Kanya Honoki

Frequent mutations of lysophosphatidic acid receptor-1 gene in rat liver tumors.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors, including LPA1 to LPA5. In the present study, to clarify an involvement of LPA1 gene alterations in the development of hepatocellular carcinomas (HCCs) we investigated the LPA1 mutations in rat HCCs induced by exogenous and endogenous liver carcinogenesis models. We induced HCCs in rats with N-nitrosodiethylamine (DEN) and a choline-deficient l-amino acid-defined (CDAA) diet. RNAs were extracted from 15 HCCs induced by DEN and 12 HCCs induced by the CDAA diet. To identify LPA1 mutations, reverse transcription (RT) - polymerase chain reaction (PCR) - single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Missense mutations were detected in 7 out of 15 HCCs (46.7%) induced by DEN. Five out of 12 HCCs (41.7%) induced by the CDAA diet also showed missense mutations. These results demonstrated that mutations in LPA1 gene occur in rat HCCs induced by DEN and the CDAA diet, suggesting that LPA1 mutations may be essentially involved in rat liver carcinogenesis.

Mutations of lysophosphatidic acid receptor-1 gene during progression of lung tumors in rats.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. In this study, mutations of lysophosphatidic acid receptor-1 (LPA1) gene were investigated to clarify the possible molecular mechanisms underlying the development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks. Genomic DNAs were extracted from paraffin-embedded tissues and exons 2-4 were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No LPA1 mutations were detected in 15 hyperplasias, but 2 out of 12 adenomas (16.7%) and 7 out of 17 adenocarcinomas (41.2%). These results suggest that mutations of LPA1 gene may be involved in the acquisition of growth advantage from adenomas to adenocarcinomas in lung carcinogenesis induced in rats by BHP.

Loss of lysophosphatidic acid receptor-3 enhances cell migration in rat lung tumor cells

Lysophosphatidic acid (LPA) indicates several biological effects, such as cell proliferation, differentiation and migration. LPA interacts with G protein-coupled transmembrane LPA receptors. In our previous report, we detected that loss of the LPA receptor-1 (Lpar1) expression is due to its aberrant DNA methylation in rat tumor cell lines. In this study, to assess an involvement of the other LPA receptor, Lpar3, in the pathogenesis of rat lung tumor cells, we measured the expression levels of the Lpar3 gene and its DNA methylation status by reverse transcription (RT)-polymerase chain reaction (PCR) and bisulfite sequencing analyses, respectively. RLCNR lung adenocarcinoma cells showed reduced expression of the Lpar3, compared with normal lung tissues. In the 5 upstream region of the Lpar3, normal lung tissues were unmethylated. By contrast, RLCNR cells were highly methylated, correlating with reduced expressions of the Lpar3. Based on these results, we generated the Lpar3-expressing RLCNR-a3 cells and measured the cell migration ability. Interestingly, the cell migration of RLCNR-a3 cells was significantly lower than that of RLCNR cells. This study suggests that loss of the Lpar3 due to aberrant DNA methylation may be involved in the progression of rat lung tumor cells.

Possible involvement of lysophosphatidic acid receptor-5 gene in the acquisition of growth advantage of rat tumor cells.

Aberrant expressions of lysophosphatidic acid (LPA) receptor genes have been reported in tumor cells. Here, we measured the expression levels of the Lpa5 gene and its DNA methylation status in rat tumor cells, and investigated cell growth effects of LPA in Lpa5 expressed cells. Real‐time reverse transcription (RT)‐polymerase chain reaction (PCR) analysis revealed that increased expressions of the Lpa5 gene were detected in rat liver‐derived hepatoma RH7777 and lung‐derived adenocarcinoma RLCNR cells. For the analysis of methylation status, bisulfite sequencing was performed with RH7777 and RLCNR cells and compared with other tumor cells and liver epithelial cells. The Lpa5 gene in Lpa5 unexpressed cells and liver epithelial cells were highly methylated in the 5′ upstream region. In contrast, the Lpa5 gene in RH7777 and RLCNR cells was unmethylated, correlating with increased expressions of Lpa5. In the assays for cell growth effects of LPA, LPA enhanced cell proliferation and motility in RH7777 and RLCNR cells. LPA also stimulated cell invasion in RLCNR, but not in RH7777 cells. In rat liver and lung tumors induced by nitroso‐compounds, 4 out of 6 hepatocellular carcinomas and 5 out of 6 lung adenocarcinomas indicated increased expressions of Lpa5 with unmethylated status. These results suggest that increased Lpa5 expressions due to aberrant DNA methylation may be involved in the acquisition of growth advantage of rat tumor cells.

Do stem-like cells play a role in drug resistance of sarcomas?

Stem cells are defined by their unique characteristics, which include their abilities to self-renew and differentiate. Normal somatic stem cells have been isolated from various tissues such as bone marrow, adipose tissue, mammary glands and the nervous system. They are considered naturally resistant to chemotherapeutic agents because they express high levels of membrane transporter molecules, detoxifying enzymes and DNA repair proteins. Several recent studies have identified the presence of side populations in various cancer tissues, the so-called ‘cancer stem cells’, which are defined as the counterparts of stem cells in tumor tissues. These cancer stem cells possess stem-like properties, such as self-renewal and differentiation abilities, as well as playing a role in tumor initiation. Most sarcomas, which are thought to originate from mesenchymal stem cells, are highly malignant and approximately 30–40% of them show local and/or distant relapse (metastasis), even in the case of relatively chemosensitive tumors such as osteosarcomas and Ewing sarcomas. Several studies have suggested the presence of stem-like cell populations in sarcomas, based on their tumorigenicity and drug resistance. This review explores the issues of drug resistance of cancer stem cells in sarcomas and the possibilities of targeting cancer stem cells for the future treatment of sarcomas.

Mutations of lysophosphatidic acid receptor genes in human osteosarcoma cells.

Objective: Lysophosphatidic acid (LPA), which is a bioactive phospholipid, interacts with specific G protein-coupled transmembrane receptors. Recently, alterations in LPA receptor genes have been reported in some tumor cells. In this study, to assess an involvement of LPA receptor genes in the development of human cancer cells, we looked for the presence of mutations in LPA receptor 1–6 (LPA1–6) genes in MG63 osteosarcoma, HT1080 fibrosarcoma, A549 lung adenocarcinoma, MCF-7 breast carcinoma, and G-361 melanoma cells. Methods: Genomic DNAs were extracted from each cell and polymerase chain reaction-single-strand conformation polymorphism analysis was performed to identify the mutations. Results: MG63 showed mutations in LPA1 and LPA3 genes, while no mutations in the LPA receptor genes were found in HT1080, A549, MCF-7 and G-361 cells. Sequence analysis revealed a CGC to CGT (Arg to Arg) transition at codon 314 in LPA1, and a GCG to GTG (Ala to Val) transition at codon 247 in LPA3. Conclusion: These results indicated that the mutations in LPA1 and LPA3 genes occur in MG63 cells, suggesting that the alterations in LPA receptor genes may play some role in the pathogenesis in human osteosarcoma cells.

Hibernoma of the axillary region: a rare benign adipocytic tumor

Hibernoma is a rare benign tumor considered to arise from remnants of fetal brown adipose tissue. It tends to occur in sites where brown fat persists beyond fetal life, such as the interscapular region, but can occur in sites where brown fat is usually absent in adults. Clinicallywell, hibernomas are slow-growing, asymptomatic tumors. However, unlike lipomas, MRI findings sometimes mislead clinicians to diagnose a malignant neoplasm. We describe a 63-year-old male with an axillary hibernoma involving the brachial neurovascular bundles and mimicking a well-differentiated liposarcoma, from which it should be distinguished.

Epithelioid Sarcoma of the Forearm Arising from Perineural Sheath of Median Nerve Mimicking Carpal Tunnel Syndrome

We report here a case of epithelioid sarcoma in the forearm of a 33-year-old male presenting with symptoms and signs of carpal tunnel syndrome originating from the direct involvement of the median nerve. Due to the slow growing of the tumor, the patient noticed the presence of tumor mass in his forearm after several months from the initial onset of the symptoms. Magnetic resonance imaging showed an 8 × 4 cm mass involving the median nerve in the middle part of the forearm, and histological analysis of the biopsy specimen revealed the diagnosis of epithelioid sarcoma. Radical surgical resection was performed in conjunction with adjuvant chemotherapy. The function of the flexors were restored by the multiple tendon transfers (EIP → FDS; ECRL → FDP; BrR → FPL; EDM → opponens) with superficial cutaneous branch of radial nerve transfer to the resected median nerve. The function of the affected hand showed excellent with the DASH disability/symptom score of 22.5, and both the grasp power and sensory of the median nerve area has recovered up to 50% of the normal side. The patient returned to his original vocation and alive with continuous disease free at 3.5-year follow-up since initial treatment.

Epiphyseal preservation and an intercalary vascularized fibular graft with hydroxyapatite composites. Reconstruction in metaphyseal osteosarcoma of the proximal tibia: a case report

Background data and objectivesWe present here a case report of a patient with metaphyseal osteosarcoma with a preserved epiphysis and reconstructed by a vascularized fibular graft and hydroxyapatite composites.MethodsThe case was a 14-year-old boy, who had osteosarcoma in the proximal tibia. After the diagnosis was confirmed by biopsy, the patient immediately received preoperative chemotherapy including high-dose Methotrexate, Cisplatin and Doxrubicin. Imagings after preoperative chemotherapy including MRI and contrasted enhanced CT confirmed no tumor penetration into the physis. Subsequently, we performed transepiphyseal resection of the proximal tibia to reserve the joint surface. The intercalary twin-barreled vascularized fibular graft was placed with hydroxyapatite composites. The patella tendon was reattached to the grafted fibular to biologically reconstruct the knee extensor mechanism. Postoperative chemotherapy was completed with the same regime as preoperative chemotherapy.OutcomesThe bony union was completed at 10xa0months after the operation. The Enneking’s functional evaluation score was 28 out of 30 points (93%). There was no evidence of local recurrence and no metastatic disease during the 42xa0months follow-up after initial diagnosis.ConclusionAn accurate evaluation of MRI and CT can give a reliable assessment of intraphyseal penetration of metaphyseal osteosarcoma. In case of no involvement of the tumor in the physis, transepiphyseal osteotomy is the optimal procedure to preserve the joint surface and superior function of the joint, especially in the proximal tibia.

Type 1 neurofibromatosis with a giant intrathoracic lesion: a case report with 25 years of follow-up.

We report a case of neurofibromatosis 1 with a huge intrathoracic lesion followed-up for 25 years. When the patient was 5 years of age, we performed a partial resection as an excisional biopsy. Microscopically, small spindle cells and collagen were spread between fat cells. A CT scan revealed a large intrathoracic mass, but tumor growth increasingly retarded as the patient grew older. During the entire 25-year follow-up period, from 5 to 30 years of age, the patient showed no neurological signs or other clinical symptoms. The findings of our case suggest that for a neurofibromatosis 1 patient, a good prognosis can be expected even if a huge destructive intrathoracic intrusion is detected. To assess the risk of malignant transformation, follow-up is required, accompanied by appropriate diagnostic modalities and physical examinations.