Clinical course and therapeutic outcomes of operatively and non-operatively managed patients with denosumab-related osteonecrosis of the jaw (DRONJ).

PURPOSE: Details regarding risk factors, onset, and outcomes for denosumab-related osteonecrosis (DRONJ) are sparse. This study examines the clinical characteristics and operative and non-operative therapeutic outcomes in patients with DRONJ not previously exposed to other antiresorptives. METHODS: A retrospective medical record review was conducted, and data were collected, including clinical findings, management, healing outcomes, and radiologic, histologic, and micro-computed tomography (CT) analyses. RESULTS: Seventeen patients were treated with denosumab, with 14.1 ± 8.3 doses before DRONJ onset. The majority of lesions were observed at sites of dental prostheses (41%) and dental extractions (35%). Sixteen patients were managed non-operatively (10/16) or operatively (6/16) with either major (5/6) or minor surgery (1/6) and included in the follow-up analysis. Complete healing was significant in patients treated with major surgery (80%) compared to the non-operative group (20%; p < 0.035). Denosumab was discontinued in 60% of non-operative patients and major surgery patients with no effect on healing. Histologic findings of 4 patients analyzed exhibited a decreased number of osteocyte lacunae, and micro-CT of 3 patients scanned revealed trabecular thickening. CONCLUSION: DRONJ lesions occurred mostly at sites of prostheses sores after a mean of 14 doses of denosumab. Major surgery demonstrated more complete healing than non-operative management, and denosumab cessation did not improve healing outcomes.

Estrogen and Progesterone hormone receptor expression in oral cavity cancer

BACKGROUND: Recent studies have shown an increase in the incidence of oral squamous cell carcinoma (OSCC) in younger patients. The hypothesis that tumors could be hormonally induced during pregnancy or in young female patients without the well-known risk factors alcohol or tobacco abuse seems to be plausible. MATERIAL AND METHODS: Estrogen Receptor alpha (ERá) and Progesterone Receptor (PR) expression were analyzed in normal oral mucosa (n =5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen. OSCCs were stratified in a young female (n = 7) study cohort and older patients (n = 46). In the young female study cohort three patients (n = 3/7) developed OSCC during or shortly after pregnancy. Breast cancer tissues were used as positive control for ERá and PR expression. RESULTS: ERá expression was found in four oral precursor lesions (squamous intraepithelial neoplasia, SIN I-III, n = 4/35, 11%) and in five OSCC specimen (n = 5/46, 11%). The five ERá positive OSCC samples were older male patients. All patients within the young female study cohort were negatively stained for both ERá and PR. CONCLUSIONS: ER expression could be regarded as a seldom risk factor for OSCC. PR expression seems to be not relevant for the development of OSCC

Standardized pretreatment inflammatory laboratory markers and calculated ratios in patients with oral squamous cell carcinoma.

Analyzing the inflammatory microenvironment has become an important issue in the management of oral squamous cell carcinoma (OSCC). Pretreatment C-reactive protein (CRP) levels, leucocytes, monocytes, lymphocytes, neutrophils, basophils, eosinophils, platelets, neutrophil-to-lymphocyte ratio (NLR), derived NLR (dNLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR) derived from the peripheral blood were analyzed. Receiver operating characteristic (ROC) curves determined a cut-off value for each parameter in 146 patients with OSCC compared with 93 controls and the results were associated with clinicopathological characteristics. CRP expression of tumors was measured by immunohistochemistry. ROC analysis determined cut-off values for CRP levels, leucocytes, monocytes, lymphocytes, neutrophils, NLR, dNLR, LMR, PLR and showed significant differences between the OSCC and control group. Compared with single laboratory tests calculated ratios were superior in measuring sensitivity and specificity of OSCC disease. NLR was significant directly associated and correlated with PLR. LMR was significant inversely associated and correlated with NLR and PLR. Immunohistochemical analysis did not show CRP expression of OSCCs. This study highlights the first analysis for cut-off values of pretreatment single laboratory tests and calculated ratios, which are strongly needed for a follow-up of cancer patients. Additionally, the calculated baselines can be used as a goal for successful immunotherapies in the future. The links between NLR, LMR, and PLR might be helpful for the clinical course (monitoring) of cancer patients and have been first described for OSCC in this study. Taken together, analyzing these data provides an additional practical guideline of further postoperative OSCC management.

Monitoring carcinogenesis in a case of oral squamous cell carcinoma using a panel of new metabolic blood biomarkers as liquid biopsies.

INTRODUCTION: One of the common malignant tumors of the head and neck worldwide with generally unfavorable prognosis is squamous cell carcinoma (OSCC) of the oral cavity. Early detection of primary, secondary, or recurrent OSCC by liquid biopsy tools is much needed. CASE PRESENTATION: Twelve blood biomarkers were used for monitoring a case of OSCC suffering from precancerous oral lichen ruber planus mucosae (OLP). After curative R0 tumor resection of primary OSCC (buccal mucosa), elevated epitope detection in monocytes (EDIM)-Apo10, EDIM-transketolase-like-1 (TKTL1), squamous cell carcinoma antigen (SCC-Ag), total serum lactate dehydrogenase (LDH), and its anaerobic isoforms (LDH-4, LDH-5) decreased to normal levels. Three and six months after surgery, transformation of suspicious mucosal lesions has been accompanied with an increase of EDIM scores, total serum LDH values, and a metabolic shift from aerobic (decrease of LDH-1, LDH-2) to anaerobic (increase of LDH-4, LDH-5) conditions. Two months later, secondary OSCC was histopathologically analyzed after tissue biopsy. Cytokeratin fraction 21-1 (CYFRA 21-1), carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9) were not affected during the clinical course of carcinogenesis. CONCLUSIONS: A combination strategy using a standardized panel of established (metabolic) blood biomarkers (TKTL1, LDH, LDH isoenzymes) is worth and can be recommended among others (apoptosis resistance-related Apo10, SCC-Ag) for early detection and diagnosis of primary, secondary, and recurrent OSCC. A tandem strategy utilizing (metabolic pronounced) routine liquid biopsies with imaging techniques may enhance diagnosis of OSCC in the future. Although we demonstrated the diagnostic utility of separated liquid biopsies in our previous study cohorts, further investigations in a larger patient cohort are necessary to recommend this combination strategy (EDIM blood test, LDH value, metabolic shift of LDH isoenzymes, and others, e.g., SCC-Ag or immunophenotyping) as a diagnostic tool for the addition to the OSCC staging system and as a routine procedure in the aftercare.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. STUDY DESIGN: Activated THP-1 cells were exposed to .5 to 50 µM of nitrogen-containing bisphosphonates and .5 to 500 µM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. RESULTS: All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 µM and 500 µM; P < .05). Low concentrations (0.5 µM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations. CONCLUSIONS: Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Papillomavirus-Associated Tumor Formation Critically Depends on c-Fos Expression Induced by Viral Protein E2 and Bromodomain Protein Brd4.

We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.

Analysis of circulating CD14+/CD16+ monocyte-derived macrophages (MDMs) in the peripheral blood of patients with oral squamous cell carcinoma.

OBJECTIVES: Monocytes/macrophages are regarded as the first line of defense in tumors. Therefore, analyzing monocyte subtypes in oral squamous cell carcinoma (OSCC) may be of value in disease monitoring and to explore immunotherapeutic strategies for cancer patients. STUDY DESIGN: Circulating peripheral blood CD14+/CD16+ monocyte-derived macrophages (MDMs) were evaluated in OSCC patients with oral squamous cell carcinoma (n = 44) compared with controls (n = 85). Moreover, epitope detection in monocytes (EDIM) technology was used to detect biomarkers Apo10 and transketolase-like-1 in CD14+/CD16+ MDMs. RESULTS: Compared with controls, no significant (P = .3646) difference (control group 9.8%, OSCC group 8.8%) in CD14+/CD16+ MDM were noted in OSCC. However, EDIM-Apo10 and EDIM-TKTL1 scores detected in the CD14+/CD16+ MDMs were increased in OSCC compared with controls (P < .0001). CONCLUSIONS: Analyzing CD14+/CD16+ MDMs represents a stable cell population for detecting biomarkers in cancer disease monitoring.

Altered macrophagic THP-1 cell phagocytosis and migration in bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Local immune dysfunction via macrophages is a proposed aetiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study aimed to clarify the effects of various bisphosphonates on macrophage function using a THP-1 monocytic model to examine migration, phagocytosis, and fibrin structure. MATERIALS AND METHODS: THP-1 cell migration was measured in the presence and absence of zoledronate, ibandronate, risedronate, alendronate, pamidronate (0.5, 5 and 50 µM) and clodronate (125, 250 and 500 µM) using the real-time xCELLigence system. Phagocytosis and actin fibre assays were performed after 72 h with zoledronate, ibandronate, alendronate and clodronate. RESULTS: Time to maximum migration for THP-1 cells was significantly reduced (p < 0.05) for high dosages of zoledronate, ibandronate and alendronate compared to controls. All dosages of clodronate and a low dose of zoledronate exhibited prolonged migrations. Phagocytic capacity was significantly reduced in high dosages of all bisphosphonates and for 5 µM zoledronate and ibandronate (p < 0.05). Low bisphosphonate exposure was accompanied by overcharged phagosoms. Altered appearance in F-actin fibrin structure was observed in bisphosphonate-exposed cells. CONCLUSIONS: All bisphosphonates altered the migration of THP-1 cells dose-dependently. Low doses also prolonged migration and altered cell morphology. These findings support the idea of a disturbed local immune function of macrophages even in jaw bone exposed to low concentrations of bisphosphonate. CLINICAL RELEVANCE: These are the first real-time results for disrupted migration and function of macrophagic THP-1 cells in high doses. Low dosages also demonstrated altered macrophage phagocytosis and cell morphology, suggesting a disturbed local immune function in BRONJ pathogenesis.

Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue.

The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3(+)), T helper cells (Th, CD3(+)/CD4(+)), regulatory T cells (Treg, CD4(+)/CD25(+)/CD127(low)), cytotoxic T cells (Tc, CD3(+)/CD8(+)), activated T cells (CD3(+)/HLA-DR(+)), and natural killer (NK) cells (CD3(-)/CD16(+)/CD56(+)). In tumor tissue, the prevalence of CD3(+), CD4(+), and CD8(+) TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses.

Glutaminolysis and carcinogenesis of oral squamous cell carcinoma.

Glutaminolysis is a crucial factor for tumor metabolism in the carcinogenesis of several tumors but has not been clarified for oral squamous cell carcinoma (OSCC) yet. Expression of glutaminolysis-related solute carrier family 1, member 5 (SLC1A5)/neutral amino acid transporter (ASCT2), glutaminase (GLS), and glutamate dehydrogenase (GLDH) was analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. SLC1A5/ASCT2 and GLS were significantly overexpressed in the carcinogenesis of OSCC compared with normal tissue, while GLDH was weakly detected. Compared with SIN I-III SLC1A5/ASCT2 and GLS expression were significantly increased in OSCC. GLDH expression did not significantly differ from SIN I-III compared with OSCC. This study shows the first evidence of glutaminolysis-related SLC1A5/ASCT2, GLS, and GLDH expression in OSCC. The very weak GLDH expression indicates that glutamine metabolism is rather related to nucleotide or protein/hexosamine biosynthesis or to the function as an antioxidant (glutathione) than to energy production or generation of lactate through entering the tricarboxylic acid cycle. Overcoming glutaminolysis by targeting c-Myc oncogene (e.g. by natural compounds) and thereby cross-activation of mammalian target of rapamycin complex 1 or SLC1A5/ASCT2, GLS inhibitors may be a useful strategy to sensitize cancer cells to common OSCC cancer therapies.

Evaluation of a biomarker based blood test for monitoring surgical resection of oral squamous cell carcinomas.

INTRODUCTION: The potential use of determination of biomarkers in blood for the monitoring of surgical removal of oral squamous cell carcinomas (OSCC) was evaluated using the epitope detection in monocytes (EDIM) technology. MATERIALS AND METHODS: In tumor specimen, elevated Apo10 and transketolase-like 1 (TKTL1) expression was analyzed by immunohistochemistry. Apo10 and TKTL1 biomarkers have been used prospectively for EDIM blood test in patients with primary and/or recurrent OSCC (n = 92) before surgery and after curative tumor resection (n = 45). RESULTS: There were highly significant (p < 0.0001) correlations found between EDIM blood scores and the tissue expression of both biomarkers measured by immunohistochemistry (Apo10: n = 89/92, 97%; TKTL1: n = 90/92, 98%). EDIMApo10 and EDIM-TKTL1 scores were positive in 92% (EDIM-Apo10: n = 85/92) and 93% (EDIM-TKTL1: n = 86/92), respectively, in patients with OSCC before surgery. The combined score EDIM-Apo10/EDIM-TKTL1 increased significantly the detection rate of tumors to 97% (n = 89/92). After surgery, the EDIM-TKTL1 and EDIMApo10 scores significantly decreased in 75.6 and 86.7% of the patients (p < 0.0001), respectively, in the aftercare. CONCLUSIONS: The correlation of TKTL1 and Apo10 immunohistochemistry with the blood test results indicates that the EDIM blood test could serve as a non-invasive diagnostic tool (liquid biopsy) to assess surgical removal of OSCC by determination of two biomarkers. CLINICAL RELEVANCE: This is the first study that has been demonstrated a reliable and successful monitoring of OSCC cancer patients by a blood test. The specific and significant decrease of EDIM-TKTL1 and EDIM-Apo10 scores after surgery could serve as a new tool for monitoring surgical removal of OSCC.

TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer.

Chronic inflammation as an important epigenetic and environmental factor for putative tumorigenesis and tumor progression may be associated with specific activation of Toll-like receptors (TLR). Recently, carcinogenesis has been suggested to be dependent on TLR7 signaling. In the present study, we determined the role of both TLR7 and TLR8 expression and signaling in tumor cell proliferation and chemoresistance in pancreatic cancer. Expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis, normal pancreatic tissue and human pancreatic (PANC1) cancer cell line was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of (un-)stimulated TLR expression on tumor cell proliferation and chemoresistance. TLR expression was increased in pancreatic cancer, with stage-dependent upregulation in advanced tumors, compared to earlier stages and chronic pancreatitis. Stimulation of TLR7/TLR8 overexpressing PANC1 cells resulted in elevated NF-κB and COX-2 expression, increased cancer cell proliferation and reduced chemosensitivity. More importantly, TLR7/TLR8 expression increased tumor growth in vivo. Our data demonstrate a stage-dependent upregulation of both TLR7 and TLR8 expression in pancreatic cancer. Functional analysis in human pancreatic cancer cells point to a significant role of both TLRs in chronic inflammation-mediated TLR7/TLR8 signaling leading to tumor cell proliferation and chemoresistance.

Serum vitamin D levels of patients with oral squamous cell carcinoma (OSCC) and expression of vitamin D receptor in oral precancerous lesions and OSCC

BACKGROUND: Resistance to programmed cell death (apoptosis) is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Vitamin D (calcitriol) may overcome apoptosis resistance in tumor cells of OSCC. Vitamin D receptor (VDR) expression in oral precancerous lesions of OSCC has not been analyzed and serum vitamin D level seems to be a predictor of cancer development. MATERIAL AND METHODS: Expression of VDR was analyzed in normal oral mucosa (n=5), oral precursor lesions(simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen (n=42) by immunohistochemistry (IHC). Moreover, serum vitamin D levels were measured by 25(OH)D3 (calcidiol) in patients with OSCC (n=42) and correlated with IHC results. RESULTS: Expression of VDR was significantly increased in precancerous and OSCC compared with normal tissue. Compared with SIN I-III lesions VDR expression significantly decreased in OSCC. Severe vitamin D deficiency was detected in our OSCC patient cohort but there was no significant correlation analyzed between serum vitamin D levels and corresponding immunohistochemically detected VDR expression in OSCC. CONCLUSIONS: Our survey provides the first evidence of VDR expression in precancerous lesions of OSCC. Apoptosis induction of VDR+ cells in oral precancerous lesions and OSCC by natural vitamin D or synthetic vitamin D compounds could be useful for chemoprevention. Moreover, systemically and/or locally applied, these compounds may act as sensitizers for apoptosis mediated by radio-, and chemotherapy treatment in OSCC

Serum vitamin D levels of patients with oral squamous cell carcinoma (OSCC) and expression of vitamin D receptor in oral precancerous lesions and OSCC.

BACKGROUND: Resistance to programmed cell death (apoptosis) is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). Vitamin D (calcitriol) may overcome apoptosis resistance in tumor cells of OSCC. Vitamin D receptor (VDR) expression in oral precancerous lesions of OSCC has not been analyzed and serum vitamin D level seems to be a predictor of cancer development. MATERIAL AND METHODS: Expression of VDR was analyzed in normal oral mucosa (n=5), oral precursor lesions (simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen (n=42) by immunohistochemistry (IHC). Moreover, serum vitamin D levels were measured by 25(OH)D3 (calcidiol) in patients with OSCC (n=42) and correlated with IHC results. RESULTS: Expression of VDR was significantly increased in precancerous and OSCC compared with normal tissue. Compared with SIN I-III lesions VDR expression significantly decreased in OSCC. Severe vitamin D deficiency was detected in our OSCC patient cohort but there was no significant correlation analyzed between serum vitamin D levels and corresponding immunohistochemically detected VDR expression in OSCC. CONCLUSIONS: Our survey provides the first evidence of VDR expression in precancerous lesions of OSCC. Apoptosis induction of VDR+ cells in oral precancerous lesions and OSCC by natural vitamin D or synthetic vitamin D compounds could be useful for chemoprevention. Moreover, systemically and/or locally applied, these compounds may act as sensitizers for apoptosis mediated by radio-, and chemotherapy treatment in OSCC.

Zoledronate but not denosumab suppresses macrophagic differentiation of THP-1 cells. An aetiologic model of bisphosphonate-related osteonecrosis of the jaw (BRONJ).

OBJECTIVES: Bisphosphonates and denosumab are antiresorptive drugs used for the treatment of osteoporosis and oncological tumors. A severe side effect is osteonecrosis of the jaw. Monocyte/macrophage dysfunction is considered to play a distinct role in osteonecrosis. THP-1 monocytic cells were used in this study to elucidate the influence of zoledronate and denosumab on phorbol-12-myrisate-13-acetate (PMA)-induced macrophage differentiation and function in real-time. MATERIALS AND METHODS: Macrophagic differentiation of the THP-1 suspension cells was measured by cell adherence in the presence or absence of different concentrations of zoledronate (0.5, 5, 50 µM) and denosumab (1, 10, 20, 40 µg/mL) using the real-time xCELLigence system. Additionally, a live/dead staining was performed by fluorescence microscopy. RESULTS: THP-1 cells demonstrated a regular initial PMA-induced differentiation to macrophages by live measurements of cell adherence and by an increase in CD68 surface expression as detected by flow cytometry. The addition of zoledronate led to cell detachment of the THP-1-derived macrophages in a dose-dependent manner in contrast to denosumab. Cell detachment was based on cell death as confirmed by live/dead staining, revealing elevated numbers of dead cells following addition of high zoledronate concentrations. However, denosumab did not deteriorate THP-1 cell viability. CONCLUSION: Our results demonstrate that zoledronate but not denosumab suppresses monocytic THP-1 cell viability after macrophagic differentiation dose-dependently. CLINICAL RELEVANCE: This is the first real-time study providing evidence for a dose-dependent immunosuppressive effect of zoledronate in contrast to denosumab on local macrophages.

Apoptosis resistance-related ABCB5 and DNaseX (Apo10) expression in oral carcinogenesis.

BACKGROUND: Apoptosis resistance is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of apoptosis resistance-related ATP-binding cassette (ABC) transporter ABCB5 [subfamily B (MDR/TAP) member 5] and DNaseX (Apo10) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry. RESULTS: Expression of ABCB5 and Apo10 were significantly increased in the carcinogenesis of OSCC compared with normal tissue. Compared with SIN I-III, ABCB5 expression was significantly decreased in OSCC. Apo10 expression did not significantly differ from OSCC compared with SIN I-III. CONCLUSIONS: This study provides the first evidence of the expression of ABCB5 and Apo10 in the multi-step carcinogenesis of OSCC. Overcoming drug resistance of ABCB5+ and Apo10+ cells in precursor lesions and tumors by natural compounds may act as sensitizers for apoptosis or could be useful for chemoprevention.

Co-expression of CD44+/RANKL+ tumor cells in the carcinogenesis of oral squamous cell carcinoma.

Receptor activator of nuclear factor-kappa (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL) signaling helps putative cancer stem cells (CSC) to maintain their stemness. Expression of CD44 and RANKL was analyzed in oral squamous cell carcinoma specimen (n = 191). Moreover, RANKL expression was measured in cancer cell lines (BICR3, BICR56) by immunohistochemistry and western blot analysis. Scanned images were digitally analyzed using ImageJ and the immunomembrane plug-in. CD44 and RANKL expression on protein level was correlated with clinical characteristics and impact on survival. RANKL was co-labeled with CD44 in immunohistochemical and immunofluorescence double labeling experiments. Although high CD44+/RANKL+ co-expression was significantly associated with clinicopathological factors and worse survival, multivariate analysis did not demonstrate high CD44+/RANKL+ co-expression as independent prognostic factor. Immunohistochemical and immunofluorescence double labeling experiments revealed RANKL expression by CD44+ cancer cells. RANKL specificity was confirmed by western blot analysis. For the first time, this study provides evidence that RANKL expression in OSCC might be associated with disease recurrence and a cell compartment measured by CD44+/RANKL+ co-expression within the mucosal epithelial basal layer cells.

Association of cancer metabolism-related proteins with oral carcinogenesis - indications for chemoprevention and metabolic sensitizing of oral squamous cell carcinoma?

BACKGROUND: Tumor metabolism is a crucial factor for the carcinogenesis of oral squamous cell carcinoma (OSCC). METHODS: Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, PFK-1, LDHA, TKTL1), mitochondrial enzymes (SDHA, SDHB, ATP synthase) were analyzed in normal oral mucosa (n = 5), oral precursor lesions (simple hyperplasia, n = 11; squamous intraepithelial neoplasia, SIN I-III, n = 35), and OSCC specimen (n = 42) by immunohistochemistry and real-time polymerase chain reaction (qPCR) analysis in OSCC cell lines. Metabolism-related proteins were correlated with proliferation activity (Ki-67) and apoptotic properties (TUNEL assay) in OSCC. Specificity of antibodies was confirmed by western blotting in cancer cell lines. RESULTS: Expression of IGF-R1, glycolysis-related proteins (GLUT-1, HK 2, LDHA, TKTL1), and mitochondrial enzymes (SDHA, SDHB, ATP synthase) were significantly increased in the carcinogenesis of OSCC. Metabolic active regions of OSCC were strongly correlated with proliferating cancer (Ki-67+) cells without detection of apoptosis (TUNEL assay). CONCLUSIONS: This study provides the first evidence of the expression of IGF-R1, glycolysis-related proteins GLUT-1, HK 2, PFK-1, LDHA, and TKTL1, as well as mitochondrial enzymes SDHA, SDHB, and ATP synthase in the multi-step carcinogenesis of OSCC. Both, hypoxia-related glucose metabolism and mitochondrial oxidative phosphorylation characteristics are associated with the carcinogenesis of OSCC. Acidosis and OXPHOS may drive a metabolic shift towards the pentose phosphate pathway (PPP). Therefore, inhibition of the PPP, glycolysis, and targeted anti-mitochondrial therapies (ROS generation) by natural compounds or synthetic vitamin derivatives may act as sensitizer for apoptosis in cancer cells mediated by adjuvant therapies in OSCC.