Risa Chaisuparat

Effect of zoledronic acid on oral fibroblasts and epithelial cells: a potential mechanism of bisphosphonate-associated osteonecrosis.

Osteonecrosis of the jaw secondary to bisphosphonate infusion (zoledronic acid‐ZA) is assumed to be a bone disease. This study investigated the effects of ZA on soft tissues using oral mucosal cells as an in vitro model of soft tissue cell death in the pathogenesis of bone necrosis. Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations of ZA (0·25–3 μmol/l), using 1 μmol/l as the expected baseline concentration. A dose–response effect on apoptosis and cell proliferation [Terminal deoxynucleotidyl transferase‐mediated dUTP‐Biotin End Labelling and Annexin V or Coulter counter and 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium), respectively] was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Gene expression analysis using RT2 Profiler polymerase chain reaction Arrays demonstrated the differential expression of multiple genes involved in apoptosis including those that encode TNF, BCL‐2, Caspase, IAP, TRAF and Death Domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression. This study demonstrated that low concentrations of ZA rapidly and directly affected the oral mucosal tissues though the induction of a gene‐regulated apoptotic process. These findings support the potential for soft tissue injury as an initiating/potentiating event for osteonecrosis.

Dual Inhibition of PI3Kα and mTOR as an Alternative Treatment for Kaposi's Sarcoma

Rapamycin (or sirolimus), the prototypical inhibitor of the mammalian target of rapamycin (mTOR) and an immunosuppressant used for the prevention of renal transplant rejection, has recently emerged as an effective treatment for Kaposis sarcoma (KS), an enigmatic vascular tumor and a model for pathologic angiogenesis. Indeed, recent work supports a role for mTOR as a central player in the transformation of endothelial cells by the KS-associated herpesvirus-encoded G protein-coupled receptor (vGPCR), the viral oncogene believed to be responsible for causing KS. However, emerging evidence that rapamycin may transiently promote the activation of Akt may limit its use as an anti-KS therapy. Here, we show that activation of Akt in endothelial cells expressing vGPCR is augmented by treatment with rapamycin, resulting in the up-regulation of several Akt proliferative and survival pathways. However, use of a novel dual phosphatidylinositol 3-kinase alpha (PI3Kalpha)/mTOR inhibitor, PI-103, effectively and independently blocked activation of both PI3K and mTOR in vGPCR-expressing endothelial cells. This resulted in more effective inhibition of endothelial cell proliferation and survival in vitro and tumor growth in vivo. Our results suggest that PI-103 may be an effective therapeutic option for the treatment of patients with KS. Moreover, as KS may serve as a model for pathologic angiogenesis, our results further provide the basis for the early assessment of PI-103 as an antiangiogenic chemotherapeutic.

Amplification of the Angiogenic Signal through the Activation of the TSC/mTOR/HIF Axis by the KSHV vGPCR in Kaposi's Sarcoma

Background Kaposis sarcoma (KS) is a vascular neoplasm characterized by the dysregulated expression of angiogenic and inflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascular endothelial growth factor (VEGF), essential for KS development. However, the origin of VEGF in this tumor remains unclear. Methodology/Principal Findings Here we report that the KSHV G protein-coupled receptor (vGPCR) upregulates VEGF in KS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate in neighboring cells multiple pathways (including AKT, ERK, p38 and IKKβ) that, in turn, converge on TSC1/2, promoting mTOR activation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1α and HIF-2α protein levels and VEGF upregulation. In a mouse allograft model for KS, specific inhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation in these cells, and abolished the ability of the vGPCR-expressing cells to promote tumor formation in vivo. Similarly, pharmacologic inhibition of HIF in this model blocked VEGF secretion and also lead to tumor regression. Conclusions/Significance Our findings provide a compelling explanation for how the few tumor cells expressing vGPCR can contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for how cytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIF by vGPCR may be a vulnerable target for the treatment of patients with KS.

Zoledronic acid directly suppresses cell proliferation and induces apoptosis in highly tumorigenic prostate and breast cancers

Background: Bisphosphonates (BPs) were designed for the prevention of skeletal-related events secondary to bone metastases. The purpose of this study was to show that zoledronic acid (ZA) directly eradicates highly tumorigenic and potentially metastatic cancer cells. Materials and Methods: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, were exposed to different concentrations of ZA (0-10 μM). Reverse transcriptase double quantitative polymerase chain reaction was used for quantitative gene expression analysis. Apoptosis and cell proliferation were determined using microscopic observation and MTS assays. Western blot was used to confirm the translational effects of apoptotic genes on protein expression. Results: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, showed multiple genes demonstrating differential expressions, including TRAF, TRADD, BCL2, CASPASES and IAP families. Increasing ZA concentrations showed a greater concentration-time response on cell proliferation and apoptosis in the highly tumorigenic cells. These results were confirmed by both reversing and enhancing the effect of ZA on cell proliferation with caspase 3, 7 or survivin siRNA, respectively. Pro-apoptotic proteins bax and caspase 2, 3, 7 and 9 were up-regulated, while the anti-apoptotic proteins bcl2, birc3 and survivin were down-regulated only in the highly tumorigenic cells. Conclusions: This explains the ability of ZA to inhibit bony metastasis in highly tumorigenic cells compared with the low- or non-tumorigenic cells through a significant decrease in cell proliferation and increase in apoptosis through gene-regulated and translational-mediated down-regulation of survivin coupled with the inhibition of caspase 3 or 7. This has significant implications toward understanding the pharmacophysiology of BPs in metastasis and supports the clinically observed effect of BPs when administered adjunctively with anticancer drugs such as cyclophosphamide/methotrexate/5-fluorouracil, epirubicin in combination with cyclophosphamide or docetaxel, and doxorubicin.

Secretory Leukocyte Protease Inhibitor (SLPI) Expression and Tumor Invasion in Oral Squamous Cell Carcinoma

Differential expression of secretory leukocyte protease inhibitor (SLPI) impacts on tumor progression. SLPI directly inhibits elastase and other serine proteases, and regulates matrix metalloproteinases, plasminogen activation, and plasmin downstream targets to influence invasion. We examined tissues from human oral squamous cell carcinoma (OSCC) for SLPI expression in parallel with proteases associated with tumor progression and evaluated their relationships using tumor cell lines. Significantly decreased SLPI was detected in OSCC compared to normal oral epithelium. Furthermore, an inverse correlation between SLPI and histological parameters associated with tumor progression, including stage of invasion, pattern of invasion, invasive cell grade, and composite histological tumor score was evident. Conversely, elevated plasmin and elastase were positively correlated with histological parameters of tumor invasion. In addition to its known inhibition of elastase, we identify SLPI as a novel inhibitor of plasminogen activation through its interaction with annexin A2 with concomitant reduced plasmin generation by macrophages and OSCC cell lines. In an in vitro assay measuring invasive activity, SLPI blocked protease-dependent tumor cell migration. Our data suggest that SLPI may possess antitumorigenic activity by virtue of its ability to interfere with multiple requisite proteolytic steps underlying tumor cell invasion and may provide insight into potential stratification of oral cancer according to risk of occult metastasis, guiding treatment strategies.

A novel soft-tissue in vitro model for bisphosphonate-associated osteonecrosis

BackgroundBisphosphonate (BP)-associated osteonecrosis of the jaw (ONJ) has been reported in patients receiving intravenous BP, particularly zoledronic acid (ZA). The purpose of this study was to develop an in vitro model representative of the effects BP has on soft tissue secondary to its release from bone. Human gingival fibroblasts and oral epithelial cell lines were exposed to various concentrations (0-10 μM) of ZA using dentine discs (DDs) as a direct carrier of BP, which were exposed for 24 hours to ZA in normal medium (NM), washed in phosphate-buffered saline (PBS) and placed in a new co-culture with the cells. The cells were allowed to proliferate until they grew over the bone discs and then the discs either were left unchelated, or were chelated using 0.001% EDTA or EGTA to release BP from the discs and to observe the cellular effects. Direct effects were determined using direct and fluorescent imaging. Apoptotic effects were determined by vital stain, terminal dUTP nick-end labeling, and annexin V studies. The effect on cell proliferation was determined by mitochondrial tetrazolium salt assay. The level of BP release was determined based on the effect of BP directly on cells, using the DDs or the supernatant fluids resulting from chelation.ResultsA dose-response effect was seen on imaging, and effects on apoptosis and cell proliferation were observed with increasing ZA concentrations liberated from the DDs, particularly after calcium cleavage and release of ZA from the DDs with a variety of chelating agents. Apoptotic effects were observed microscopically after chelation at 24 hours. Release of ZA was confirmed by extracting medium from non-chelated and chelated cell culture models with DDs and applying this medium to untreated fresh cell cultures, providing appropriate controls.ConclusionsThe results from this study demonstrate that low concentrations of ZA released from bone can rapidly and directly affect the oral mucosal tissues, initially through the induction of apoptosis and long term through the inhibition of cell proliferation. These findings provide an in vitro model for a soft-tissue mechanistic component in the initiation and/or progression of ONJ.

Expression and alterations of the PTEN / AKT / mTOR pathway in ameloblastomas

OBJECTIVES Recently, an allelic loss of phosphatase and tensin homologue (PTEN) was shown to occur in ameloblastomas. In carcinogenesis, loss of PTEN allows for overactivity of the phosphatidylinositol-3-kinase/protein kinase B (PI3K / AKT) pathway inducing an upregulation of mammalian-target of rapamycin (mTOR) and its downstream effector ribosomal-subunit-6 kinase (S6K); allowing for uncontrolled cell proliferation, apoptosis inhibition and cell cycle deregulation. METHODS Thirty ameloblastomas and five dental follicles were studied, looking at the immunohistochemical expression of total PTEN and AKT, as well as their phosphorylated (p) active forms, and the downstream effector and indicator of mTOR activity p70 ribosomal-subunit-6 kinase (pS6K). Also assessed was the expression of extracellular-signal-regulated kinase (ERK), which cross talks with AKT. RESULTS Total PTEN was absent in 33.3% of ameloblastomas, while its stabilized, phosphorylated(ser380 / thr382 / thr383) form was absent in 83.3% of tumors. In contrast, AKT was expressed in 83.3% of ameloblastomas, showing high expression of the p-thr(308)AKT and p-ser(473) AKT forms in 93.3% and 56.6% of cases, respectively. Further, the mTOR activated pS6K(ser240 / 244) was detected in 86.7% of ameloblastomas, while ERK was overexpressed in 70.0% of the cases. CONCLUSION Immunohistochemical analysis of aberrant signaling in the PI3K/AKT/mTOR pathway in ameloblastomas may represent a valuable tool for elucidating pathogenesis, aggressiveness and selecting optimal therapeutics.

A clinicopathological study of malignant odontogenic tumours.

Chaisuparat R, Sawangarun W & Scheper M A 
(2012) Histopathology 61, 107–112

The Akt/mTOR pathway is activated in verrucous carcinoma of the oral cavity

BACKGROUND The Akt/mTOR pathway is activated in many malignancies, including oral squamous cell carcinoma (OSCC). However, the role of the Akt/mTOR pathway in oral verrucous carcinoma (OVC), a low-grade variant of OSCC, remains unknown. Thus, the objective of this study was to investigate the activation level of important markers of the Akt/mTOR pathway in OVC and to compare the results with OSCC samples. METHODS The expression of p-Akt (Thr308), p-Akt (Ser473), and p-RPS6 was evaluated by immunohistochemistry in 30 OSCC cases, 18 OVC cases, and 30 control cases (normal epithelium overlying fibromas). Statistical analysis was performed to determine the differences in protein expression between samples. RESULTS All OVC cases were positive for p-Akt (Thr308), p-Akt (Ser473), and p-RPS6. There were significant differences in expression level of all studied proteins between OVC and control, as well as between OVC and OSCC. However, OVC showed significant lower staining scores than OSCC. CONCLUSIONS Our findings demonstrate that the Akt/mTOR pathway is upregulated in OVC, indicating a role for this pathway in the development and progression of this malignancy.

Activation of the Akt/mTOR pathway in dentigerous cysts, odontogenic keratocysts, and ameloblastomas

OBJECTIVE The aim of this study was to investigate the Akt/mTOR pathway in dentigerous cysts (DCs), odontogenic keratocysts (OKCs), and ameloblastomas. STUDY DESIGN A total of 90 cases were studied (30 DCs, 30 OKCs, and 30 ameloblastomas). Patient records on age, sex, lesion location, symptoms, and radiographic and histopathologic features were collected. The phosphorylation of components of the Akt/mTOR pathway [p-Akt (Ser473), p-Akt (Thr308), and phosphorylated-ribosomal protein S6 (p-RPS6)] was studied using immunohistochemistry. Correlations with clinical features were analyzed using the Spearman rank test. RESULTS Over 90% of OKCs and ameloblastomas and 60% of DCs stained positive for p-Akt (Ser473). Phospho-Akt (Thr308) was positive in 73% of ameloblastomas, 40% of OKCs, and 20% of DCs. Phospho-RPS6 was detected most frequently in OKCs (83%), followed by ameloblastomas (76%) and DCs (53%). No correlations were noted between the immunohistochemical findings and the clinicopathologic parameters. CONCLUSIONS The Akt/mTOR pathway is upregulated in DCs, OKCs, and ameloblastomas. This pathway may be involved in the development of these lesions.