Leukoplakia Cell Research Articles

In situ immunohistochemical detection of intracellular Mycoplasma salivarium in the epithelial cells of oral leukoplakia.

BackgroundMycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue.ObjectiveOur aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry.DesignWe produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry.ResultsWe obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivariumDNA in the epithelial cells of leukoplakia.ConclusionIntracellular M. salivarium was identified in the epithelial cells of leukoplakia.

MECHANISM OF GENESIS OF NEOPLASIA AT DEFEAT OF CERVICAL EPITHELIUM BY HIGH-RISK HUMAN PAPILLOMAVIRUS

Abstract. The 1044 biopsies from HPV-positive cervical epithelia were tested by cytological, histological, immunomorphological methods and PCR. One of the mechanisms of genesis of cervical cancer is damage of mitotic apparatus owing to association E7 HPV with NuMA 1, leads to infringement of distribution of chromosomes and virus DNA between daughter cells, shown pathological mitosis and integration of virus DNA. Infringements of differentiation of cells at CIN testified to their delay in pre-mitotic phases of cellular cycle unlike SCC which cells, having finished mitosis, are capable to the differentiation, accompanied by infringement of intercellular adhesion and neoanginesis. Genesis koilocytes and the cells of leukoplakia it is caused by defeat HPV basal cells.

Study of Immune Effector Cells in Leukoplakia and Oral Cancer

Aim: Immunosuppresion in oral squamous cell carcinoma (OSCC) is related to high degree of recurrence and believed to develop from premalignant lesion. Leukocytes especially T cell subsets are important in immune surveillance during malignant transformation. This study has been planned to observe changes in systemic immune response in premalignant and malignant oral lesions. Method: The proportions of Neutrophils, Monocytes, Lymphocytes, total T cells, T cell subsets including αβ/γδ T cells, Cytotoxic T cells, Helper T cells, Naive/ Effector/Memory T cells, Regulatory T cells and NK-T subpopulations were analysed in peripheral circulation of healthy donors (N= 49), Leukoplakia (N=20) and OSCC patients (N=100) by flowcytometry. Results: In comparison with healthy donors, decreased Lymphocytes, Naive Helper cells, NK T subpopulations and increased Effector cells were observed in Leukoplakia patients. Similarly, decreased Lymphocytes, NK T subpopulations and increased Neutrophils, Monocytes, Helper and Regulatory T cells were observed in OSCC patients as compared to healthy controls. Moreover, Lymphocytes were decreased and Regulatory T cells were increaed during the progression of Leukoplakia to OSCC. Further, in relation with clinicopathological parameters, Cytotoxic cells were found to be reduced with increasing histological grade. Also, Helper cells were found to be decreased in patients with tobacco and alcohol habit and also with increasing tumor size. Further, in univariate survival analysis, increased incidence of relapse was observed in patients with low γδ and NK T cells. In multivariate survival analysis, low γδ T cells emerged as poor prognosticator for disease free survival and high Regulatory T cells emerged as poor prognosticator for predicting overall survival. Conclusion: Altered systemic immune response was seen during malignant transformation and also found to be associated with patient’s survival. Thus, investigation of circulating Leukocyte and T cell subsets seems to be useful for predicting patient’s survival and to identify immunosuppressed patients who may be benefited with immunotherapy.

Abstract 2580: Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells.

Abstract Oral leukoplakia is a precancerous lesion that may progress to squamous cell carcinoma of the head and neck (HNSCC). The long latency period to invasive cancer provides an opportunity for intervention at the precancerous stage. The inability of surgical resection to prevent the recurrence of oral leukoplakia speaks clearly to the need to develop an efficacious regimen for the chemoprevention of this disease. Tobacco smoke constituents and ethanol, the major risk factors for oral leukoplakia and HNSCC, are metabolized by cytochrome P450 1B1 (CYP1B1) to form potentially carcinogenic species. Our previous data indicate that knockdown of CYP1B1 in human oral leukoplakia cells (MSK-Leuk1) by shRNA decreases cell motility and proliferation, relative to control cells overexpressing empty vector. Homoeriodictyol (HED), a flavonoid found in citrus fruits, is a selective inhibitor of CYP1B1 (IC50 0.24 μM). As a next step, we hypothesized that pharmacologic inhibition of CYP1B1 by HED would attenuate cell motility and proliferation in a manner similar to genetic knockdown. Because HED is commonly found in our diet, its toxicity is expected to be low, making it a promising agent for preventive or therapeutic intervention. The ability of HED to inhibit CYP1B1 activity in MSK-Leuk1 cells was established by incubating cells with either vehicle (0.01% ethanol) or HED (5-10 μM) for 72 hrs and quantifying 7-ethoxyresorufin o-deethylation (EROD), a measure of the activity of CYP1B1 and other enzymes of the CYP1 subfamily. HED decreased EROD activity in a dose-dependent manner, with 40% inhibition observed at 10 μM, as compared to vehicle-treated controls. Western blot analyses revealed that CYP1B1 levels were unaltered following HED treatment. The effect of CYP1B1 inhibition on cell motility was examined by exposing MSK-Leuk1 cells to HED (0-10 μM) for 96 hrs followed by a wound-healing assay, in which the ability of cell monolayers to close a gap was monitored for 23 hrs. HED inhibited cell motility in a concentration dependent manner, with maximum inhibition of gap closure reached at 10 μM (6.7 fold relative to vehicle). To assess the effect of CYP1B1 inhibition on cell growth, MSK-Leuk1 cells were exposed to HED (0-10 μM) for 5 days and proliferation was analyzed (Fluorescent DNA Quantitation kit). A direct association was observed between HED dose and inhibition of proliferation, with a 33% decrease observed at 10 μM. In summary, inhibition of CYP1B1 activity by HED resulted in attenuation of the motility and proliferation of cultured MSK-Leuk1 cells. These data confirm that CYP1B1 is a promising molecular target for intervention in HNSCC and provide strong support for the further development of CYP1B1 inhibitors as efficacious agents for the prevention of HNSCC in patients with oral leukoplakia. (Supported by NCI fellowship 1 F32 CA144199-01). Citation Format: Ekaterina G. Shatalova, Margie L. Clapper. Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2013-2580

Expression of P53 Protein in Premalignancies and Squamous Cell Carcinoma of the Oral Cavity

Introduction: Expression of p53 gene is one of the common findings in premalignancies and malignancies of the oral cavity. The aim of the study was to know whether P53 protein is overexpressed in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. The aim of the present study was to determine the overexpression of P53 protein in histological sections of oral submucous fibrosis, oral leukoplakia and oral squamous cell carcinoma. Materials and methods: Eighty-five histopathologically diagnosed. formalin-fixed, paraffin embedded tissue samples were divided into study group and control group. The study group was further subdivided into three groups: Group 1 consisted of 25 samples of oral submucous fibrosis: group 2 consisted of 25 samples of oral leukoplakia and group 3 consisted of 25 samples of oral squamous cell carcinoma. The control group consisted of 10 normal oral mucous membrane tissue samples. The samples were subjected to immunohistochemisty using indirect immunoenzyme Labelled StreptAvidin Biotin (LSAB) method. The results were analyzed statistically by Chi-square (X 2 ) test of significance Results: 8125 samples of group 1.5;25 samples of group 2. 8125 samples of group 3 and none of the samples of the control group were positive for P53 protein. P53 staining was seen mostly in the basal layer of the samples of group I while in the samples of groups 2 and 3 the staining was more diffuse. Conclusion: Among P53 may be considered a tumor marker as a definite number of samples were positive for P53

Nodular leukoplakia and squamous cell carcinoma

Nodular leukoplakia and squamous cell carcinoma

In situ gelatinolytic activity and immunoexpression of metalloproteinases 2 and 9 in oral leukoplakia and oral squamous cell carcinomas

In situ gelatinolytic activity and immunoexpression of metalloproteinases 2 and 9 in oral leukoplakia and oral squamous cell carcinomas

CRP as an Inflammatory Marker in Oral Rinses

Objective: 1) Learn if CRP stimulates production of IL-6 in human leukoplakia cells (MSK-Leuk1). 2) Learn if pioglitazone attenuates CRP stimulation of IL-6 in leukoplakia cells. 3) Establish an oral rinse CRP reference range in controls that can be used in an ongoing trial of leukoplakia patients treated with pioglitazone. Method: ELISA will be used to measure IL-6 release from MSK-Leuk1 treated with varying concentrations of CRP and pioglitazone. ELISA will be used to measure CRP in oral rinses of control patients. Two-sided t test and ANOVA are used for analysis of results with significance level of 5%. Results: When treated with CRP (0.001-10 ug/mL), MSK-Leuk1 cells increased IL-6 release in a dose-dependent manner ( P < .05). When treated with CRP (0.01-10 ug/mL) and with pioglitazone (10 uM) MSK-Leuk1 cells released a statistically significantly decrease in IL-6 as compared to CRP treatment ( P < .05) Oral rinses were easily collected and CRP was measured by ELISA to establish controls for an ongoing study of pioglitazone treatment of leukoplakia and CRP as an affective inflammatory marker. Conclusion: CRP is now shown to play an active roll in increasing the inflammatory cascade in leukoplakia cells, and pioglitazone attenuates this stimulation as measured by IL-6. The next step is to test CRP in already collected oral rinses of leukoplakia patients before and after treatment with pioglitazone.

Abstract 601: PPARδ mediated p21 induction in aerodigestive preneoplastic cell lines

Abstract PPARδ activating strategies are currently being tested preclinically and clinically in aerodigestive cancer prevention. Thiazolidinedione drugs like pioglitazone are clinically used for the treatment of diabetes and activate PPARδ as a primary target. P21/WAF1 is a cell cycle protein that is a cyclin dependent kinase inhibitor that reguylates cell cycle progression at the G1 phase; therefore its induction may be associated with cell cycle downregulation in precancerous cells experiencing increased proliferation. In our present study we examined the effects of pioglitazone on proliferation in human leukoplakia cells (MSK 1) and transformed lung (BEAS 2B) cells. We found that pioglitazone significantly inhibited cell proliferation in both cell lines in a dose dependent fashion at concentrations between 0 and 40 uM, as judged by MTT assays from 1-5 days. We found p21/WAF 1 induction by western blot within the first eight hours after the intitiation of treatment with pioglitazone, which were preceded by measureable increases in p21 induction on q PCR. In an analysis of an Affymetrix database of human head and neck tumors (41 tumors compared to 13 normal oral mucosa samples), we found significant dysregulation in several p21 activated kinases. We conclude the PPARδ activator, pioglitazone, can activate p21, which is associated with decreased proliferation in 2 aerodigestive preneoplastic cell lines. We also conclude that this gene may be a potential hypothesis driven biomarker in translational studies of pioglitazone as a chemoprevention agent for aerodigestive cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 601. doi:1538-7445.AM2012-601

Abstract 1588: Synergistic apoptosis by combination of natural compound EGCG and resveratrol in head and neck cancer: Potential role for AKT-dependent signaling

Abstract Natural dietary agents have drawn great attention for cancer prevention because of their wide safety margin and ready availability. Green tea is one such agent that is widely used for chemoprevention (Amin et al. 2009, Kim et al., 2010). Recent clinical trials suggest that green tea alone might not be sufficient for chemoprevention of head and neck cancer (HNC) (Tsao et al., 2009). Therefore, identification of compounds which exhibit synergistic effects with green tea is warranted. In the current study, we investigated combination of green tea component EGCG and resveratrol (a major constituent of red wines and grapes), and found that their combination strongly induced apoptosis as measured by annexin-V staining and supported by cleavage of PARP and caspase 3. The combination of resveratrol (15 and 20 μM, which alone induce 10-15% apoptosis) with EGCG (30-80 μM, which also induce <10% apoptosis) strongly increased apoptosis (to 60-80%) in multiple HNC cell lines. Data analysis for the combination using CalcuSyn software suggests that the combination of EGCG and resveratrol has synergistic apoptotic effects with combination index between 0.4-0.7. Interestingly, a premalignant oral leukoplakia cell line was sensitive to relatively lower doses of the combination (5 and 10 μM resveratrol, 10 and 15 μM EGCG) than fully transformed cancer cell lines, suggesting that the combination of EGCG and resveratrol might be a suitable combination for prevention of HNC. To further elucidate the mechanism of synergistic apoptosis, we found that combination of EGCG and resveratrol strongly inhibited AKT and ERK phosphorylation along with its several downstream targets including p-mTOR, p-S6 and p-4EBP1. Retroviral transduction of constitutively active AKT significantly inhibited apoptosis induced by the combination (p=.003). We also found that the combination of EGCG and resveratrol strongly inhibited p-AMPK, a kinase that regulates autophagy, a protective mechanism during energy stress. The fact that inhibition of autophagy triggers apoptosis (Xu Y et al., 2011) suggests that inhibition of p-AMPK might also play a role in EGCG- and resveratrol-induced apoptosis. Furthermore, strong inhibition of anti-apoptotic Mcl-1 and survivin by the combination was observed as a consequence. Taken together, our studies identify a novel combination of two natural dietary agents, EGCG and resveratrol, which induces synergistic anti-tumor effects in both premalignant and fully transformed HNC cells by targeting multiple signal transduction pathways. The signaling pathways modulated by the combination of EGCG and resveratrol are critical for development of HNC, thus our study provides an important rationale for future preclinical and clinical development. (Supported by U01CA101244, R01CA112643, and P50CA128613 to DMS) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1588. doi:1538-7445.AM2012-1588

Induction of apoptosis and up-regulation of cellular proliferation in oral leukoplakia cell lines inside electric field

In dentistry, metallic alloys are used for dentures, restorative materials, and orthodontic devices. Electric voltages up to 950 mV may occur between different dental alloys in the oral cavity. This study aimed to investigate physiologic reactions of oral leukoplakia cells in vitro to electric fields. A human leukoplakia cell line (MSK-LEUK1), cultivated in keratinocyte growth medium (KGM-2) supplemented with growth factors in 5% CO(2) humidified air at 37°C, was exposed to electric field strength of 1-20 V/m for 24 hours in a custom-made pulse chamber. The cells were then analyzed for proliferation with the use of BrdU assay and for apoptosis with the use of TUNEL assay. Findings were assessed with the use of fluorescent microscopy. Ultrastructural changes were studied by transmission electron microscopy. Electric field strength of 1-10 V/m led to up-regulation of cell proliferation rate from 10.64% to 44.06% (P = .0001). The apoptotic index increased significantly (P = .0001) from 20.03% at 1 V/m to 46.56% at 10 V/m. Individual cell keratinization was seen in leukoplakia cells treated with 16 V/m. Oral galvanism induces subcellular changes in oral precancer cells in vitro that closely simulate some of the morphologic features of oral squamous cell carcinoma cells in vivo.

Immunohistochemical evaluation of bcl-2 oncoprotein in normal oral mucosa, leukoplakia and oral squamous cell carcinoma

Immunohistochemical evaluation of bcl-2 oncoprotein in normal oral mucosa, leukoplakia and oral squamous cell carcinoma

Diagnostic Reliability of Fine Needle Aspiration Cytology against Histopathology for the Diagnosis of Oral Squamous Cell Carcinoma and Oral Leukoplakia

The purpose of this study was to determine the possibility of using fine needle aspiration cytology (FNAC) as a primary diagnostic test in oral leukoplakia and squamous cell carcinoma. This study consisted of clinically diagnosed 15 cases of leukoplakia and 15 cases of oral squamous cell carcinomas. FNAC and biopsy were done on all the cases. A cytological and histopathological correlation was undertaken to determine the proportion of cancers. A 23-gauge sterile disposable needle was attached to a disposable syringe and introduced into the lesion at the proposed biopsy site in one movement. In leukoplakias, the center of the lesion or erythroplakic areas and, in squamous cell carcinomas, proliferative areas and edges of the ulcers were chosen. In leukoplakia group, out of 15 biopsy samples, one (6.67%) sample was negative and 14 (93.33%) were positive. Whereas out of 15 FNAC samples, 14 (93.33%) were negative and one (6.67%) sample was positive. In squamous cell carcinoma, out of 15 biopsy samples, no sample was negative and all (100.00%) were positive. Whereas out of 15 FNAC samples, two (13.33%) were negative and 13 (86.67%) sample were positive. It is noted that FNAC can be employed as a sound diagnostic tool for rapid diagnosis of oral squamous cell carcinoma. It may be particularly useful in cases, where formal biopsy procedure is difficult or contraindicated due to medical reasons or in cases of advanced malignancy. FNAC has been shown to be reliable and safe technique in the diagnosis of malignant in the head and neck. When the aspirations are performed by cytopathologists, it is easy to perform a rapid staining of the first smear and within 10 to 15 minutes to ensure that the material is sufficient and diagnosable and to suggest a preliminary diagnosis.

Modulation of growth and angiogenic potential of oral squamous carcinoma cells in vitro using salvianolic acid B

BackgroundOur previous studies showed that Salvianolic acid B (Sal B) inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters and such anti-cancer effects might be related to the inhibition of angiogenesis. This study was aimed to further investigate the anti-proliferative effect of Sal B on the most common type of oral cancer, oral squamous cell carcinoma (OSCC) and the possible mechanisms of action with respect to angiogenesis inhibition.MethodsTwo well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC4, and premalignant leukoplakia cells were treated with different concentrations of Sal B. Cytotoxicity was assessed by MTT assay. cDNA microarray was utilized to evaluate the expression of 96 genes known to be involved in modulating the biological processes of angiogenesis. Real-time reverse transcription-polymerase chain reaction analysis was conducted to confirm the cDNA microarray data.ResultsSal B induced growth inhibition in OSCC cell lines but had limited effects on premalignant cells. A total of 17 genes showed a greater than 3-fold change when comparing Sal B treated OSCC cells to the control. Among these genes, HIF-1α, TNFα and MMP9 are specifically inhibited, expression of THBS2 was up-regulated.ConclusionsSal B has inhibitory effect on OSCC cell growth. The antitumor effect can be attributed to anti-angiogenic potential induced by a decreased expression of some key regulator genes of angiogenesis. Sal B may be a promising modality for treating oral squamous cell carcinoma.

O63. Comparison of degranulated mast cell counts in oral leukoplakia and oral squamous cell carcinoma

O63. Comparison of degranulated mast cell counts in oral leukoplakia and oral squamous cell carcinoma

Differential Inhibition of Protein Translation Machinery by Curcumin in Normal, Immortalized, and Malignant Oral Epithelial Cells

Curcumin has shown some promise in the prevention of oral carcinogenesis by mechanism(s) that are still not completely resolved. Messenger RNA translation is mediated in eukaryotes by the eIF4F complex composed of eukaryotic translation initiation factors eIF4E, eIF4G, and eIF4A. Overexpression of some of these components or the inactivation of initiation repressor proteins (4E-BP1) has been implicated in cancer development including oral carcinogenesis by affecting cell survival, angiogenesis, and tumor growth and invasion. In this study, we examined the possibility that curcumin affects the translational machinery differently in normal, immortalized normal, leukoplakia, and malignant cells. Curcumin treatment in vitro inhibited the growth of immortalized oral mucosa epithelial cells (NOM9-CT) and the leukoplakia cells (MSK-Leuk1s) as well as in the UMSCC22B and SCC4 cells derived from head and neck squamous cell carcinoma. Curcumin only exerted minor effects on the growth of normal oral epithelial cells (NOM9). In the immortalized, leukoplakia, and cancer cells, curcumin inhibited cap-dependent translation by suppressing the phosphorylation of 4E-BP1, eIF4G, eIF4B, and Mnk1, and also reduced the total levels of eIF4E and Mnk1. Our findings show that immortalized normal, leukoplakia, and malignant oral cells are more sensitive to curcumin and show greater modulation of protein translation machinery than the normal oral cells, indicating that targeting this process may be an important approach to chemoprevention in general and for curcumin in particular.

Morphometric computer-assisted image analysis of oral epithelial cells in normal epithelium and leukoplakia

There are very few studies documenting morphometric parameters of normal oral mucosa and leukoplakia. The present study was undertaken to establish the morphometric parameters of the parabasal and spinous cells of normal oral epithelium. Analysis of changes occurring in these cells in leukoplakia was also done. This study was conducted on tissue sections of clinically normal oral mucosa and leukoplakia. Morphometric analysis was done for parabasal and spinous cells. Statistical analysis was done using one way ANOVA and Mann-Whitney test. Morphometric parameters were greater in the spinous cells than in parabasal cells in normal oral mucosa. Leukoplakia showed greater cellular and nuclear parameters than normal mucosa. Normal oral epithelium showed site-wise difference in cell and nuclear measurements. Nuclear parameters showed a statistically significant change than cellular parameters in dysplasia. These changes were expressed in the earliest stage of transformation to dysplasia.

Analysis of cell proliferation rate in Oral Leukoplakia and Oral Squamous Cell Carcinoma

Objectives: Assessment of the cell proliferation rate in tissues can be one of the markers for impending malignancy in precancers. The state of activation and the proliferation activity of the cells can be assessed by the frequency of silver stained Nucleolar Organiser regions (AgNOR) within the nuclei which is significantly higher in malignant cells. The present study was carried out to analyze the distribution of the AgNOR in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), and in their various histological grades, and to assess if the AgNOR distribution could give information on the malignant potentiality in premalignant lesions and aggressiveness of the malignant lesions. Study design: The study specimens comprised of 35 archival cases, of which 15 cases were of OL and 20 cases of OSCC. The specimens were stained by hematoxylin and eosin and modified silver staining method of Ploton et al. for the Nucleolar Organiser Regions. The specimens were analyzed independently by the two observers and was further statistically analysed. Results: The mean AgNOR count in OL was 2.80 ±0.50 and in cases of OSCC was 5.71± 1.08. The mean AgNOR count in OL cases of mild dysplasia was 2.59 ±0.66, in moderate dysplasia was 2.92± 0.43 and in severe dysplasia was 2.79. The mean AgNOR count in cases of well differentiated OSCC was 5.73± 1.62 and in cases of moderately differentiated OSCC was 5.67±1.19. Conclusion: The mean AgNOR count was higher in cases of OSCC as compared to cases of OL, and the AgNOR counts increased with the increase in the grades of dysplasia indicating a higher proliferative rate with increase in dysplasia.

Effects of Podoplanin on cell proliferation and cell cycle in oral leukoplakia cells

To investigate the effects of Podoplanin on cell proliferation and cell cycle in oral leukoplakia cells. Oral leukoplakia cell line (Leuk-1) transfected with pCMV-Podoplanin (A4-1) or pCMV (B4-1) was used in this study. The levels of mRNA and protein of Podoplanin were detected by real-time PCR and Western boltting in A4-1, B4-1 and Leuk-1 cells. The localization of Podoplanin was detected by confocal microscope. Cell growth and proliferation was examined by methyl thiazolyl tetrazolium (MTT) assay and cell cycle was detected by flow cytometry (FCM). The value of Podoplanin mRNA level in A4-1 cells was 0.022, which was significantly higher than the values in B4-1 (0.001) and Leuk-1 cells (0.002), P < 0.05. The gray scale of Podoplanin protein in A4-1 cells was significantly higher than in the control groups (P < 0.05). The expression of Podoplanin was observed in cell plasm and membrane of A4-1, B4-1 and Leuk-1 cells. But the expression level of Podoplanin in A4-1 cells was higher than in control groups. A4-1 cells grew faster than Leuk-1 cells. The proliferation rate after 3 days of culture in A4-1 cells was 40.4% higher than that in B4-1 cells (P < 0.05). The G₂-M phase (24.89 +/- 4.55)% and PI (0.57 +/- 0.06) of A4-1 cells were significantly higher than that in B4-1 cells [(4.13 +/- 5.24)%, (0.41 +/- 0.04)] and Leuk-1 cells [(4.69 +/- 7.42)%, (0.40 +/- 0.02)], P < 0.05. Over expression of Podoplanin accelerated the growth and proliferation of oral leukoplakia cells. Podoplanin may be one of key genes in the malignant transformation of oral leukoplakia.

In vitro and Clinical Studies of Gene Therapy with Recombinant Human Adenovirus-p53 Injection for Oral Leukoplakia

Oral leukoplakia is a well-recognized precancerous lesion of squamous cell carcinoma. When accompanied with abnormal p53 expression, it suffered a higher risk of canceration. The present study was carried out to test whether the recombinant human adenovirus-p53 could introduce wild-type p53 gene to oral leukoplakia cells and induce cell cycle arrest and apoptosis. We select p53(-) oral dysplastic keratinocyte POE-9n, to observe the growth inhibition, cell cycle change, apoptosis-induced effects, and elaborate the corresponding molecular mechanism of recombinant adenovirus-p53 on POE-9n cells. Meanwhile, we evaluate the feasibility, safety, and biological activity of multipoints intraepithelial injections of recombinant adenovirus-p53 in 22 patients with dysplastic oral leukoplakia. Exogenous p53 could be successfully transduced into POE-9n cells by recombinant adenovirus-p53. The optimal infecting titer in this study was multiplicity of infection (MOI) = 100. Recombinant adenovirus-p53 could strongly inhibit cell proliferation, induce apoptosis, and arrest cell cycle in stage G(1) in POE-9n cells by inducing p21(CIP/WAF) and downregulating bcl-2 expression. In the posttreatment patients, p53 protein and p21(CIP/WAF) protein expression were significantly enhanced, yet bcl-2 protein presented low expression. Sixteen patients showed clinical response to the treatment, and 14 patients showed obvious histopathologic improvement. Intraepithelial injections of recombinant human adenovirus-p53 were safe, feasible, and biologically active for patients with dysplastic oral leukoplakia.