Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells.

Human papillomavirus (HPV) infection is a high-risk factor for cervical intraepithelial neoplasm (CIN) but the association between the quantitative HPV DNA load and the severity of CIN remains controversial. We conducted a community study to investigate the correlation between the two. Potential study subjects were selected through Pap smear screening in Kaohsiung County, Taiwan. Ninety-one subjects with either their first case of inflammation or zCIN1 by biopsy confirmation were assigned to a case group; 175 normal subjects with negative findings by Pap smears or biopsies were assigned to a control group. Cervical HPV load was detected with Hybrid Capture II assay for high-risk HPV infection, with nested PCR for high-and low-risk HPV infection, and with type-specific PCR for HPV type 16 (HPV-16). Individuals with positive high-risk HPV infection had an increased risk of developing CIN. Compared with HPVnegative subjects, the odds ratios were 32.2 [95% confidence interval (95% CI), 10.4-99.5] for subjects with CIN1, 37.2 (95% CI, 7.4-187.6) for subjects with CIN2, and 68.3 (95% CI, 14.1-328.5) for subjects with zCIN3 after adjusting for other confounding factors. The similar trend was also found among the HPV-16 -negative individuals. In addition, high-risk HPV DNA load levels were highly correlated with the different grades of CINs in the overall population (Spearman's correlation coefficient r = 0.67, P < 0.0001, n = 266) and the HPV-16 -negative population (Spearman's correlation coefficient r = 0.58, P < 0.0001, n = 234). We concluded that high-risk HPV infection, irrespective of HPV-16 infection, was highly and positively associated with the development of CIN. (Cancer Epidemiol Biomarkers Prev 2005;14(11):2544 -9)

Although cooking emission from high-temperature frying has been deemed a Group 2A carcinogen by the International Agency for Research on Cancer, little is known about its impact on cervical tumorigenesis. To investigate the precancerous consequence of cooking oil fumes on cervical intraepithelial neoplasm (CIN), a community-based case-control study, which takes all known risk factors into consideration, was conducted in Taiwan. From 2003 to 2008, in a Pap smear screening and biopsy examination network, 206 pathology-verified women with inflammations/atypical squamous cells of undetermined significance or CIN grade-1 (CIN1) and 73 with CIN223 (defined as low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous intraepithelial lesions (HGSIL), respectively); and 1,200 area-and-age-matched controls with negative cytology were recruited. Multinomial logistic regression was applied in the multivariate analysis to determine the likelihood of contracting LGSIL or HGSIL. The risks of the two lesions increased with the increase of carcinogenic high-risk human papillomavirus DNA load, with a clear dose-response relationship. Chefs were observed to experience a 7.9-fold elevated HGSIL risk. Kitchens with poor fume ventilation during the main cooking life-stage correlated to a 3.7-fold risk of HGSIL, but not for LGSIL. More than 1 hr of daily cooking in kitchens with poor fume conditions appeared to confer an 8.4-fold HGSIL risk, with an 8.3-fold heterogeneously higher odds ratio than that (aOR 5 1.0) for LGSIL. Similar risk pattern has been reproduced among never-smoking women. Our findings demonstrate the association between indoor exposure to cooking fumes from heated oil and the late development of cervical precancerous lesions. This final conclusion needs to be verified by future research.

Cervical cancer screening guidelines do not comprehensively define what constitutes high risk. This study developed and validated simple risk-scoring schemes to improve Papanicolaou smear screening for women at high risk. Four cumulative risk score (CRS) schemes were derived respectively for the development of cervical intraepithelial neoplasia grade 1 (CIN1) and grade 2 or worse (CIN21) using community-based case-control data (n 5 1523). By calculating the area under the receiver operating characteristic (AU-ROC) curve, these schemes were validated in a Papanicolaou smear follow-up cohort (n 5 967) and a hospital-based cytology screening population (n 5 217). A high DNA load of high-risk human papillomavirus (HR-HPV) was the main predictor for CIN1 and CIN21, although age, married status combined with the number of sexual partners, active and passive smoking and age at sexual debut also affected associated lesions. In the training set, only the HPVtesting-contained CIN21 CRS scheme presented an excellent discrimination for identifying CIN21 (AU-ROC 5 0.866). Using a CRS cutoff value of 4 to identify CIN21, the sensitivity and specificity of predicting CIN21 for the 3-and 5-year follow-ups were 100% and 90.8%, and 83.3% and 90.4%, respectively, in the validation cohort. In the hospital-based validation population, the CRS scheme showed comparable discrimination for CIN21 detection (sensitivity 88.2% and specificity 84.6%). Women with CRS 4 had a 5.4% and 9.1% of 3-and 5-year cumulative incidence, respectively, and a 40.5-fold hazard ratio of developing CIN21. In conclusion, combined with HR-HPV testing and verified risk factors, a simple CRS scheme could effectively improve the implementation of CIN21 screening.Carcinoma of the cervix uteri was the third most common malignancy and the fourth leading cause of cancer death in women worldwide in 2008.1 In low-resource countries, this neoplasm is the principal cause of cancer deaths among women aged 30 to 50 years.2 Prevention efforts for this carcinoma have centered on screening and detecting women at risk using Papanicolaou cytologic examinations of cervical cells and treating related premalignant lesions. In countries with wide screening coverage at frequent intervals, the incidence of invasive cervical cancer has decreased appreciably.3 However, in many countries-particularly low-and middle-income developing countriesexisting programs resulted in limited benefits to morbidity and mortality of this cancer.4,5 A low cytologic screening rate (estimated at 19%, on average) has been specified as a major reason for this unsuccessful public health care in these areas. 5,6 Recent studies have reported that poor women with higher exposure to smoking, unsafe sex and other cervical cancer risk factors are less likely to receive effective screening.

Cervical cancer is one of the most common cancer among women. Early detection and prompt treatment would be the best management options. DNA methylation is one of the epigenetic regulation mechanisms leading to gene silencing in neoplastic cells. Silencing of tumor suppressor gene resulted from aberrant methylation has been detected in a majority of human cancers. Here, we applied methylation-specific polymerase chain reaction (MSP) to examine the methylation state of three tumor suppressor genes (rar-beta, p16 and cdh1) and an inflammatory-related cox-2 gene in different stages of cervical intraepithelial neoplasia (CIN). Our analyses revealed that cox-2 gene is in unmethylated form from CIN I to carcinoma specimens. Besides, no significant increase in methylation levels of rar-beta and cdh1 genes was observed. However, the high frequency of aberrant hypermethylation of p16 gene (13.2% in normal specimens ; 18.2% in CIN I; 35.7% in CIN II; 31.6% in CIN III and 15.4% in carcinoma) suggesting that p16 is progressively increased during the development of malignant stages in cervical intraepithelial neoplasia, especially in the absence of HPV infection. The result of bisulfite sequencing indicated that the 10 CpG sites of p16 gene are all methylated in ten individuals. In conclusion, this study identifies promoter methylation analysis of p16 on cervical cell specimens may be an additional tool for current cervical cytomorphology based screening. Citation Format: Sheng Hsiung Lin, Chun Chieh Yu, Ming Tsang Wu, Ruei Nian Li. Promoter methylation status of tumor suppressor genes and an inflammatory-related COX-2 gene in cervical intraepithelial neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B98.