Ioannis Gkouveris

Erk1/2 activation and modulation of STAT3 signaling in oral cancer

Constitutive activation of the signal transducer and activator of transcription 3xa0(STAT3) signaling pathway possesses confirmed oncogenic potential in oral squamous cell carcinomaxa0(OSCC). Crosstalk with other molecular pathways contributes to STAT3 regulation in cancer. The effects of mitogen-activated protein kinasesxa0(MAPKs) and particularly extracellular signal-regulated kinase 1/2xa0(Erk1/2) on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examined the effects of Erk1/2 modulation on STAT3 signaling and cell growth in OSCC cells. Constitutive expression levels of phosphorylated (tyrosine and serine) and total STAT3, Erk1/2 and cyclinxa0D1 were assessed in OSCC cell lines. Erk1/2 modulation was achieved by pharmacological agents; siRNA silencing against Erk1/2 was also performed. Cell proliferation and viability were assessed. Erk1/2 inhibition with either U0126 treatment or specific siRNA silencing resulted in decreases in p-serxa0STAT3 and cyclinxa0D1 levels and increases in p-tyr STAT3 in OSCC cells. Moreover, Erk1/2 inhibition resulted in a dose-dependent reduction in OSCC cell growth and viability. Erk1/2 induction had the opposite effects. Taken together, these results are supportive of an active crosstalk between the oncogenic Erk1/2 and STAT3 pathways in OSCC, the significance of which requires further investigation.

Zoledronate Impairs Socket Healing after Extraction of Teeth with Experimental Periodontitis

Osteonecrosis of the jaws (ONJ) is a rare but severe complication of antiresorptive medications, such as bisphosphonates, used in the treatment of bone malignancy or osteoporosis. Tooth extraction and dental disease have been strongly associated with ONJ development. Here, we investigated molecular and cellular markers of socket healing after extraction of healthy or teeth with experimental periodontitis (EP) in Wistar-Han rats treated with zoledronic acid (ZA). We included 4 experimental groups: vehicle-treated animals with extraction of healthy teeth or teeth with ligature-induced EP and ZA-treated animals with extraction of healthy teeth or teeth with EP. Animals were pretreated with vehicle or ZA for a week, and EP was induced. Four weeks later, the second maxillary molars were extracted; sockets were allowed to heal for 4 wk; animals were euthanized; and maxillae were isolated. Radiographically, extraction sockets in groups 1, 2, and 3 demonstrated normal healing. Contrary incomplete socket healing was noted after extraction of teeth with EP in ZA-treated rats of group 4. Histologically, persistent inflammation and extensive osteonecrosis were seen in group 4. Disorganization of the collagen network, collagen type III predominance, and lack of collagen fiber insertion in the necrotic bone were associated with impaired socket healing. Cells positive for MMP-9, MMP-13, and α-SMA expression were present at the areas of epithelial invagination and adjacent to osteonecrotic bone. Importantly, human biopsies from patients with ONJ showed similar findings. Our data emphasize the importance of dental disease and tooth extraction in ONJ pathogenesis and help delineate an altered profile in wound-healing markers during ONJ development.

Role of JNK signaling in oral cancer: A mini review

JNKs (c-Jun N-terminal kinases) belong to mitogen-activated protein kinases’ family and become activated by several growth factors, stress, radiation, and other extracellular signals. In turn, JNK activation results in phosphorylation of downstream molecules involved in many normal cellular processes. Nevertheless, recent data have linked JNK signaling with several pathological conditions, including neurodegenerative diseases, inflammation, and cancer. The role of JNK in cancer remains controversial. Initially, JNK was thought to play a rather oncosuppressive role by mediating apoptosis in response to stress stimuli, inflammatory, or oncogenic signals. However, a number of studies have implicated JNK in malignant transformation and tumor growth. The contradictory functions of JNK in cancer may be due to the diversity of JNK upstream and downstream signaling and are under intensive investigation. This review summarizes current literature focusing on the significance of JNK pathway in cancer development and progression, particularly addressing its role in oral cancer. Understanding the complexity of JNK signaling has the potential to elucidate important molecular aspects of oral cancer, possibly leading to development of novel and individualized therapeutic strategies.

ERK1/2, JNK and STAT3 activation and correlation with tumor differentiation in oral SCC

Signal transducer and activator of transcription 3 (STAT3) and mitogen activated protein kinases (MAPKs), including ERK and JNK, have been implicated in oral squamous cell carcinoma (OSCC) development and progression. Our purpose was to evaluate the levels of activated STAT3, ERK1/2 and JNK by immunohistochemistry in OSCC and to investigate possible correlations of these molecules with each other as well as with the degree of tumor differentiation. Immunohistochemical assessment of the phosphorylated levels of STAT3(tyrosine/ serine), ERK1/2 and JNK was performed in 60 OSCC, including well, moderately and poorly differentiated tumors. Semiquantitative scoring system was used, by calculating intensity of immunostaining, percentage of positive cells and combined scores. Statistics included Fishers test, Students T-Test and Kruskal-Wallis analysis, Spearmans correlation coefficient and multivariate logistic regression analyses. Immunohistochemical levels of both pSTAT3(tyr) and pERK1/2 showed statistically significant differences between well and poorly differentiated tumors with the latter receiving higher mean percentage, intensity and total scores. On the other hand, pJNK showed statistically significantly higher intensity levels in moderately compared to poorly differentiated tumors. pSTAT3(ser) immunoexpression did not appear to correlate with tumor differentiation. Between different molecules, more pronounced, pERK1/2 levels exhibited statistically significant positive correlation with pSTAT3(ser), pSTAT3(tyr) and pJNK expression. ERK1/2 and STAT3 activation (as assessed by tyrosine but not serine phosphorylation) could contribute to a less differentiated phenotype in OSCC, while JNK activation may have an opposite, although possibly less pronounced, effect. Positive correlations between MAPK and STAT3 levels may indicate a direct crosstalk and/or regulation by common upstream pathways.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14u2009±u20095%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33u2009±u200920%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1u2009±u20090.6% vs. control mice (0.5u2009±u20090.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

p38 Expression and Modulation of STAT3 Signaling in Oral Cancer

p38 protein belongs to Mitogen-activated protein kinases family that link extracellular stimuli with intracellular responses participating in numerous of fundamental cell processes. Persistent activation of STAT3 has been associated with cell proliferation, differentiation and apoptosis in oral squamous cell carcinoma (OSCC). This study examines the effects of p38 modulation on STAT3 signaling and cellular activities in OSCC cells and investigates possible correlation of p38 expression with tumor degree of differentiation. Phospho-p38 immunostaining was performed in 60 OSCC including well, moderately and poorly differentiated tumors. Semiquantitative analysis was used, by calculating intensity, percentage and combined scores. Protein expression levels of STAT3 (total, tyrosine and serine phosphorylated), p38 and cyclin D1 were assessed in two OSCC cell lines. p38 inhibition was achieved by pharmacological agent(SB2023580). Cell proliferation and viability rates were also evaluated. Phospho-p38 immunoexpression was intense in almost all tumor specimens, nevertheless did not correlate with tumor differentiation. Inhibition of p38 with SB203580 did not appear to affect tyrosine or serine phosphorylated STAT3 as well as cyclin D1 levels in both cell lines. Moreover, p38 inhibition resulted in mild dose-dependent decreases in cell growth and viability in both cell lines. p38 is highly expressed in OSCC but does not seem to mediate the oncogenic STAT3 pathway. However, changes found in proliferation and viability may suggest that p38 functions as potent regulator of HNSCC. Understanding the complexity of p38 signaling and cross-talk between other major molecules, may guide the development of novel pharmacologic therapies for cancer treatment and prevention.

A preliminary immunohistochemical study of signal transducer and activator of transcription (STAT) proteins in primary oral malignant melanoma

OBJECTIVEnPrimary oral malignant melanoma (POMM) is a rare type of malignancy with a very poor prognosis, the molecular pathogenesis of which remains elusive. The aim of this study was to assess the expression status of signal transducer and activator of transcription (STAT) proteins in POMM.nnnSTUDY DESIGNnSix POMMs were included in the study. Total protein levels of STAT1, STAT3, and STAT5a, as well as the tyrosine phosphorylated (activated) form of STAT3 (pSTAT3), were assessed immunohistochemically.nnnRESULTSnImmunohistochemical evaluation of total STAT3 revealed diffuse and strong cytoplasmic and nuclear expression in the majority of tumor cells of all cases, whereas activated pSTAT3 had mostly mild nuclear expression in 5%-40% of malignant melanocytes in all cases. Evaluation of STAT1 and STAT5a identified mainly mild cytoplasmic expression in the absence of nuclear localization.nnnCONCLUSIONnThe identification of aberrant STAT3 expression and activation in oral malignant melanocytes supports a possible role of this molecule in POMM. In contrast, STAT5a has only limited cytoplasmic expression, mitigating against its involvement in POMM. Also, STAT1s low levels may have implications for POMM sensitivity to interferon-based therapeutic strategies, considering the role of this molecule in cutaneous melanoma immunotherapy.

Alterations in the expression of DNA damage response-related molecules in potentially preneoplastic oral epithelial lesions

OBJECTIVESnThe aim of this study was to evaluate the expression levels of DNA damage response (DDR) markers in potentially preneoplastic oral epithelial lesions (PPOELs).nnnSTUDY DESIGNnImmunohistochemical expression of DDR markers (γΗ2 ΑΧ, pChk2, 53 BP1, p53, and phosphorylated at Ser 15 p53) was assessed in 41 oral leukoplakias, ranging from hyperplasia (H) to dysplasia (D) and in comparison with oral squamous cell carcinoma (OSCC) and normal mucosa (NM). Statistical and receiver operating characteristic curve analysis were performed.nnnRESULTSnγH2 AX immunoexpression demonstrated a gradual increase and upper layer extension from NM to H to higher D degrees to OSCC. pChk2 expression was minimal in NM, relatively low in PPOELs, with an increasing tendency from H to D, and higher in OSCC. 53 BP1 demonstrated higher levels in OSCC than in NM, whereas its expression in PPOELs was heterogeneous, gradually increasing according to D. p53 demonstrated progressively higher levels and upper layer extension from H to D to OSCC. Phosphorylated p53 was absent in NM and relatively low in PPOELs and OSCC.nnnCONCLUSIONSnDDR markers expression is variable in PPOELs, showing a tendency to increase along with dysplasia. Activated DDR mechanisms may play an important protective role at early stages of oral carcinogenesis, but probably suffer progressive deregulation, eventually failing to suppress malignant transformation.

Development of Medication-Related Osteonecrosis of the Jaw After Extraction of Teeth With Experimental Periapical Disease

PURPOSEnMedication-related osteonecrosis of the jaw (MRONJ) is a rare but severe side effect of antiresorptive medications. Most animal models use tooth extraction as an instigating local factor to induce MRONJ, with varied results. However, these teeth are healthy and absent of dental disease, a rare finding that does not reflect clinical practices. The authors hypothesized that extraction of teeth with periapical inflammation would lead to MRONJ in rats treated with high-dose bisphosphonates.nnnMATERIALS AND METHODSnRats were pretreated with zoledronic acid (ZA) for 1xa0week. Pulp exposure (PE) was established by exposing the pulpal chamber of the first and secondxa0molars. Experimental periapical disease (EPD) was induced by PE and bacterial inoculation into pulp chambers of the first and second mandibular molars. The mandibular molars were extracted 4xa0weeks after PE or EPD, and animals were euthanized 4xa0weeks after tooth extraction. Extraction sockets were assessed clinically, radiographically, and histologically.nnnRESULTSnClinically, radiographically, and histologically, socket healing was observed in all vehicle-treated animals and in ZA-treated animals after extraction of healthy teeth or teeth with PE. In contrast, bone exposure, lack of socket healing, and osteonecrosis were present in most ZA-treated animals after extraction of teeth with EPD. Bacterial presence was noted in areas of osteonecrotic alveolar bone.nnnCONCLUSIONnThese data support a synergistic contribution of severe dental disease and tooth extraction to MRONJ pathogenesis. Importantly, this model is amenable to manipulation of methodologic conditions for the dissection of parameters involved in MRONJ pathogenesis.

Clinically Relevant Doses of Sclerostin Antibody Do Not Induce Osteonecrosis of the Jaw (ONJ) in Rats with Experimental Periodontitis: SCLEROSTIN ANTIBODY DOES NOT INDUCE ONJ

Antiresorptive agents, such as bisphosphonates and denosumab, are frequently used for the management of osteoporosis. Indeed, both medications decrease the risk of osteoporotic fractures; however, these medications are associated with rare but potentially severe side effects, such as osteonecrosis of the jaw (ONJ). ONJ, defined as an area of exposed bone in the maxillofacial region that lasts for 8 weeks, often presents with significant pain and infection and can lead to serious complications. Interestingly, other treatments for osteoporosis have been developed, such as antibodies against the osteocyte‐secreted protein, sclerostin. Sclerostin functions to inhibit the Wnt signaling cascade, leading to inhibition of bone formation. In clinical trials, a sclerostin antibody (romosozumab, Amgen Inc., UCB Brussels) increases bone formation and lowers the risk of osteoporotic fractures. However, in conjunction with increased osteoblastic activity, a reduction in bone resorption markers is observed. This antiresorptive effect raises the concern of possible ONJ development in patients treated with sclerostin antibodies. Here, utilizing ligature‐induced experimental periodontitis (EP), we evaluated the effects of sclerostin inhibition on the development of ONJ‐like lesions in ovariectomized rats. Beginning 8 weeks post‐ovariectomy, rats were treated for 22 weeks with weekly injections of vehicle (Veh), 200u2009μg/kg zoledronic acid (ZA), a potent bisphosphonate at 100‐fold the osteoporosis dose, or 5u2009mg/kg sclerostin antibody (Scl‐Ab) at the osteoporotic dose. EP was initiated at week 12 and maintained for the remainder of the study. Scl‐Ab treatment transiently increased serum P1NP, a bone formation marker, increased BV/TV, and decreased eroded surfaces in lumbar vertebrae. ZA‐treated rats developed histologic features of ONJ, whereas Veh‐treated controls did not. Scl‐Ab animals lost less periodontal bone in sites with EP. However, these animals presented with no histologic signs of ONJ. In conclusion, sclerostin inhibition enhanced structural bone parameters, without inducing ONJ‐like lesions, in ovariectomized rats with EP.