Novel Histopathological Findings of Micro Bone Fragments and Epithelial Response in the Oral Mucosa in Bisphosphonate-Related Osteonecrosis of the Jaw.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) occurs in the jawbone and interfacing oral mucosa of patients treated with bisphosphonates. Herein, we report novel histopathological findings in the oral mucosa of a surgical specimen obtained from a 61-year-old man with BRONJ. The resected jawbone and adjacent oral mucosa were separated for histological examination. The mucosal tissue was examined using Von Kossa staining and immunohistochemical (CK5/6, p63) staining of non-decalcified paraffin sections. Pseudoepitheliomatous hyperplasia (PEH), a microscopic feature of the mucosal epithelium in BRONJ, was observed in soft tissue specimens, concomitant with inflammatory cell infiltration. Von Kossa staining revealed small fragments of necrotic bone, tens to hundreds of micrometers in size, scattered within the connective tissues; the PEH forefront contacted some of the bone fragments. Immunohistochemical staining demonstrated that occasionally, the PEH not only contacted but also encompassed the bone fragments. To our knowledge, this is the first report of presence of micro bone fragments and their association with PEH in the oral mucosa in BRONJ.

Predictive Value of Neutrophil-Lymphocyte Ratio as a Marker in Antiresorptive Agent-Related Osteonecrosis of the Jaw: A Retrospective Analysis.

Antiresorptive agent-related osteonecrosis of the jaw (ARONJ), a multifactorial disease, can drastically affect a patient's quality of life. Moreover, disease progression to severe acute inflammation can hinder treatment. Therefore, we aimed to investigate the diagnostic value of the neutrophil−lymphocyte ratio (NLR) and platelet−lymphocyte ratio (PLR) in predicting the risk of acute inflammation in patients with ARONJ. In total, 147 patients with ARONJ were enrolled between 1 January 2011 and 31 December 2019. They were divided into two groups according to their baseline NLR (high NLR vs. low NLR) or PLR (high PLR vs. low PLR) to analyze the relationship between NLR and PLR and the outcomes of acute inflammatory events. An optimal NLR cut-off value of 2.83 was identified for hospitalization for an inflammatory event. Logistic regression analysis showed that NLR > 2.83 was associated with an increased risk of hospitalization for an inflammatory event. A PLR cut-off value of 165.2 was identified for hospitalization for an inflammatory event. However, logistic regression analysis showed that PLR > 165.2 was not significantly associated with hospitalization for an inflammatory event. Our study findings suggest that the NLR has diagnostic value in predicting the risk of hospitalization for inflammatory events among patients with ARONJ.

Role of Phosphodiesterase2A in Proliferation and Migration of Human Osteosarcoma Cells.

BACKGROUND/AIM: The prognosis of patients with osteosarcoma is poor; therefore, new treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of the 11 families (PDE1-PDE11) of the phosphodiesterase superfamily that regulates the intracellular concentrations and effects of cAMP and cGMP. This in vitro study was performed to investigate the role of PDE2 in human oral osteosarcoma HOSM-1 cells. MATERIALS AND METHODS: PDE2 expression was measured by a cAMP-PDE assay and real-time-PCR. The effects of the PDE2-specific inhibitors, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), 8-bromo-cAMP, and 8-bromo-cGMP on cell proliferation and migration were assessed. RESULTS: PDE2 activity and PDE2A mRNA expression were detected in HOSM-1 cells. Cell proliferation was inhibited by EHNA and 8-bromo-cAMP but not by 8-bromo-cGMP. Cell migration was stimulated by EHNA and 8-bromo-cGMP, but it was inhibited by 8-bromo-cAMP. CONCLUSION: Cell proliferation is regulated by PDE2-cAMP signaling and cell migration is regulated by PDE2-cGMP signaling in HOSM-1 cells.

Evaluation of a New Hydroxyapatite Nanoparticle as a Drug Delivery System to Oral Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: Due to its abilities of substance adsorption and intracellular transportation, hydroxyapatite is a potential carrier in drug delivery systems (DDS). This in vitro study investigated whether newly-developed, highly-dispersive calcined hydroxyapatite nanoparticles with an average grain diameter of 20 nm (nano-SHAP) were suitable as a DDS for the drugs zoledronic acid (ZA), cisplatin, and carboplatin. MATERIAL AND METHODS: The effects of drug-bearing nano-SHAP on cell proliferation were assessed using three human oral squamous cell carcinoma cell lines (HSC-4, KOSC, and SAS) and one human breast cancer cell line (MCF-7). RESULTS: Nano-SHAP alone did not affect proliferation of any cell line until a concentration of 1 µg/ml was reached. Although the effective concentration of ZA in ZA-bearing nano-SHAP differed, it inhibited cell proliferation better than ZA alone. Cisplatin and carboplatin-bearing nano-SHAP had the same effect as these drugs alone. CONCLUSION: The nano-SHAP system is of potential use as a drug delivery system.

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells.

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N(6)-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.

Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells.

The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.

Phosphodiesterase 4 regulates the migration of B16-F10 melanoma cells.

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. Eleven PDE gene families (PDE1-11) have been identified and several PDE isoforms are selectively expressed in various cell types. PDE4 family members specifically hydrolyze cyclic AMP (cAMP). Four genes (PDE4A-D) are known to encode PDE4 enzymes, with additional diversity generated by the use of alternative mRNA splicing and the use of different promoters. While PDE4 selective inhibitors show therapeutic potential for treating major diseases such as asthma and chronic obstructive pulmonary disease, little is known concerning the role of PDE4 in malignant melanoma. In this study, we examined the role of PDE4 in mouse B16-F10 melanoma cells. In these cells, PDE4 activity was found to be ∼60% of total PDE activity. RT-PCR detected only PDE4B and PDE4D mRNA. Cell growth was inhibited by the cAMP analog, 8-bromo-cAMP, but not by the specific PDE4 inhibitors, rolipram and denbufylline, which increased intracellular cAMP concentrations. Finally, migration of the B16-F10 cells was inhibited by the PDE4 inhibitors and 8-bromo-cAMP, while migration was increased by a protein kinase A (PKA) inhibitor, PKI(14-22), and was not affected by 8-pCPT-2'-O-Me-cAMP, which is an analog of exchange protein activated by cAMP (Epac). The inhibitory effect of rolipram on migration was reversed by PKI(14-22). Based on these results, PDE4 appears to play an important role in the migration of B16-F10 cells, and therefore may be a novel target for the treatment of malignant melanoma.

Expression and role of phosphodiesterase 5 in human malignant melanoma cell line.

BACKGROUND: Eleven phosphodiesterase (PDE) gene families (PDE1-11) have been identified, and some PDE isoforms are selectively expressed in various cell types. Previously, we reported PDE1, PDE3 and PDE4 expressions in human malignant melanoma cells. However, the expression and role of PDE5 in malignant melanoma cells is not clear. Therefore, we characterized PDE5 in human malignant melanoma MAA cells. MATERIALS AND METHODS: PDE5 activity and PDE5A mRNA expression were investigated in MAA cells. The full open reading frames for human PDE5A1 were sequenced. Effects of PDE5 inhibitors on cell growth were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays. RESULTS: PDE5 activity and PDE5A1 mRNA expression were detected in MAA cells. The nucleotide sequence of PDE5A1 was identical to that of human PDE5A1, previously published. Two PDE5 inhibitors inhibited the growth of cells. CONCLUSION: PDE5A1 mRNA is expressed and may play an important role in the growth of human malignant melanoma MAA cells.

Phosphodiesterase 3 (PDE3): structure, localization and function.

Cyclic adenosine 3'5'-monophosphate (cAMP) and cyclic guanosine 3'5'-monophosphate (cGMP) are critical intracellular messengers involved in transduction of signals generated by a wide variety of extracellular stimuli, including growth factors, cytokines, peptide hormones, light and neurotransmitters. These messengers modulate many fundamental biological processes, including myocardial contractility, platelet aggregation, vascular smooth muscle relaxation, proliferation and apoptosis, etc. Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cAMP and cGMP, and are important in regulating intracellular concentrations and biological actions of these signal-transducing molecules. These enzymes contain at least 11 highly regulated and structurally related gene families (PDE1-11). In this review, we will discuss some general information of PDEs and then focus on PDE3 gene family, including the molecular biology, structure, function and potential as therapeutic targets. Furthermore, we show the possibilities of PDE3 as therapeutic targets in malignant tumor cells and salivary gland.

Characterization of phosphodiesterase 1 in human malignant melanoma cell lines.

BACKGROUND: Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one] from Dictyostelium discoideum exhibits antiproliferative activity in mammalian cells. We have previously shown that phosphodiesterase 1 (PDE1) is a pharmacological target of DIF-1, but there are no reports of PDE1 in human malignant melanoma cells. Therefore, we characterized PDE1 in human malignant melanoma MAA cells. MATERIALS AND METHODS: PDE1 mRNA expression was investigated in MAA cells. The full open reading frames for human PDE1C1 and PDE1C3 were cloned. Cell growth was determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. RESULTS: PDE1C mRNA expression was detected in MAA cells. The nucleotide sequence of PDE1C1 was identical to that of human PDE1C1, previously published. At nucleotide 2246 in PDE1C3, A was replaced by G, but this did not change the encoded amino acid. Cell growth was inhibited by the PDE1 inhibitor vinpocetin. CONCLUSION: PDE1C mRNA is expressed and may play an important role in human malignant melanoma MAA cells.

A role for cyclic nucleotide phosphodiesterase 4 in regulation of the growth of human malignant melanoma cells.

The activity, expression and function of phosphodiesterase 4 (PDE 4) were investigated in the HMG human gingiva-derived malignant melanoma cell line. A specific PDE4 inhibitor, rolipram, inhibited PDE activity in homogenates of HMG cells, and PDE4B and 4D mRNAs were detected by RT-PCR in RNA from HMG cells. Two specific PDE4 inhibitors, rolipram and Ro-20-1724, and an adenylate cyclase activator, forskolin, increased intracellular cAMP in HMG cells. Cell growth induced by rolipram, Ro-20-1724, and forskolin was inhibited by the H-89 protein kinase A (PKA) inhibitor. However, in contrast to effects of H-89, two other PKA inhibitors, KT5720 and PKI, did not inhibit rolipram-induced cell growth. A cAMP analogue that selectively activates Epac, 8-pCPT-2'-O-Me-cAMP, also promoted the growth of HMG cells. These findings suggested that PDE4, PDE4B and/or 4D regulate cell growth through cAMP targets in the HMG malignant melanoma cell line. There have been no previous studies of positive regulation of cell growth by PDE4 inhibition, suggesting that it may be possible to target PDE4 in therapy for human malignant melanoma.

Expression of cyclic nucleotide phosphodiesterase 3A in isolated rat submandibular acini.

Phosphodiesterase (PDE) 3 has been characterized in isolated rat submandibular acini. PDE3 activity was detected in homogenates of isolated rat submandibular acini; little or no PDE3 activity was found in ducts. About 62% of PDE3 activity in the acini was recovered in the supernatant fractions; 38% in particulate fractions. In the acini, but not ducts, PDE3A mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The PDE3-specific inhibitor, cilostamide, increased the ratio of apomucin mRNA/18s rRNA, as quantified by real-time RT-PCR. Our results indicate that PDE3A may be important in regulating cAMP pools that control acini functions.

Characterization of the human mandibular osteoblastic osteosarcoma cell line HOSM-2 after long-term culture.

We have been subculturing a human mandible-derived osteosarcoma cell line (HOSM-2) for approximately 15 years, and have compared the characters of early generations, which did not exhibit tumorigenicity, to those in the later generations. The shape and doubling time of the cells did not change during long-term culture. The number of chromosomes, however, changed from 59-81 in the 6th generation (modal number: 70) to 54-59 (modal number: 56 and 57), and the chromosomal structure also changed. In addition, the cell line in the later generations showed tumorigenicity in nude mice, and Codon 306 of the p53 gene was mutated to a stop codon due to a point mutation. HOSM-2 cells expressed osteoblast markers, thus confirming them to be osteoblastic osteosarcoma cells. These results showed that changes in certain genes in the HOSM-2 cells led to tumorigenicity in nude mice following long-term culture. In addition, as a mandible-derived cell line with characteristics different from those of limb-derived osteosarcoma cell lines, HOSM-2 cells may be a valuable model for mandibular osteosarcoma and osteoblasts.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is a pharmacological target of differentiation-inducing factor-1, an antitumor agent isolated from Dictyostelium.

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10-40 micro M dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5-20 micro M inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Magnetic resonance imaging of synovial proliferation in temporomandibular disorders with pain.

OBJECTIVE: The objective of this study was to assess the clinical significance of synovial proliferation in patients with painful temporomandibular disorders based on magnetic resonance imaging findings. METHODS: The current study was conducted in 100 joints of 100 patients with unilateral painful temporomandibular disorders. One hundred joints on the contralateral side of patients with unilateral disease were used as nonpain group. Areas in the articular space that showed a low signal intensity on T1-weighted imaging, a high signal intensity on T2-weighted imaging, and high signal intensity on gadolinium-enhanced fat-suppressed T1-weighted imaging were judged to be regions of synovial proliferation. RESULTS: Synovial proliferation alone was observed in 8.0% of the pain group, but in none of the nonpain group. Synovial proliferation + effusion was observed in 33.0% of the pain group and in 7.0% of the nonpain group. Effusion alone was observed in 7.0% of the pain group and in 3.0% of the nonpain group. The mean visual analog scale value of pain was in the order of synovial proliferation alone > synovial proliferation + effusion > effusion alone. The incidence rates of anterior displacement of the disk were 100% for synovial proliferation alone, 93.9% for synovial proliferation + effusion, 57.1% for effusion alone, and 57.7% for "without synovial proliferation/effusion." CONCLUSIONS: Strong correlations were observed between synovial proliferation, pain, and disk displacement. It is considered that evaluating effusion alone provides only limited information on the disease state in painful temporomandibular disorders. Thus, it is essential to include enhanced T1-weighted imaging as a means to judge the disease state as well as to assess disease progression.

Phosphodiesterase 4 in osteoblastic osteosarcoma cells as a potential target for growth inhibition.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that HOSM-1 cells, an osteosarcoma cell line established from human mandible, expressed mRNA for osteoblastic markers, such as alkaline phosphatase, osteonectin, osteocalcin and parathyroid hormone receptor, thus exhibiting an osteoblastic phenotype. We have investigated a possible role of cyclic nucleotide phosphodiesterases (PDEs) in osteosarcoma cells. RT-PCR analysis revealed that HOSM-1 cells expressed mRNA for PDE4A, 4B and 4C. In addition, rolipram, a specific inhibitor of PDE4, inhibited HOSM-1 cell proliferation. The finding that PDE4 is involved in proliferation of osteosarcoma cells suggests the possibility that PDE4 may be a new target for antitumor therapy.

Effect of the phosphodiesterase 4 inhibitor, rolipram, on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 cell line.

We have evaluated effects of a phosphodiesterase (PDE) 4 inhibitor on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 clone 8 (10T1/2) cell line. 10T1/2 cells were cultured in minimum essential medium (MEM) and 10% fetal bovine serum with or without 1 microM retinoic acid and/or the PDE 4 inhibitor, rolipram, and harvested at specific intervals before measurement of alkaline phosphatase activity, cAMP production in response to parathyroid hormone, osteocalcin synthesis and expression, and phosphodiesterase activity. Retinoic acid-increased alkaline phosphatase activity, and slightly enhanced cAMP production in response to parathyroid hormone. However, it did not affect osteocalcin synthesis and expression. In the presence of retinoic acid, PDE 4 activity was not changed. A PDE 4 inhibitor, rolipram, and cAMP analog, 8-bromo-cAMP dramatically increased retinoic acid's ability to induce alkaline phosphatase activity. This is the first report that PDE 4 may be involved in regulation of retinoic acid-increased alkaline phosphatase activity.

Expression and role of phosphodiesterase 3 in human squamous cell carcinoma KB cells.

Phosphodiesterase (PDE) 3s have been characterized in human squamous cell carcinoma KB cells. PDE3 activity was detected in homogenates of KB cells. PDE3A and 3B mRNAs were detected by RT-PCR in RNA from KB cells; the nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. Immunoblotting with anti-PDE3 antibodies detected both PDE3A- and 3B-immunoreactive proteins in KB cells. The PDE3-specific inhibitor, cilostamide, inhibited the proliferation of KB cells. Our results indicate that PDE3s may be important regulators of the growth of KB cells. Therefore, PDE3 inhibitors may be potential new drugs for antiproliferative therapies in squamous cell carcinoma in the head and neck.

Characterization of phosphodiesterase 3 in human malignant melanoma cell line.

Little is known concerning the expression, distribution and function of phosphodiesterase (PDE) 3s in malignant tumor cells, including human malignant melanoma HMG and osteosarcoma HOSM-1 cells. PDE3 activity was detected in homogenates of HMG cells; however, much less activity was found in HOSM-1 cells. In HMG cells, most of the PDE3 activity was in the particulate fraction. PDE3A and 3B mRNAs were detected by RT-PCR in RNA from HMG cells only. The nucleotide sequences of the fragments were identical to those of human PDE3A and 3B. The PDE3-specific inhibitors, trequinsin and cilostamide, did not inhibit the proliferation of HMG or HOSM-1 cells. Although two PDE3 isoforms may be expressed in human malignant melanoma cells, their functional importance is not known.