Lung cancer has long been recognized as an important world heath concern due to its high incidence and death rate. The failure of treatment strategies, as well as the regrowth of the disease driven by cancer stem cells (CSCs) residing in the tumor, lead to the urgent need for a novel CSC-targeting therapy. Here, we utilized proteome alteration analysis and ectopic tumor xenografts to gain insight on how gigantol, a bibenzyl compound from orchid species, could attenuate CSCs and reduce tumor integrity. The proteomics revealed that gigantol affected several functional proteins influencing the properties of CSCs, especially cell proliferation and survival. Importantly, the PI3K/AKT/mTOR and JAK/STAT related pathways were found to be suppressed by gigantol, while the JNK signal was enhanced. The in vivo nude mice model confirmed that pretreatment of the cells with gigantol prior to a tumor becoming established could decrease the cell division and tumor maintenance. The results indicated that gigantol decreased the relative tumor weight with dramatically reduced tumor cell proliferation, as indicated by Ki-67 labeling. Although gigantol only slightly altered the epithelial-to-mesenchymal and angiogenesis statuses, the gigantol-treated group showed a dramatic loss of tumor integrity as compared with the well-grown tumor mass of the untreated control. This study reveals the effects of gigantol on tumor initiation, growth, and maintain in the scope that the cells at the first step of tumor initiation have lesser CSC property than the control untreated cells. This study reveals novel insights into the anti-tumor mechanisms of gigantol focused on CSC targeting and destabilizing tumor integrity via suppression of the PI3K/AKT/mTOR and JAK/STAT pathways. This data supports the potential of gigantol to be further developed as a drug for lung cancer.

Background: Mucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade. Methods: LINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation. Results: LINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001). Conclusions: Our findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction.

CCNA1 Promoter Methylation: a Potential Marker for Grading Papanicolaou Smear Cervical Squamous Intraepithelial Lesions CCNA1 promoter methylation;cervical cancer;HPV;duplex MS-PCR;Pap smear; Background: From our previous study, we established that cyclin A1 (CCNA1) promoter methylation is strongly correlated with multistep progression of HPV-associated cervical cancer, suggesting potential use as a diagnostic maker of disease. Objectives: The purpose of the present study was to assess the prevalence of CCNA1 promoter methylation in residual cervical cells isolated from liquid-based cytology that underwent hrHPV DNA screening for cervical cancer, and then to evaluate this marker for diagnostic accuracy using parameters like sensitivity, specificity, predictive values and likelihood ratio. Methods: In this retrospective study, histopathology was used as the gold standard method with specimens separated into the following groups: negative (n=31), low-grade squamous intraepithelial lesions (LSIL, n=34) and high-grade squamous intraepithelial lesions or worse (HSIL+, n=32). The hrHPV was detected by Hybrid Capture 2 (HC2) and CCNA1 promoter methylation was examined by CCNA1 duplex methylation specific PCR. Results: The results showed the frequencies of CCNA1 promoter methylation were 0%, 5.88% and 83.33%, while the percentages of hrHPV were 66.67%, 82.35% and 100% in the negative, LSIL and HSIL+ groups, respectively. Although hrHPV infection showed high frequency in all three groups, it could not differentiate between the different groups and grades of precancerous lesions. In contrast, CCNA1 promoter methylation clearly distinguished between negative/LSIL and HSIL+, with high levels of all statistic parameters. Conclusion: CCNA1 promoter methylation is a potential marker for distinguishing between histologic negative/LSIL and HSIL+using cervical cytology samples.

The incidence of oral squamous cell carcinoma (OSCC) continues to increase each year. Clinical examination and biopsy usually detect OSCC at an advanced stage that is difficult to treat, leading to poor prognosis. DNA methylation pattern is tissue specific and has emerged as a biomarker for the detection of cancers of tissue origin. Herein, we aimed to discover a novel site-specific methylation marker for OSCC. We selected OSCC datasets analyzed using the IlluminaHumanMethylation27 BeadChip from the Gene Expression Omnibus repository of the National Center for Biotechnology Information using a bioinformatics approach. From 27,578 CG dinucleotide (CpG) sites, the CpG site with the highest difference in methylation level between healthy and cancerous cells was selected for further validation. A total of 18 mucosal tissue samples were collected from nine healthy controls and nine from OSCC subjects and subjected to microdissection for cell purification, followed by DNA extraction, bisulfite conversion, and pyrosequencing. Additionally, epithelial cells were collected from 2 cohorts including oral rinse from healthy controls, oral rinse and oral swab from OSCC subjects and oral rinse from oropharyngeal squamous cell carcinoma (SCC) were examined for their methylation status using real-time polymerase chain reaction (PCR). Among the 27,578 differentially methylated CpG sites, cg01009664 of the thyrotropin-releasing hormone (TRH) gene showed the greatest difference in methylation level between healthy and cancerous cells. Validation of the TRH gene using pyrosequencing revealed a methylation percentage of 7% ± 3.43% in healthy cells in contrast to 63% ± 19.81% in cancerous cells. Screening of epithelial cells using real-time PCR showed that the DNA methylation level was significantly higher in oral swab and rinse samples collected from OSCC and oropharyngeal SCC subjects than those from healthy controls (p < 0.001). In addition, when using a cutoff at 3.31 ng/μL, the TRH methylation biomarker was able to distinguish OSCC and oropharyngeal SCC subjects from healthy controls with high level of area under the curve, sensitivity and specificity. We demonstrated cg01009664 of TRH as a potential biomarker for OSCC and oropharyngeal SCC screening using oral rinse and swab techniques.

Alu Hypomethylation in Smoke-Exposed Epithelia and Oral Squamous Carcinoma Alu element;hypomethylation;oral cancer;smoke-exposed epithelia; Background: Alu elements are one of the most common repetitive sequences that now constitute more than 10% of the human genome and potential targets for epigenetic alterations. Correspondingly, methylation of these elements can result in a genome-wide event that may have an impact in cancer. However, studies investigating the genome-wide status of Alu methylation in cancer remain limited. Objectives: Oral squamous cell carcinoma (OSCC) presents with high incidence in South-East Asia and thus the aim of this study was to evaluate the Alu methylation status in OSCCs and explore with the possibility of using this information for diagnostic screening. We evaluated Alu methylation status in a) normal oral mucosa compared to OSCC; b) peripheral blood mononuclear cells (PBMCs) of normal controls comparing to oral cancer patients; c) among oral epithelium of normal controls, smokers and oral cancer patients. Materials and Methods: Alu methylation was detected by combined bisulfite restriction analysis (COBRA) at 2 CpG sites. The amplified products were classified into three patterns; hypermethylation ($^mC^mC$), partial methylation ($^uC^mC+^mC^uC$), and hypomethylation ($^uC^uC$). Results: The results demonstrate that the $%^mC^mC$ value is suitable for differentiating normal and cancer in oral tissues (p=0.0002), but is not significantly observe in PBMCs. In addition, a stepwise decrease in this value was observed in the oral epithelium from normal, light smoker, heavy smoker, low stage and high stage OSCC (p=0.0003). Furthermore, receiver operating characteristic (ROC) curve analyses demonstrated the potential of combined $%^mC$ or $%^mC^mC$ values as markers for oral cancer detection with sensitivity and specificity of 86.7% and 56.7%, respectively. Conclusions: Alu hypomethylation is likely to be associated with multistep oral carcinogenesis, and might be developed as a screening tool for oral cancer detection.