BACKGROUNDHigh-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and ...effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line.METHODSWe evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells.RESULTSThe GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways.CONCLUSIONSThe GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.
Our previous study showed the local production of γ-aminobutyrate (GABA) in hypertrophic-zone chondrocytes of the rat tibial growth plate, an important long bone growth site. The aim of this study ...was to identify the presence of GABA receptors in growth plate chondrocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Chondrocytes expressed both GABA^sub A^ and GABA^sub B^ receptor subunit mRNAs as well as the corresponding proteins necessary for the assembly of functional receptors. The GABA^sub A^ receptor subunits detected included α1-α4, α6, β1-β3, and δ, and both R1 and R2 subunits of GABA^sub B^ receptors were detected. All receptor subunits were expressed in chondrocytes of the proliferative and hypertrophic zones. These results suggest that GABA is an autocrine/paracrine factor that regulates the physiological state of the growth plate. Subsequent studies with the mouse chondrogenic cell line ATDC5 showed the presence of mRNAs and the corresponding proteins for GABA^sub A^ receptor α1, β2, and β3 subunits and GABA^sub B^ receptor R1 and R2 subunits. GABA, muscimol (a GABA^sub A^ receptor agonist), and baclofen (a GABA^sub B^ receptor agonist) increased 5-bromodeoxyuridine (BrdU) incorporation into ATDC5 cells. The effect of muscimol was blocked by bicuculline (a GABA^sub A^ receptor antagonist), and the effect of baclofen was blocked by CGP 35348 (a GABA^sub B^ receptor antagonist). These results suggest that GABA contributes to the ATDC5 cell proliferation via GABA^sub A^ and GABA^sub B^ receptors and these mechanisms may be involved in cartilaginous cell growth.PUBLICATION ABSTRACT
Chondrosarcoma is a malignant bone tumor that produces cartilaginous neoplastic tissue. Owing to the absence of an effective adjuvant therapy, high-grade chondrosarcoma has a poor prognosis. ...Therefore, it is important to develop an effective adjuvant therapy to prevent the recurrence and metastasis. Mammalian target of rapamycin (mTOR), a central regulator of cell growth, metabolism, proliferation, and survival, is considered an important target for anticancer drug development. The mitogen activated protein kinase (MAPK) pathway is another highly implicated cellular pathway in cancer and is thought to have compensatory effects in response to the inhibition of the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR signaling pathway. We investigated the mechanism of anti-proliferative effect of the mTOR inhibitor rapamycin and MAPK/ERK (MEK) inhibitor PD 0325901, and the combined effect of rapamycin and PD 0325901 on human chondrosarcoma cell line (OUMS-27). Combination therapy with rapamycin and PD 0325901 showed a stronger anti-proliferative effect on OUMS-27 cells than rapamycin monotherapy. We confirmed that the dual inhibition of the PI3K/Akt/mTOR and RAF/MEK/ERK signaling pathways had synergistic anti-proliferative effects in OUMS-27. Our results suggest that combination therapy of mTOR and MEK inhibitor could be an effective therapeutic approach against chondrosarcoma.
The association between melatonin system and the spontaneous development of the spinal deformities in the Hereditary Lordoscoliotic Rabbit, the natural animal model for idiopathic scoliosis, was ...studied.
To examine the implication for melatonin and its receptor in the spinal deformities of the natural animal model, the Hereditary Lordoscoliotic Rabbit.
We previously reported radiologic and histologic studies investigating the etiology of spinal deformities in a breed of Japanese White Rabbit, the Hereditary Lordoscoliotic Rabbit. These animals develop thoracic lordoscoliosis during growth and as such can be used as a model for human idiopathic scoliosis. Although previous studies in chickens have established that pinealectomy produces scoliosis, the cause of the condition is yet to be fully elucidated.
Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were measured by radioimmunoassay and compared with those of Japanese White Rabbits (controls). The expression of melatonin receptor in the rabbit was detected by homology cloning to access the number of the melatonin receptor mRNA in the rabbit spinal cord by quantitative reverse-transcribed polymerase chain reaction.
Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were significant higher than those of controls in each period until 20 weeks. We detected the expression of melatonin receptor mRNA in rabbit spinal cord. However, no significant quantitative differences were found in the level of expression of melatonin mRNA in the spinal cord between Hereditary Lordoscoliotic Rabbits and controls.
In relation to the present study, we suggest that causes of spinal deformities in the Hereditary Lordoscoliotic Rabbit may be the result of the contribution of melatonin receptors as well as that of altered serum melatonin levels in the Hereditary Lordoscoliotic Rabbit. Further studies will be required to investigate the expression of melatonin receptor in other tissues of the Hereditary Lordoscoliotic Rabbit as well as to delineate the role of melatonin in the pathogenesis of idiopathic scoliosis.
Changes in the fiber-type ratio and diameter during limb lengthening in 10 adult rabbits were studied using histochemical techniques. Changes in the ratio and diameter of muscle fibers (classified as ...type 1, 2A, or 2B fibers) in tibialis anterior muscles were examined after 20% gradual distraction of the tibia. There was an increase in the number of type 1 fibers and a decrease in type 2B fibers after tibial lengthening. Moreover, the average diameter of the type 1 fibers increased, whereas that of the type 2B fibers decreased. The diameters of muscle fibers measured immediately after completion of the lengthening showed a tendency to recover to normal levels within 1 month; however, the ratio profile of the muscle fibers changed both immediately and 1 month after lengthening. The above results demonstrate that stretched skeletal muscle adapts differently to bone lengthening according to the type of muscle fibers present, resulting in qualitative changes in the fiber-type profile.
The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABAB receptor ...is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABAB receptor subunits in rat spermatids by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABAB(1) immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatids. In contrast, GABAB(2) immunoreactivity was not observed in spermatids during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABAB(2) immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABAB(1) were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABAB(2) immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.
Gamma-aminobutyrate (GABA) is a major inhibitory neurotransmitter in the adult mammalian brain. GABA is also considered to be a multifunctional molecule that has different situational functions in ...the central nervous system, the peripheral nervous system, and in some nonneuronal tissues. GABA is synthesized primarily from glutamate by glutamate decarboxylase (GAD), but alternative pathways may be important under certain situations. Two types of GAD appear to have significant physiological roles. GABA functions appear to be triggered by binding of GABA to its ionotropic receptors, GABA(A) and GABA(C), which are ligand-gated chloride channels, and its metabotropic receptor, GABA(B). The physiological, pharmacological, and molecular characteristics of GABA(A) receptors are well documented, and diversity in the pharmacologic properties of the receptor subtypes is important clinically. In addition to its role in neural development, GABA appears to be involved in a wide variety of physiological functions in tissues and organs outside the brain.
γ‐Aminobutyric acid (GABA), which is a major inhibitory neurotransmitter in the brain, is also found in many peripheral nonneuronal tissues, including male reproductive organs. However, the ...distribution of GABAergic cells in various organs is not known. The GAD67‐GFP knock‐in mouse is a useful model for studying the distribution and morphology of GABAergic neurons in the brain. We examined the male reproductive organs of GAD67‐GFP knock‐in mice by fluorescence microscopy and found cells with strong green fluorescent protein (GFP) signal exclusively in the epithelium of the initial segment and proximal caput of the epididymis. The characteristic cell morphology suggested that these were narrow cells. These GFP‐positive narrow cells also expressed GAD67 and GABA. Reverse transcription polymerase chain reaction (RT‐PCR) analysis showed that the predominant glutamic acid decarboxylase (GAD) isoform expressed in the epididymis is GAD67. RT‐PCR analysis also revealed that mRNAs encoding the GABAA and GABAB receptor subunits necessary for the assembly of functional receptors are expressed in the epididymis. GABAA receptor subunit mRNAs detected in the proximal epididymis included α2, β1, γ1, and γ3, and both the R1 and R2 subunit mRNAs of GABAB receptors were detected. Immunohistochemical analysis of GABAA receptor subunit proteins revealed that α2, β1, and γ subunits expressed in spermatozoa, whereas we did not detect these GABAA receptor subunits in epithelial cells. GABAB receptors were produced by narrow cells and spermatozoa of GAD67‐GFP knock‐in and wild‐type Jcl:ICR mice. Our data suggest that the GABA system might have important functional roles in narrow cells and on spermatozoa in the lumen.
High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant ...therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line.
We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca
currents and evaluated the changes in intracellular Ca
concentration via Ca
channels, which are related to the GABA
receptor in high-grade chondrosarcoma cells.
The GABA
receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABA
receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca
via GABA
receptor-related Ca
channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways.
The GABA
receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca
channels in high-grade chondrosarcoma cells.
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