• Farnesol-Induced Apoptosis in Oral Squamous Carcinoma Cells is Mediated by MRP1 Extrusion and Depletion of Intracellular Glutathione

      Intapa, Chaidan; Jabra-Rizk, Mary Ann (2013)
      Farnesol is a key intermediate in the sterol biosynthesis pathway in eukaryotic cells that has exhibited significant anti-cancer and antimicrobial activity. We have shown that farnesol triggers apoptosis in oral squamous carcinoma cells (OSCCs) and in the fungal pathogen Candida albicans via a classical apoptotic process. However, the exact mechanism of farnesol cytotoxicity in eukaryotic cells has not been fully elucidated. In the cell, hydrophobic xenobiotics conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. This process results in the formation of glutathione S-conjugates which act as substrates for export by ATP-binding cassette transporters (ABC transporter) and are extruded from the cell. This study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with multidrug resistance-associated protein 1 (MRP1) - mediated extrusion of glutathione-farnesol conjugates and oxidized glutathione results in total glutathione depletion, oxidative stress and ultimately cell death. The combined findings demonstrated that farnesol exposure resulted in significant decrease in intracellular glutathione levels concomitant with intracellular Reactive oxygen species (ROS) accumulation and decrease in cell viability. However, addition of exogenous glutathione maintained intracellular levels and enhanced cell viability. Furthermore, gene and protein expression studies demonstrated significant up-regulation of MRP1 in cells treated with farnesol. However, MRP1 blocking and monoclonal antibody specific inhibition of MRP1 enhanced cell tolerance to farnesol. This is the first study describing the involvement of MRP1-mediated glutathione efflux as a mechanism for farnesol-induced apoptosis in OSCCs. Understanding of the mechanisms underlying farnesol-cytotoxicity in eukaryotic cells may lead to the development of this redox-cycling agent as an alternative chemotherapeutic target.
    • Novel effects of piRNAs and pfeRNAs in lung somatic cells

      Gable, Tyler; Mao, Li, M.D.; Schneider, Abraham (2017)
      Aberrant expression and function of PIWI-interacting RNAs (piRNAs) and piRNA-Likes (pilRNAs or piR-Ls) have been reported in various cancers. The majority of current reports have identified or assumed roles of these pilRNA that require association with PIWI family proteins to affect either transposable element silencing or mRNA transcript silencing through base pair matching. However, new reports are describing pilRNA which are capable of regulating physiological and pathological conditions through interactions with non-PIWI and non-PIWI-related proteins and whose interaction is necessary for the function of the binding partner protein. Specific cases examined in lung tissue have also found evidence that such interactions may be occurring at critical phosphorylated residues on the target proteins and suggests an early mechanism through which such interactions may occur. Therefore, we hypothesized that somatic expression of piRNA and piRNA-Like non-coding RNAs play active and dynamic roles in the progression of lung squamous cell and adenocarcinoma via phosphorylation-site interactions of PIWI-independent mechanisms. We began our investigations with RNA sequencing profiles of differentially expressed piRNA and pilRNA in lung somatic cells. Guided by expression data, we explored the phenotype and mechanism of action of potentially significant pilRNA species. We began with our study investigating a piR-L species which induces chemoresistance to cisplatin-based therapy by inhibiting apoptosis in lung squamous cell carcinoma. Next, we examined mitochondrial piRNA57125 which associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis. Finally, we performed a phosphorylation-wide sncRNA screen which reveals Protein Functional Effector sncRNAs (pfeRNAs) in human lung somatic cells. Collectively, these studies have supported the hypothesis that pilRNA and pfeRNA do play critical roles in the progression of lung squamous cell and adenocarcinomas and may, in fact, do so through phosphorylation-site interactions. As a result of these studies, we have re-named these pilRNA as pfeRNA, a more encompassing and descriptive terminology which is described below.
    • Role of Survivin and Hexokinase II in the regulation of Autophagy and Apoptosis in Oral Squamous Cell Carcinoma

      Desai, Prapti; Scheper, Mark; Meiller, Timothy F. (2012)
      Survivin, an inhibitor of apoptosis and hexokinase II, a key enzyme in glycolysis are under-expressed in normal oral tissue; while they are over-expressed in oral squamous cell carcinoma (OSCC). Previous studies in our lab have shown a direct correlation between survivin and hexokinase II, using bromo-pyruvic acid, a known hexokinase II inhibitor. Additionally, it has also been shown that there is an increase in the expression of these two biomarkers with the increase in severity of premalignant lesions. These data were obtained from our patient database at Oral Pathology Consultants (OPC) which was compared to the Maryland Cancer Registry (MCR) for a cancer match. Based on this previous work, we decided to study two of the cancer hallmarks in OSCCs; dysregulation of apoptosis and altered cellular metabolism, and molecules associated with them; survivin and hexokinase II. Interestingly, survivin was found to be over-expressed in 80% of OSCCs. Hence, our hypothesis was that in cells during survivin over-expression, treatments with DNA damaging agents will lead to induction of autophagy in OSCCs. To prove this hypothesis, two well known DNA damaging drugs, cisplatin and paclitaxel were used. The basic aim of the study was to down-regulate survivin and study the effect of this down-regulation on autophagy proteins, beclin-1 and mammalian light chain protein kinase, LC3 (autophagy marker). Using a premalignant cell line (Leuk-1) and malignant cell lines (SCC 9 and SCC 25), we have shown down-regulation of survivin using cisplatin and paclitaxel. Survivin is known to inhibit caspases, hence its down-regulate is likely to activate the process of apoptosis. Our western blot results represent down-regulation of beclin-1 as well as the active form of LC3, LC3-II. However, beclin-1 forms a complex with anti-apoptotic proteins, Bcl-2/xL and its down-regulation with chemotherapeutic drugs disturbs the complex, increasing the loss of mitochondrial membrane integrity and ultimately apoptosis. Also, LC3 is a high turn-over protein which can be incorrectly reflected on a western blot. However, further validation of the processes activated due to beclin-1 and LC3 down-regulation will raises a strong possibility of cross-talk between autophagy and apoptosis. Further, literature studies have shown that activation of one signaling pathway regulates key players at transcription and translational levels, which in turn activate or down-regulate other signaling pathways. Our findings support the novel role of the survivin/hexokinase II axis in the regulation of autophagy and apoptosis, in both dysplastic oral lesions and in OSCC.  
    • The Diagnostic Utility of Cancer Stem Cell Marker CD44 in Early Detection of Oral Cancer

      Alsalem, Bader; Meiller, Timothy F. (2019)
      The incidence of Oral Squamous Cell Carcinoma (OSCC) is expected to increase in the coming decades. In recent years, cancer biomarkers have emerged as a promising diagnostic and prognostic tool for various cancers. The cluster differentiation antigen (CD44) is among the most frequently identified cancer stem cell markers in solid tumors. Using immunohistochemistry labeling in previously diagnosed specimens, we aimed to analyze the utility of CD44 in early diagnosis of OSCC. Biopsy specimen subgroups of lateroventral tongue revealed an increased proportion of cells staining positive for CD44 in the epithelial samples of OSCC compared with erosive lichen planus and oral dysplastic lesions. CD44, in combination with other oral biomarkers, therefore, has the potential to assist in the early diagnosis of OSCC. Future studies with larger sample sizes and multiple biomarkers should be carried out, utilizing a pre-determined IHC staining and interpretation strategy to promote reproducibility of evidence.