Hsp90 inhibitor induces autophagy and apoptosis in osteosarcoma cells.

Heat shock protein 90 (Hsp90) is constitutively expressed at 2­10­fold higher levels in tumor cells compared to normal cells, suggesting that it may be critically important for tumor cell growth and survival. These features make Hsp90 a potential target for anticancer drug development. Inhibition of Hsp90 activity not only results in rapid degradation of Hsp90 client proteins but also induces apoptosis of various tumor cells. Hsp90 also plays an important role in autophagy. An Hsp90 inhibitor induces autophagy through inhibition of mTOR. It is still under debate whether chemotherapy­induced autophagy in tumor cells is a protective response or is invoked to promote cell death. The aim of this study was to examine the effects of the Hsp90 inhibitor, geldanamycin (GA), on KTHOS osteosarcoma cells. We further examined whether a combination of GA and the autophagy inhibitor 3­methyladenine (3­MA) enhanced GA­induced apoptosis in KTHOS cells. GA had an inhibitory effect on cell proliferation and inhibited the Akt/mTOR signaling pathway in KTHOS cells. GA alone induced autophagy and apoptosis in KTHOS cells, but treatment with a combination of GA and 3­MA suppressed autophagy and induced apoptosis to a much greater extent than GA alone in these cells. It was considered that the autophagy inhibitor 3­MA suppressed a protective mechanism induced by Hsp90 inhibitor in tumor cells and induced apoptosis. Therefore, the combination of an Hsp90 inhibitor and an autophagy inhibitor may be an effective treatment for osteosarcoma because this combination effectively induces apoptotic pathways.

Parosteal osteoma of the clavicle.

Introduction. Osteoma is a benign, slowly growing, asymptomatic, osteogenic neoplasm. Osteoma of a bone other than the skull and facial bones is extremely rare. An extremely rare case of parosteal osteoma is reported. Case Presentation. A 51-year-old woman presented with a large mass in the left supraclavicular fossa. Radiographs and computed tomography revealed a well-defined, 9 × 6 cm, lobed mass in the midportion of the clavicle. Magnetic resonance imaging revealed that it had the same density as cortical bone. An open biopsy was performed to rule out malignant bone tumours, and parosteal osteoma was diagnosed. Four years after the biopsy, the patient was asymptomatic. Conclusion. A rare case of parosteal osteoma of the clavicle was described. Open biopsy is required to rule out a malignant bone tumour, even if parosteal osteoma is suspected based on the clinical course and imaging findings.

A large amount of microdamages in the cortical bone around fracture site in a patient of atypical femoral fracture after long-term bisphosphonate therapy.

A breast cancer patient developed an atypical femoral fracture after 9 years of bisphosphonate therapy for the treatment of multiple bone metastases. We histopathologically analyzed the femoral cortical bone at the fracture site and the iliac cancellous bone. Four months prior to the fracture, the patient had experienced pain in the right femur and underwent plain radiography and bone scintigraphy which revealed cortical thickening and radioisotope accumulation at each site, respectively. The patient had also experienced a non-traumatic fracture at the same site on the contralateral side 2 years earlier. Based on these findings, atypical femoral fracture was diagnosed and intramedullary nailing performed. A cortical bone specimen taken from near the fracture site during surgery showed marked microdamages, and analysis of the iliac cancellous bone specimen revealed severely suppressed bone turnover. These findings suggest that microdamage and severely suppressed bone turnover are associated with atypical femoral fracture reported in this patient with long-term bisphosphonate therapy.

The combination of rapamycin and MAPK inhibitors enhances the growth inhibitory effect on Nara-H cells.

The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy, and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) is well known to induce autophagy. Autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents, and blocking autophagy can trigger apoptosis. In the present study, we demonstrate that an mTOR inhibitor, rapamycin, induces autophagy in the Nara-H malignant fibrous histiocytoma (MFH) cell line through the activation of ERK1/2. Rapamycin-induced apoptosis was enhanced following the inhibition of the MEK/ERK pathway. In the Nara-H cells, we examined the effects of rapamycin treatment on cell proliferation and on the phosphorylation of the mTOR pathway components and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without the MEK inhibitor, U0126, on the induction of apoptosis by using fluorescence microscopy. Rapamycin inhibited Nara-H cell proliferation and decreased the phosphorylation of the mTOR pathway in the Nara-H cells. Rapamycin induced the apoptosis of Nara-H cells, and this apoptosis was enhanced by U0126. Simultaneously, phospho-ERK1/2 was activated by rapamycin. The present study demonstrates that rapamycin induces autophagy in Nara-H cells by activating the MEK/ERK signaling pathway, and the rapamycin-induced apoptosis can be enhanced by the MEK inhibitor, U0126. These results suggest that self­protective mechanisms involving mTOR inhibitors in Nara-H cells are prevented by the inhibition of the MEK/ERK pathway. The combination of an mTOR inhibitor (e.g., rapamycin) and an MEK inhibitor (e.g., U0126) may offer effective treatment for MFH, as this combination effectively activates apoptotic pathways.

Polyostotic fibrous dysplasia with epiphyseal involvement in long bones: a case report.

Fibrous dysplasia (FD) is an uncommon, but well-known benign skeletal disorder. In cases affecting long bones, FD is commonly recognized to locate in the diaphyses or the metaphyses and to spare the epiphyses. In this paper, we present a rare case of polyostotic FD in a 13-year-old girl with unilateral multiple epiphyseal lesions arising in the femur, the tibia, and the fibula with the growth plates.

Massive heterotopic ossification around the ankle in a patient with macrodactyly of the foot: a case report.

A 40-year-old man with macrodactyly of the left great toe presented with progressive enlargement of the ipsilateral ankle and increasing pain after minor trauma. Radiographs and computed tomographic scans of the foot and ankle revealed macrodactyly of the great toe and a large heterotopic ossified mass in the anteromedial aspect of the ankle. The large osseous lesion in the ankle was operatively excised. The lesion was easily excised en bloc after releasing the adhesion and histologically diagnosed as normal mature bone with osteoarthritic changes. Massive heterotopic ossification developing around the ankle in a patient with macrodactyly has not been previously reported. The current case was classified as hyperostotic macrodactyly. Operative treatment relieved the pain and improved the range of the motion of the ankle.

Intra-articular nodular fasciitis of the knee: a rare cause of recurrent hemarthrosis.

A 20-year-old man presented with pain and recurrent hemarthrosis in the right knee. Magnetic resonance imaging of the knee showed a lesion with homogeneous low signal intensity on T1-weighted images and a heterogeneous, low to high signal intensity on T2-weighted images. At arthroscopy, the mass was located between the posterior cruciate ligament and the posterior knee joint capsule. The tumor was excised through a posterior approach and histologically diagnosed as a nodular fasciitis. Intra-articular nodular fasciitis is a very rare clinicopathologic entity. The current case showed the unique clinical feature of recurrent hemarthrosis at initial presentation, which has not been previously reported.

Inhibition of induced autophagy increases apoptosis of Nara-H cells.

Inhibition of the mTOR signaling pathway promotes initiation of autophagy. However, recent studies indicate that autophagy is a self-defense mechanism of cancer cells that are subjected to anti-tumor agents and that blocking autophagy can trigger apoptosis. Here, we examined the effects of an mTOR inhibitor, temsirolimus, on a malignant fibrous histiocytoma (MFH) cell line, Nara-H cells, and the effect of suppressing autophagy on the induction of apoptosis in these MFH cells. In Nara-H cells, we examined the effects of temsirolimus treatment on cell proliferation using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and on phosphorylation of mTOR pathway components and autophagy using Western blot-based assays. Furthermore, we examined the effects of temsirolimus with or without 3-methyladenine (3-MA) on induction of apoptosis using fluorescent microscopic analysis. In Nara-H cells, temsirolimus treatment inhibited cell proliferation, suppressed phosphorylation of mTOR pathway components, and induced autophagy as assessed by LC-3 II expression. Moreover, treatment with a combination of temsirolimus and 3-MA induced apoptosis in Nara-H cells. Apparently, simultaneous inhibition of autophagy and mTOR induced apoptosis in Nara-H cells because inhibition of autophagy prevented the cells from protecting themselves from the effects of the inhibition of mTOR. Therefore, a combination therapy that includes an mTOR inhibitor and an autophagy inhibitor (temsirolimus and 3-MA, respectively) may effectively treat MFH by inducing apoptosis in tumor cells.

The role of MAPK pathway in bone and soft tissue tumors.

AIM: Expression of mitogen-activated protein kinase (MAPK) signaling and its role in cell proliferation of the bone malignancies, osteosarcoma (OS) and malignant fibrous histiocytoma (MFH) were investigated. MATERIALS AND METHODS: Gene expression and protein levels of RAF1 and MEK1/2 in 6 human sarcoma cell lines and 7 surgically obtained OS specimens were assessed by RT-PCR and immunohistochemistry, respectively. MEK inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-aminophynyltio) butadiene], was used for cell proliferation assays. RESULTS: RAF1 and MEK 1/2 mRNA was detected in all cell lines and OS specimens. RAF1, MEK 1/2 and p-MEK protein was also expressed in the cells, as was MEK1/2 in OS specimens. Treatment with U0126 resulted in dose- and time-dependent inhibition of cell proliferation and suppression of p-ERK expression, opposite to promotion of p-MEK. CONCLUSION: U0126 blocks MAPK signaling and decreases cell proliferation in OS and MFH. Thus, selective MAPK inhibitors might be therapeutically advantageous in the treatment of bone and soft tissue sarcomas.

Ischiogluteal bursitis: a report of three cases with MR findings.

Ischiogluteal bursitis is an uncommon disorder which can be confused with neoplastic conditions in the buttock. Three cases of ischiogluteal bursitis in a 57-year-old man, a 73-year-old woman and a 73-year-old man are presented. All patients presented with a gradually increasing, painful buttock mass. Magnetic resonance imaging (MRI) revealed a soft tissue mass around the ischial tuberosity and showed various features in the three cases. Two patients underwent excision of the lesion, which was histologically diagnosed as ischiogluteal bursitis. One patient was conservatively treated and the symptoms gradually decreased. MRI was very useful in diagnosing and detecting the lesion. Ischiogluteal bursitis should be considered in the differential diagnosis of a buttock mass.

Inhibition of PKCalpha activation in human bone and soft tissue sarcoma cells by the selective PKC inhibitor PKC412.

BACKGROUND: PKC412, formerly CGP41251, N-benzoylstaurosporine, was initially developed as a selective protein kinase C (PKC) inhibitor, and it preferentially inhibits conventional PKC family members. In this study, the expression of PKCa was examined in human osteosarcoma and MFH cell lines, and the inhibitory effect of PKC412 on the proliferation of the cell lines was evaluated. MATERIALS AND METHODS: Three human osteosarcoma cell lines (KTHOS, MG63 and KHOS) and four human MFH cell lines (TNMY1, GBS-1, Nara-F and Nara-H) were used. The expression of PKCalpha and phosphorylated PKCalpha were analyzed using both Western blotting analysis and immunocytochemical analysis. The effect of PKC412 on cell proliferation was evaluated using the MTS assay technique. RESULTS: PKC412 inhibited cell proliferation of all seven cell lines in a dose- and time-dependent manner. Both Western blotting analysis and immunocytochemical analysis revealed that not only PKCalpha but also phosphorylated PKCalpha were expressed in all cell lines incubated with the culture medium without any stimuli. PKC412 suppressed phosphorylation of PKCalpha in all cell lines at a concentration of 1 microM. CONCLUSION: The inhibition of cell proliferation of the human osteosarcoma and MFH cell lines by PKC412 might be due to reduced PKCalpha activity. This suggests PKC412 might be a potent chemotherapeutic agent for human sarcomas.

Intra-tendinous ganglion in the long head of the biceps humeri.

We present details of a case of intra-tendinous ganglion arising from the long head of the biceps at an unusual location. MRI scans have important implications for surgical planning and treatment. After excision of the ganglion, the tendon remaining could be repaired. Five months after surgery, there was no sign of recurrence.

Establishment and characterization of a KIT-positive and stem cell factor-producing cell line, KTHOS, derived from human osteosarcoma.

Osteosarcoma is a malignant bone tumor that commonly affects adolescents and young adults. In the present study a human osteosarcoma cell line, KTHOS, was established from a primary osteosarcoma lesion in the distal femur of a 16-year-old girl. After 106 passages, the KTHOS cell line retained the biological characteristics of osteosarcoma. The KTHOS cells had spindle to pleomorphic cytoplasm with round to ovoid nuclei containing multiple prominent nucleoli, as expected based on the mesodermic origin of osteoblasts. The KTHOS cells were immunoreactive for osteocalcin, osteonectin, stem cell factor (SCF), and KIT (CD117). Reverse transcriptase-polymerase chain reaction indicated that the KTHOS cell line expressed mRNA for SCF and KIT. The KTHOS cells produced relatively high amounts of soluble SCF as determined by enzyme-linked immunosorbent assay. The results suggest that cell proliferation of the KTHOS cell line might be involved in autocrine and/or paracrine loops of the SCF/KIT signaling system. The KTHOS cell line is a novel human osteosarcoma cell line that releases SCF and expresses KIT. This cell line can be used for studies to explore the mechanisms for oncogenesis of human osteosarcomas.

Inhibitory effect of STI571 on cell proliferation of human malignant fibrous histiocytoma cell lines.

BACKGROUND: Malignant fibrous histiocytoma (MFH) is one of the most common high-grade sarcomas in bone and soft tissue and, due to its chemo-resistance, the prognosis of the disease is poor. ST1571 is a tyrosine kinase inhibitor that was initially developed as a BCR/ABL inhibitor for chronic myeloid leukemia patients. STI571 also selectively inhibits platelet-derived growth factor receptors (PDGFRs) and c-kit. We examined the expression of PDGFRs and c-kit in human MFH cell lines, and the effect of STI571 on cell proliferation. MATERIALS AND METHODS: Four human MFH cell lines (TNMY1, GBS-1, Nara-F and Nara-H) were used. mRNA expression of the receptor tyrosine kinases (PDGFRs and c-kit) was analyzed using reverse transcription-polymerase chain reaction, and the inhibitory effect of STI571 on cell proliferation was analyzed using the MTS assay technique. RESULTS: PDGFRalpha mRNA was expressed in TNMY1 and GBS-1, and PDGFRbeta and c-kit mRNAs were expressed in TNMY1, GBS-1 and Nara-F. All three of these mRNAs were absent in Nara-H. STI571 inhibited cell proliferation of TNMY1, GBS-1 and Nara-F in a dose- and time-dependent manner, but cell proliferation of Nara-H was not inhibited by ST1571 at concentrations of 10 microM or less. CONCLUSION: STI571 significantly inhibited proliferation of the three human MFH cell lines that expressed mRNAs of target receptor tyrosine kinases. The inhibitory effect of ST1571 on cell proliferation in these three cell lines might be due to decreased tyrosine kinase activity. STI571 might be a potent chemotherapeutic agent for human MFHs.

Expression of transforming growth factor beta isoforms and their receptors in malignant fibrous histiocytoma of soft tissues.

PURPOSE: Transforming growth factor beta (TGF-beta) is a multifunctional growth factor that variably affects proliferation, differentiation, and extracellular matrix formation. Little information is currently available on the TGF-beta expression in malignant fibrous histiocytoma (MFH). The aims of the present study were to investigate the expression of TGF-beta isoforms and their receptors in human MFH specimens. EXPERIMENTAL DESIGN: The expression of TGF isoforms, and TGF-beta receptors (TGF-beta R1 and -beta R2) were immunohistochemically evaluated in 43 paraffin-embedded MFH specimens. Furthermore, the correlation of the TGF-beta and receptor expression with tumor proliferative activity assessed by MIB-1 indices was analyzed. RESULTS: Positive immunoreactivity for TGF-beta1, -beta2, and -beta 3 was identified in tumor cells of 42, 40, and 38 of the 43 MFHs, respectively. In each TGF-beta isoform immunostaining, the specimens were divided into two groups based on the number of positive tumor cells: those with low (<25%) and those with high (>==25%) immunoreactivity. There were no statistically significant differences in the MIB-1 indices between the two groups. Positive immunoreactivity for TGF-beta R1 and -beta R2 was identified in tumor cells of 36 and 24 of the MFHs, respectively. The specimens were divided into two groups based on their receptor expression patterns: those with both TGF-beta R1- and -beta R2-positive immunoreactivity (n = 23), and those with both or either TGF-beta R1- and -beta R2-negative immunoreactivity (n = 20). The MIB-1 indices in the both-TGF-beta R1- and -beta R2-positive group were significantly higher than those in the other group (P = 0.0102). There was no significant difference in pulmonary metastasis ratios between the two groups. CONCLUSIONS: These findings strongly suggest an association of the TGF-beta ligand/receptor system with a significantly higher MIB-1 index in human MFHs. Investigation of the TGF-beta R1 and -beta R2 coexpression might be useful in predicting tumor behavior of MFHs.

Expression of betacellulin, heparin-binding epidermal growth factor and epiregulin in human malignant fibrous histiocytoma.

BACKGROUND: Heparin-binding epidermal growth factor (HB-EGF), betacellulin (BTC) and epiregulin (EPR) are members of the EGF system and involved in the cell growth of various epithelial malignancies. There have been no reports on the HB-EGF, BTC and EPR expression in mesenchymal malignancies of fibrohistiocytic origin including malignant fibrous histiocytoma (MFH). MATERIALS AND METHODS: We investigated the expression of HB-EGF, BTC, EPR and EGF-receptor (EGF-R) in 43 human MFH tissue samples using immunohistochemical techniques. RESULTS: Positive immuno-reactivity for HB-EGF, BTC, EPR and EGF-R was identified in 28 (65%), 7 (16%), 43 (100%) and 36 (84%) out of the 43 MFH cases analyzed, respectively. Coexpression of HB-EGF/BTC, BTC/EPR and HB-EGF/EPR was observed in 6 (14%), 7 (16%) and 28 (65%) of the MFHs, respectively. Coexpression of HB-EGF/EGF-R, BTC/EGF-R and EPR/EGF-R was observed in 25 (58%), 6 (14%) and 36 (84%) of the MFHs, respectively. CONCLUSION: These results revealed that HB-EGF, BTC and EPR are expressed not only by epithelial tumor cells, but also by MFH cells. It is suggested that HB-EGF and EPR might be more important tumor growth regulators of MFH through autocrine or paracrine pathways, when compared with BTC.

Coexpression of hepatocyte growth factor and its receptor c-Met correlates with high MIB-1 proliferative index in malignant fibrous histiocytoma.

Hepatocyte growth factor (HGF) is a multifunctional cytokine that variably affects cell motility, proliferation, and morphogenesis. Little information is currently available on the HGF and its receptor c-Met expression in malignant fibrous histiocytoma (MFH). We immunohistochemically investigated the HGF and c-Met expression in 43 MFH tissue specimens. Furthermore, the correlation of the HGF and c-Met expression with tumor proliferative activity assessed by MIB-1 indices was analyzed. Our results showed that positive cytoplasmic immunoreactivity for HGF and c-Met was identified in tumor cells in 36 (84%) and 20 (47%) of the 43 MFH cases analyzed, respectively. Coexpression of HGF and c-Met was observed in 20 (47%) of the 43 MFHs, and was correlated with high MIB-1 proliferative indices (p = 0.0446). These findings strongly indicate that the HGF/c-Met signaling system plays an important role in promoting cell proliferation of human MFHs via an autocrine loop.

Apophysitis of the ischial tuberosity mimicking a neoplasm on magnetic resonance imaging.

We present multimodality imaging features of an ischial tuberosity apophysitis in a 13-year-old boy who was an active baseball pitcher. Roentgenography of the pelvis and computed tomography showed mild irregularity in the inferior margin of the left ischial tuberosity. T1-weighted MRI showed a wide area with low signal intensity in the left ischial body; T2-weighted fat-suppression images showed areas with markedly high signal intensity in the ischial apophysis and body and the surrounding periosteum; contrast-enhanced T1-weighted fat-suppression MRI showed that the ischial body, surrounding periosteum, and origin of the hamstring muscles strongly enhanced; technetium-99m scintigraphic scans showed increased isotope uptake in the entire ischial body. Histological specimens obtained from the bone showed increased osteoblastic activity, edema, and proliferation of benign spindle cells and small vessels in the bone marrow spaces. In the present case, because MR imaging demonstrated extensive signal abnormalities involving the apophysis, periosteum, and intramedullary portion of bone, a neoplasm could not be excluded, and a biopsy was undertaken.