Two populations of TSPO binding sites in oral cancer SCC-15 cells.

Oral cancer mortality and morbidity rates remain high. The main inducer of oral cancer is cigarette smoke (CS). Translocator protein 18kDa (TSPO) was shown to play a role in carcinogenesis. We characterized TSPO binding sites in human oral cancer cell line SCC-15 and examined effect of CS on TSPO binding. We exposed SCC-15 human squamous cells to cigarette smoke. [3H]PK 11195 binding results were assessed in cells confluent for one day. To characterize the number of population sites, a custom written Matlab program compared Pearson linear correlation coefficients between all points in the Scatchard plot. Using [3H]PK 11195 as a radio ligand, we found that TSPO binding sites are not uniform, but separated into two sub-populations, one with high affinity (respective Kd and Bmax values of 1.40±0.08nM and 1586±48 fmol/mg protein), another with lower affinity (respective Kd and Bmax values of 61±5nM and 26260±1050 fmol/mg protein). We demonstrate rapid decrease in TSPO binding to the high affinity site induced by exposure to CS; specifically, significant 36% decrease in binding after 30min CS exposure (p<0.05), and 69% decrease after 2h CS exposure (p<0.05). Association between TSPO and CS exposure may contribute to understanding the underlying mechanism of oral carcinogenesis.

Tumor Growth and Cell Proliferation Rate in Human Oral Cancer.

BACKGROUND AND AIMS: Oral squamous cell cancer (SCC) has a high rate of morbidity and an overall 5-year survival rate for patients of 50%, statistics that have not changed in the last half century. A better understanding of the biological nature of this aggressive disease is mandatory. The two most studied human oral cancer cell lines-SCC-15 and SCC-25-share some biological characteristics but differ in others and may serve as a platform for further oral SCC analysis. We compared their basic carcinogenic characteristics, cellular proliferation rate and tumor growth, and discussed results according to available data from the literature and our own previous studies. METHODS: We examined doubling time both in vitro and in vivo of the SCC-15 and SCC-25 cell lines. After seeding the exact same number of cells in a six-well dish we counted them daily. To confirm doubling time differences in cells, we progressed to an in vivo model in nude mice using male 9- to 10-week-old BALB/c-nu/nu mice. RESULTS: In both models (in vitro and in vivo) SCC-15 multiplied faster than SCC-25 cell line. In vivo the difference was more than double and in vitro this change was 24%. CONCLUSION: Both SCC-15 and SCC-25 cell lines are suitable for further exploration of the oral carcinogenesis process. Based on our currently presented results and on the available literature, it seems that SCC-15 has an increased potential for local tumor growth and cell proliferation, whereas SCC-25 has a higher potential for invasion and metastasis.

Carbonyl levels and survival rates in oral cancer cells exposed to cigarette smoke.

BACKGROUND: Cigarette smoke (CS) is the main inducer of oral cancer, increasing prevalence 4-7 times. MATERIALS AND METHODS: We examined SCC-25 and SCC-15 suitability for studying CS effects on oral cancer cells, measuring carbonyl levels for free radical-mediated CS effect on survival and time/CS dependence. RESULTS: Protein oxidation increased significantly during CS exposure. At all time points, carbonyl levels increased six-fold (p<0.001) in both cell lines. Cell viability decrease was time-dependent. Longer CS exposure led to higher cell mortality. At 120 min, SCC-25 cell survival reduction was 43.7%, (p<0.01). Propidium iodide (PI) assay results matched the Trypan blue assay showing a time-dependent cell viability decrease following CS exposure. At 120 min, cell survival reduction was 37% (p<0.05). CONCLUSION: Cell death is mediated by CS free radicals with pathological process occurring first. Oral cancer cell models SCC-25 and SCC-15 are suitable for studying CS-induced free radical-related damage, potentially leading to the pathogenesis of oral cancer.

Cigarette smoke-induced effects on the cell cycle in oral cancer cells: reduction of G2/M fraction.

BACKGROUND: Cigarette smoke (CS) is the main inducer of oral cancer, increasing its prevalence by 4-7 times. MATERIALS AND METHODS: We examined the suitability of cell models SCC-25 and SCC-15 for studying effects of CS on oral cancer cells and whether CS significantly affects the cell cycle using fluorescence-activated cell sorting assays. RESULTS: There was significant change in the fraction of SCC-15 cells in pre-G1 state following CS exposure. At 60 and 90 min, increase in the pre-G1 cell fraction was 118% (p<0.05) and 135% (p<0.01) respectively. The G2/M cell fraction was significantly lower following CS exposure. At 90 and 120 min following CS exposure, the G2/M fraction levels had declined by 44% (p<0.05) and 34% (p<0.01) respectively. Results for SCC-25 cells were similar. At 90 and 120 min following CS exposure, the pre-G1 fraction of the cells increased by 230% and 550%, respectively (p<0.01). At 120 min of CS exposure, the fraction of G2/M cells was lower by 47% (p<0.05) compared to controls. CONCLUSION: CS profoundly affects the cell-cycle distribution in both SCC-15 and SCC-25 oral cancer cellular models. Such effects have been associated with DNA damage and carcinogenesis. Both models are useful for studying oral cancer pathogenesis.

DNA fragmentation induced by cigarette smoke in oral cancer cells.

BACKGROUND/AIM: Cigarette smoke (CS) is the main inducer of oral cancer, increasing the prevalence by 4-7 times. We examined induction of apoptosis by CS exposure of SCC-25 and SCC-15 oral cancer cells. MATERIALS AND METHODS: After controlled exposure to CS of various durations and at different time points we measured DNA fragmentation to assay apoptotic levels. RESULTS: SCC-15 cells showed a 70% (p<0.05) increase in apoptotic levels immediately after 30 min of exposure to CS. Twenty-four hours after 30-min CS exposure a further increase in apoptotic levels to 178% (p<0.05) could be observed. However, SCC-15 cells showed a decrease in apoptotic levels immediately after 180-min exposure to CS. CS-exposed SCC-25 cells did not show such CS-related effects. CONCLUSION: SCC-15 and SCC-25 oral cancer cells respond differently to CS regarding apoptotic cell death levels. In this respect, SCC-15 cells are sensitive to CS, while SCC-25 cells are not. Further comparisons between these cells may give insight regarding relationships between CS, apoptosis and invasiveness of oral cancer.

Cigarette Smoke Decreases Salivary 18 kDa Translocator Protein Binding Affinity ­ in Association with Oxidative Stress

Reactive oxygen species (ROS) generated by cigarette smoke may contribute to lung and oral cancer. The 18 kDa Translocator protein (TSPO) has been reported to be affected by ROS as well as to participate in ROS generation at mitochondrial levels, and has been implicated in pro-apoptotic and anti-carcinogenic functions. The present study reports the presence of TSPO in the cellular fraction of human saliva. In cells collected from untreated saliva, the specific TSPO ligand [(3)H]PK 11195 showed saturable binding with high affinity, with mean B(max) and K(d) values of 6,471 +/- 501 fmol/mg protein and 6.2 +/- 0.5 nM, respectively. Our study further indicates that the cellular fraction of human saliva possesses TSPO with binding characteristics similar to that of cells from other tissues of human origin. Following exposure of saliva to cigarette smoke a three-fold decrease in the affinity of salivary TSPO to its specific ligand, [(3)H]PK 11195 (p < 0.01) occurred in the cellular fraction of the saliva, in comparison to sham treated control, without significant accompanying changes in TSPO B(max), TSPO protein levels, or general protein levels. The changes in affinity of TSPO from the cellular fraction of saliva exposed to cigarette smoke were accompanied by changes in the mean levels of protein oxidation products (carbonyls) and lipid peroxides, which were three-fold higher (p < 0.01) and two-fold higher (p < 0.01), respectively, compared to those of sham treated controls. Thus, our study shows that TSPO is present in the cellular component of saliva. Interestingly, in vitro this cellular TSPO is affected by exposure of the whole saliva to cigarette smoke, in negative correlation with oxidative stress.

Oral cancer, cigarette smoke and mitochondrial 18kDa translocator protein (TSPO) - In vitro, in vivo, salivary analysis.

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Deltapsi(m)), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p<0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133+/-643 fmol/mg protein and 6956+/-549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p<0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p<0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p<0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p<0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.