethyl alcohol for modified LBC. DNA was extracted from cells for HPV detection and genotyping using real-time PCR and reverse line blot hybridization., Results: Nine samples (2.3%) were unsatisfactory specimens, 74.8% (294/393) were negative for intraepithelial malignancies (NILM) and 22.9% (90/393) exhibited squamous intraepithelial lesions (SIL). In the latter category, 13.7% of samples (54/393) contained atypical squamous cells of undetermined significance (ASCUS), 6.9% (27/393) were classified as low-grade SIL (LSIL) and 2.3% (9/393) as high-grade SIL (HSIL). A total of 331 from 393 swab samples were suitable for detection of HPV infection. Among these, 34.1% (113/331) were positive. HPV 58 (15.9%) was the most common genotype, followed by HPV 18 (14.2%) and HPV 16 (11.5%). The severity of abnormal cells was significantly associated with HPV infection. Dual staining with p16/Ki-67 was performed on 130 samples: in 30.8% (40/130) of samples positive staining was significantly associated with severity of abnormal cells. Agreement between cytology, p16/Ki67 dual-staining and high-risk HPV detection was 100% in HSIL samples. Interestingly, eight apparently NIML cases might have contained abnormal cells, since they were positive by both p16/Ki67 dual-staining and high-risk HPV detection., Conclusion: Anal specimens screened using modified LBC with 95% ethyl alcohol solution as the fixative are suitable for screening anal precancerous lesions by cytology, HPV testing and p16/Ki-67 dual staining.

Alterations of the P53 gene and human papillomavirus (HPV) infection are associated with development of oral squamous cell carcinoma (OSCC). We aimed to identify mutation of P53 exon 8 codon 282 in OSCC and correlate these with HPV infection as well as histopathological grade of OSCC. Samples of known HPV infection status were studied including oral lesion cells, formalin-fixed paraffin embedded (FFPE) tissues from OSCC and exfoliated oral cells of matched age-sex controls. P53 exon 8 mutation was detected using the polymerase chain reaction (PCR). Mutation of codon 282 was identified by allele-specific oligonucleotide typing (ASO) using EvaGreen real-time PCR. The PCR products were analyzed by gel electrophoresis and melting curve analysis. Mutation of P53 exon 8 was seen in 81.7% and 69.6% of FFPE OSCC tissues and oral lesion cells, respectively. This was significantly higher than in controls (16.7%). Frequency of mutation did not differ between HPV-positive samples (62.5% and 81.8% in oral lesion cells and FFPE tissue samples, respectively) and HPV-negative samples (73.3% and 81.5% in oral lesion cells and FFPE tissue samples, respectively). This finding is similar to P53 codon 282 mutation that was found only in FFPE tissues (35.0%) and oral lesion cells (32.6%) from both HPV-positive and negative OSCC. Interestingly, frequency of mutation was higher in well-differentiated OSCC with HPV-infection (28.1%) than without HPV (14.8%). This result demonstrated a mutation hot spot in P53 associated with oral carcinogenesis and might be useful to guide chemotherapeutic modality for HPV-associated OSCC in northeast Thailand.

Objective: High-risk human papillomavirus (HR HPV) was associated with the development of cervical cancer. Asymptomatic Chlamydia trachomatis (C. trachomatis) infection is the most common bacterial, sexually-transmitted infection. This study aimed to investigate the association of C. trachomatis in positive HR HPV and the cytological results from liquid-based cytology (LBC)., Methods: 150 residual LBC specimens were collected; all of which had undergone cytology and HPV testing by Cobas. The samples were established as C. trachomatis using real-time PCR (RT-PCR) with Cryptic F/Cryptic R primers., Results: Of 150 positive HPV findings, the most common (72.7%, 109/150) were the 12 other HR HPVs (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The cervical cytology of those positive HR HPVs were mostly negative (70.0%, 105/150).  The C. trachomatis infections in positive HR HPV were 16% (24/150) HPV. The analysis of the abnormal cytology revealed that 41.6% had C. trachomatis co-infection (C. trachomatis and HPV infection) viz., LSIL (20.8%), HSIL (12.5%), and ASC-US (8.3%). A comparison with positive HPV without C. trachomatis co-infection revealed that the highest prevalence was for LSIL, ASC-US, and HSIL (11.1%, 10.3%, and 6.4%, respectively). There was no difference between the abnormalities and negative cervical cytology with negative and positive C. trachomatis co-infection in HR HPV positive (p = 0.174)., Conclusion: C. trachomatis infection was not significantly associated HR-HPV and abnormal cytology. This study confirms the increasing rate of C. trachomatis infection in asymptomatic women so routine screening for these infections has been suggested to (a) prevent complications such as the chronic pelvic pain associated with prolong infection and (b) reduce sexual transmission of the infection.

Background: The incidence of oral cancers associated with human papillomavirus (HPV) has been increasing in recent years. Therefore, it is necessary to elucidate HPV prevalence in oral cells and exposure to risk factors in various age groups., Methods: Oral rinse samples from healthy individuals in northern Thailand were investigated for HPV prevalence and genotyped using the polymerase chain reaction (GP5+/6+ primers) and DNA sequencing of the PCR products., Results: Samples were collected from 594 participants between 4 and 60 years of age. HPV was detected in 3.7% of samples. The prevalence of HPV-positive cases was 8.6% in the 31-50 age group. HPV prevalence increased with age and was the highest (9.2%) in the 41-50 age group, but decreased (to 3%) in the 51-60 age group. Risk factors significantly associated with HPV-positive cases included alcohol consumption, coffee drinking, sexual activity, and having children. HPV 16 and 18 were common genotypes, especially in the 31-50 age group, and were associated with having sexual activity (odds ratio 19.0 [95% CI: 2.5-142.5]). At follow-up of some individuals in the 4-10 age group, a 9-year-old child was found to be positive for HPV18., Conclusions: These results suggest that HPV can be acquired at a young age and the prevalence peaks in the middle age class among healthy individuals in northern Thailand, especially in the 31-50 age group., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Human papillomavirus (HPV), Epstein-Barr virus (EBV) and herpes simplex virus (HSV) cause sexually transmitted diseases (STDs) that are frequently found in men who have sex with men (MSM) with human immunodeficiency viral (HIV) infection., Methods: This study investigated the prevalence of infection and anatomical site distribution of these viruses in asymptomatic MSM. DNA, extracted from cells collected from the anorectum, oropharynx and urethra of 346 participants, was investigated for the presence of EBV, HPV and HSV using real-time PCR. Demographic data from the participants were analyzed., Results: All three viruses were found in all sampled sites. EBV was the commonest virus, being detected in the anorectum (47.7% of participants), oropharynx (50.6%) and urethra (45.6%). HPV and HSV were found in 43.9% and 2.9% of anorectum samples, 13.8% and 3.8% of oropharynx samples and 25.7% and 2% of urethra samples, respectively. HPV infection of the anorectum was significantly associated with age groups 21-30 (odds = 3.043, 95% CI = 1.643-5.638 and P = 0.001) and 46-60 years (odds = 2.679, 95% CI = 1.406-5.101 and P = 0.03). EBV infection of the urethra was significantly correlated with age group 21-30 years (odds = 1.790, 95% CI = 1.010-3.173 and P = 0.046). EBV/HPV co-infection of the anorectum (odds = 3.211, 95% CI = 1.271-8.110, P = 0.014) and urethra (odds = 2.816, 95% CI = 1.024-7.740, P = 0.045) was also associated with this age group. Among HIV-positive MSM, there was a significant association between age-group (odds = 21.000, 95% CI = 1.777-248.103, P = 0.016) in HPV infection of the anorectum. A failure to use condoms was significantly associated with HPV infection of the anorectum (odds = 4.095, 95% CI = 1.404-11.943, P = 0.010) and urethra (odds = 7.187, 95% CI = 1.385-37.306, P = 0.019). Similarly, lack of condom use was significantly associated with EBV infection of the urethra (odds = 7.368, 95% CI = 1.580-34.371, P = 0.011)., Conclusion: These results indicate that asymptomatic MSM in Northeast Thailand form a potential reservoir for transmission of STDs, and in particular for these viruses.

Human papillomavirus (HPV) E2 and L1 proteins are expressed in cervical cells during the lytic stage of infection. Overexpression of p16 INK4A is a biomarker of HPV-associated cervical neoplasia. This study investigated antibodies to HPV16 E2, HPV16 L1, and p16 INK4A in sera from women with no squamous intraepithelial lesion (No-SIL) of the cervix, low-grade SIL, high-grade SIL, and cervical squamous cell carcinoma (SCC). HPV DNA was detected by polymerase chain reaction. Anti-E2, -L1, and -p16 INK4A antibodies in sera were determined by western blot. Among 116 samples, 69 (60%) were HPV DNA-positive. Percentages seropositive for anti-E2, -L1, and -p16 INK4A antibodies were 39.6, 22.4, and 23.3%, respectively. Anti-E2 antibody was significantly correlated with HPV DNA-positive cases. Eighty-seven women (75%) were regarded as infected with HPV, having at least one positive result from HPV DNA, L1, or E2 antibody. Antibody to p16 INK4A was associated with HPV infection (odds = 5.444, 95% CI 1.203-24.629, P = 0.028) and precancerous cervical lesions (odds = 5.132, 95% CI 1.604-16.415, P = 0.006). Interestingly, the concurrent detection of anti-E2 and -p16 INK4A antibodies was significantly associated with HPV infection (odds = 1.382, 95% CI 1.228-1.555, P = 0.044). These antibodies might be good candidate biomarkers for monitoring HPV-associated cervical lesion development to cancer.

Human papillomavirus (HPV) infection is associated with several genetic alterations including oncogene amplification, leading to increased aggression of tumors. Recently, a relationship between HPV infection and oncogene amplification has been reported, but this finding remains controversial. This study therefore investigated relationships between HPV infection and amplification of genes in the epidermal growth factor receptor (EGFR) signaling cascade in oral squamous cell carcinoma (OSCC). Extracted DNA from 142 formalin-fixed paraffin-embedded (FFPE) OSCC tissues was performed to investigate the copy number of EGFR, KRAS, c-myc and cyclin D1 genes using real-time polymerase chain reaction (RT-PCR) and compared with calibrators. A tissue microarray of OSCC tissues was used for detection of c-Myc expression and HPV infection by immunohistochemistry and HPV E6/E7 RNA in situ hybridization, respectively. HPV infection was also investigated using PCR and RT-PCR. Of the 142 OSCC samples, 81 (57%) were HPV-infected cases. The most frequently amplified gene was c-myc (55.6%), followed by cyclin D1 (26.1%), EGFR (23.9%) and KRAS (19.7%). Amplification of c-myc was significantly associated with levels of its protein product. EGFR amplification was also significantly associated with amplification of genes in the signaling cascade: KRAS (50.0%), c-myc (34.2%) and cyclin D1 (46.0%). Interestingly, HPV infection was significantly associated with amplification of both EGFR (76.5%) and cyclin D1 (73.0%). Only cyclin D1 amplification was significantly associated with severity of OSCC histopathology. HPV infection may play an important synergistic role in amplification of genes in the EGFR signaling cascade, leading to increased aggression in oral malignancies.

Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a 5-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPV infection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand., (© 2016 Wiley Periodicals, Inc.)

Human papillomavirus (HPV) infection modulates several host cytokines contributing to cancer development. Oncostatin M (OSM), an IL-6 family cytokine, acts to promote cell senescence and inhibit growth. Its dysregulation promotes cell survival, cell proliferation and metastasis in various malignancies. The effect of HPV on OSM dysregulation has not been investigated. To elucidate this, immunohistochemistry was used on formalin-fixed, paraffin-embedded oral squamous cell carcinoma (OSCC) tissues: HPV-positive (50) and HPV-negative (50) cases. Immortalized human cervical keratinocytes expressing HPV16E6 (HCK1T, Tet-On system) were used to demonstrate the role of HPV16E6 in OSM expression. In addition, a vector containing HPV16E6/E7 was transiently transfected into oral cancer cell lines. Cell viability, cell-cycle progression and cell migration were evaluated using flow cytometry and a wound healing assay, respectively. The results showed various intensities of OSM expression in OSCC. Interestingly, the median percentages of strongly stained cells were significantly higher in HPV-positive OSCCs than in HPV-negative OSCCs. To explore the role of HPV oncoproteins on OSM expression, the expression of HPV16E6 in the HCK1T Tet-On condition was induced by doxycycline and HPV16E6 was found to significantly upregulate levels of OSM mRNA and protein, with concomitant upregulation of c-Myc. In addition, the levels of OSM mRNA and protein in E6/E7 transiently transfected oral cancer cells also gradually increased in a time-dependent manner and these transfected cells showed greater viability and higher migration rates and cell-cycle progression than controls. This result demonstrates that HPV16 oncoproteins upregulate OSM and play an important role to promote OSCC development.

Background: Persistent infection with EBV has been linked to the development of malignancies including HPV-associated cervical carcinoma. However, the role of EBV in HPV-associated cervical cancer is still poorly understood., Objective: To determine the possible contributing role of EBV in HPV-associated cervical carcinogenesis according to HPV genotypes, HPV genome status and EBV localization., Study Design: Cervical tissues, including 82 with no squamous intraepithelial lesions (noSILs), 85 low-grade SILs (LSILs), 85 high grade SILs (HSILs) and 40 squamous cell carcinoma samples (SCC) were investigated using PCR and dot blot hybridization for EBV detection and PCR and reverse line blot hybridization for HPV genotyping. The amplification of papillomavirus oncogene transcripts assay and in situ hybridization were used to determine HPV physical status and EBV EBER localization, respectively., Results: EBV was detected increasingly from noSIL (13.4%), LSIL (29.4%) to HSIL (49.4%) samples. The prevalence of HPV-EBV co-infection was significantly higher in any grade of lesion than in noSIL samples (p<0.05) including noSIL (1.2%; 95% confidence intervals [CI]=0.0-3.6%, relative risk [RR]=1), LSIL (18.8%, 95% CI=10.5-27.1%, RR=15.4), HSIL (41.2%, 95% CI=30.7-51.6%, RR=33.8) and SCC (30.0%, 95% CI=15.8-44.2%, RR=24.6). Interestingly, HPV-EBV co-infection was more common in cases with episomal forms of high-risk (HR) HPV whereas HPV alone was more common in cases with integrated HR-HPV. In addition, EBER staining demonstrated that EBV was mainly present in infiltrating lymphocytes., Conclusion: Infiltrating EBV-infected lymphocytes may play a role in cancer progression of cervical lesion containing episomal HR-HPV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNA-21 (miR-21) is recognized as an oncomir and shows up-regulation in many types of human malignancy. The aim of this study was to investigate the association of miR-21 expression associated with HPV infection in normal and abnormal cervical tissues. Cervical tissue samples with different cytological or histopathological grades were investigated for HPV by PCR and for miR-21 and programmed cell death, protein 4 (PDCD4) expression using quantitative real-time PCR (qRT-PCR). Laser capture microdissection (LCM) of stromal and epithelial tissues and in situ hybridization (ISH) using locked nucleic acid (LNA) probes were performed on a subset of fixed specimens. Cell line experiments were conducted on fibroblasts stimulated in culture media from HeLa cells, which were then assessed for miR-21, PDCD4, IL-6 and α-SMA expression by qRT-PCR. Twenty normal cervical cell, 12 cervicitis, 14 cervical intraepithelial neoplastic I (CIN I), 22 CIN II-III and 43 cervical squamous cell carcinoma (SCC) specimens were investigated. miR-21 levels were significantly lower in normal than in abnormal tissues. The expression of miR-21 in HPV negative normal cytology was significantly lower than in HPV positive samples in abnormal tissue and SCC. The miR-21 expression was significantly higher in HPV negative cervicitis than HPV negative normal cells. LCM and ISH data showed that miR-21 is primarily expressed in the tumor-associated stromal cell microenvironment. Fibroblasts treated with HeLa cell culture media showed up-regulated expression of miR-21, which correlated with increased expression of α-SMA and IL-6 and with down-regulation of PDCD4. These results demonstrate that miR-21 is associated with HPV infection and involved in cervical lesions as well as cervicitis and its up-regulation in tumor-stroma might be involved in the inflammation process and cervical cancer progression.

Andrographolide (Androg) has been reported to contain antiviral and antitumor activities, but the effects of Androg on human papillomavirus (HPV) infection and cervical cancer have not been elucidated. This study investigated the effects of Androg and its derivatives, namely, 14-deoxy-11,12-didehydroandrographolide (14-DDA) and 3,19-isopropylidene andrographolide (IPAD), on HPV16 pseudovirus (HPV16PsV) infectivity, HPV16 E6 oncogene expression and cervical cancer cell apoptosis. The result demonstrated that all compounds inhibited HPV16PsV infection and that 14-DDA showed the highest potency. Only Androg suppressed long control region (LCR) transcription activity of HPV16 in transiently transfected C33A cells and significantly inhibited E6 oncogene expression in SiHa cells in a dose-dependent manner. A twofold subcytotoxic concentration of IPAD exhibited an inhibitory effect on E6 oncogene expression at 48-h posttreatment. Interestingly, p53 protein was restored in a downstream process and was detected earlier by IPAD treatment than by Androg treatment. This result corresponded to the level of cell apoptosis and cell cycle arrest at the G2/M phase. E6 oncogene expression was also suppressed in CaSki cells treated with Androg and IPAD leading to cell apoptosis. These findings imply that Androg and its derivatives have different activities and may be effective agents for HPV prevention and cervical cancer treatment.

To understand the potential role in cervical cancer development of the three most common high-risk human papillomavirus (HR-HPVs) in Thai women, HPV genotypes and viral genome statuses in different cervical lesions were investigated. Cervical tissues consisting of no cervical intraepithelial neoplasia (84 cases), grade I cervical intraepithelial neoplasia (176 cases), grade II-III cervical intraepithelial neoplasia (91 cases), and squamous cell carcinoma (66 cases) were subjected for HPV genotyping by polymerase chain reaction (PCR) and reverse line blot hybridization assay and for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. HPV prevalence was 28.6% in no cervical intraepithelial neoplasia, 40.3% in grade I cervical intraepithelial neoplasia, 70.3% in grade II-III cervical intraepithelial neoplasia and 86.4% in squamous cell carcinoma cases. The three most common HR-HPV types were HPV 16, 58, and 18 which were distributed in all cervical lesions. HPV physical statuses could be investigated in 4 no cervical intraepithelial neoplasias, 2 grade I cervical intraepithelial neoplasias, 28 grade II-III cervical intraepithelial neoplasias and 31 squamous cell carcinomas. The integrated-derived transcripts were found 3.6% in grade II-III cervical intraepithelial neoplasia and 48.4% in squamous cell carcinoma, whereas no viral genome integration was found in the group of no cervical intraepithelial neoplasia or grade I cervical intraepithelial neoplasia samples. The frequencies of HR-HPV integration in squamous cell carcinoma were found 40%, 100%, 20% of HPV 16, 18, and 58. This study indicates the oncogenic potential ability of the three most common HR-HPVs associated with cervical cancer progression., (© 2014 Wiley Periodicals, Inc.)

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il- 10 and TNF-α expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-α mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

Minor trauma to the uterine cervix is supposed to induce local immunity to prevent cervical lesions caused by human papillomavirus (HPV) infection. This study aimed to investigate the local cervical immunity in women with low grade squamous intraepithelial lesion (LSIL) and effects of abrasion after cryosurgery or Pap smear. One hundred women with LSIL and known results of HPV detection were recruited. HPV positive women were randomly divided according to abrasion into cryotherapy and Pap smear observation groups. Cervical tissues and cervico-vaginal lavage (CVL) were collected at 6 and 12 months after allocation. The levels of cytokines at first recruitment were compared with cytokine levels at 6 months after abrasions. The mRNA of IFN-γ , TNF-α and IL-10 in cervical tissues and these cytokines secreted in CVL were determined using real time PCR and ELISA, respectively. Anti-HPV16 IgG and IgA antibodies in CVL were assessed by western blotting. At first recruitment of women with LSIL (100 cases), IL-10 mRNA and cytokine in HPV positive group (60 cases) was significantly higher than negative group (40 cases). IFN-γ and TNF-α mRNA level in both groups were comparable but their secretions in CVL were significantly increased in HPV negative group. After abrasion for 6 months in HPV-positive women, all mRNA and secreted cytokines were changed, but no significant difference was observed between cryotherapy and observation groups. When individuals were compared between first recruitment and after abrasion for 6 months, IFN-γ mRNA and anti-HPV16 L1 IgA antibodies were significantly increased in the cryotherapy group. The results suggest that modulation of local cervical immunities by abrasion might promote different effects in clearance of HPV-related cytological abnormalities.

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., (Copyright © 2012 UICC.)

HPV 58 is detected commonly in cervical cancer in East Asian countries. To evaluate the HPV 58 physical state, the amplification of papillomavirus oncogene transcripts (APOT) and hybridisation assays were established. Episome- and integrate-derived transcripts were confirmed by direct sequencing. Twenty-nine HPV 58 positive samples from various cervical lesions were used. The results showed that the episome-derived transcripts were recognised as two major specific amplified products (1040 and 714 bp). Two splice donor sites were mapped to the 5' splice site of the E1 gene on SD898 and SD899 and spliced to the 3' acceptor site of the E4 gene on SA3353, SA3356 and SA3365. The episome-derived transcripts were found 100% in normal cervical epithelia and low-grade lesions (9/9 cases) while the integrate-derived transcripts were detected in 13.3% of high-grade lesions (2/15 cases) and in 20% of carcinomas (1/5 cases). HPV 58 integration sites were found on chromosomes 4q21, 12q24 and 18q12. Using the established APOT assay, the results revealed not only novel information on the HPV 58 transcription patterns of episomal transcripts, but also integration site. The APOT assay is a reliable and useful tool for the detection of the HPV 58 physical state and its oncogene expression., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: The 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., Methods: A 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., Results: The 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., Conclusions: HPV16As 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.

Background: The pattern of infection in cervical lesions with respect to HPV subtype has not been systematically studied in Thai women. The aim here was to determine HPV prevalence, genotype, and infection pattern in cervical lesions and to estimate the potential efficacy of an HPV prophylactic vaccine., Design: Formalin-fixed paraffin-embedded cervical tissue blocks of 410 Thai patients from 8 institutes in 4 regions of Thailand (northern, southern, north-eastern, and central) were studied. The samples included 169 low grade squamous intraepithelial lesions (LSILs), 121 high grade squamous intraepithelial lesions (HSILs), and 120 squamous cell carcinomas (SCCs). HPV-DNA was amplified by PCR using consensus primers GP5+ and GP6+. The HPV genotype was then determined by reverse linear blot assay that included 37 HPV-specific 5'-amino-linked oligonucleotide probes. Patterns of infection were classified as single infection (one HPV type), double infection (two HPV types), and multiple infection (three or more HPV types)., Results: The mean age of the subjects was 42 years. The prevalence of HPV infection was 88.8%. The highest HPV prevalence was found in the southern region (97.1%) and the lowest in the central region (78.6%). HPV-DNA was detected in 84.6% of LSILs, 90.1% of HSILs, and 93.3% of SCCs. A total of 20 HPV genotypes were identified. The five most common high risk HPV were HPV16 (83.2%), HPV18 (59.3%), HPV58 (9.3%), HPV52 (4.1%), and HPV45 (3.8%). In double and multiple infection patterns, the most common genotypes were HPV16/18 (27.8%) and HPV11/16/18 (54.9%). HPV6 was found only in LSIL and never in combination with other subtypes. HPV11 was most common in LSIL., Conclusion: There is no difference of HPV type distribution in women from 4 regions of Thailand with prominent HPV16 and HPV18 in all cases. The bivalent and quadrivalent vaccines have the potential to prevent 48.6 % and 74.5% of cervical cancers in Thai women. The potential of cancer prevention would rise to 87.6% if other frequent HR-HPV types (HPV58, 52, and 45) were also targeted by an HPV vaccine.

Thailand is in the process of developing a national cervical screening program. This study examined p16INK4a staining and HPV prevalence in abnormal cervical samples with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL), to evaluate the efficacy of combined HPV and p16INK4a detection to predict CIN II-III. Totals of 125 ASCUS and 87 LSIL cases were re-evaluated by Pap test and cervical cells of ASCUS and LSIL cases were prepared on slides for p16INK4a detection by immunocytochemistry. HPV genotyping of DNA extracts was performed by GP5+/6+ PCR and reverse line blot hybridization. Histopathologic tests were performed to identify cervical lesion. Total of 212 cases were diagnosed to normal (20), ASCUS (112), LSIL (78) and HSIL (2). HPV was detected in ASCUS (49/112, 43.8%), LSIL (60/78, 76.9%) and HSIL (2/2, 100%) cases. The majority of HPV positive samples typed for high-risk HPV. 55.7% (107/192) of abnormal cases (ASCUS, LSIL and HSIL) were positive p16INK4a. For the 111 HPV DNA positive cases, 34 of 49 (69.4%) ASCUS cases and 49 of 60 (81.7%) LSIL cases were p16INK4a positive. 140 biopsies were taken and histological classified: CIN negative (65 cases), CIN I (56 cases) and CIN II-III (19 cases). HPV DNA detection predicted CIN II-III with sensitivity and specificity of 84% and 49%, whereas p16INK4a staining showed higher sensitivity (89.5%) and specificity (56.2%). The prediction of CIN II-III was significantly better by combination of positive HPV DNA and p16INK4a with 93.8% sensitivity and 59.2% specificity. Detection of HPV DNA combined with p16INK4a in cervical cells can predict CIN II-III and may improve the screening diagnosis of Thai women at risk for CIN II-III or cancer.

Objective: To compare the clearance rate of HPV infection among women aged older than 30 years with biopsy-confirmed cervical low-grade squamous intraepithelial lesions (LSIL) 1 year after cryotherapy with the spontaneous clearance rate (observation)., Method: HPV DNA typing by polymerase chain reaction and reverse line blot hybridization were used to identify 14 high-risk types and 23 low-risk types. HPV DNA sequencing was also used for other types., Result: Between December 2007 and March 2009, 100 women were recruited to the study and 60 cases had positive results on HPV testing. Twenty-nine patients were randomly allocated to the cryotherapy group and 31 to the observation group. At 1 year, 89.7% (26/29; 95% CI, 78.6-100%) of the cryotherapy group and 90.3% (28/31; 95% CI, 79.9-100%) of the observation group had negative results on HPV testing (0.6% difference; 95% CI, -15.8 to 14.6%, P=0.94)., Conclusion: Cryotherapy failed to increase the clearance of prevalent HPV infections among women with LSIL, although in both arms the clearance rates were above 80%. However, in coupling with visual inspection with acetic acid as a single visit approach, its effect on prevention of HSIL and cervical cancer is still promising. Therefore, cryotherapy should not be withdrawn from such programs., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

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.