Biomedical subjects

Takeshi Sakamoto

Publications and source records attributed to Takeshi Sakamoto.

At least 19 recordsLinked to original sources

PubMed2006

Kinetic mechanism of myosinV-S1 using a new fluorescent ATP analogue.

We have used a new fluorescent ATP analogue, 3'-(7-diethylaminocoumarin-3-carbonylamino)-3'-deoxyadenosine-5'-triphosphate (deac-aminoATP), to study the ATP hydrolysis mechanism of the single headed myosinV-S1. Our study demonstrates that deac-aminoATP is an excellent substrate for these studies. Although the deac-amino nucleotides have a low quantum yield in free solution, there is a very large increase in fluorescence emission ( approximately 20-fold) upon binding to the myosinV active site. The fluorescence emission intensity is independent of the hydrolysis state of the nucleotide bound to myosinV-S1. The very good signal-to-noise ratio that is obtained with deac-amino nucleotides makes them excellent substrates for studying expressed proteins that can only be isolated in small quantities. The combination of the fast rate of binding and the favorable signal-to-noise ratio also allows deac-nucleotides to be used in chase experiments to determine the kinetics of ADP and Pi dissociation from actomyosin-ADP-Pi. Although phosphate dissociation from actomyosinV-ADP-Pi does not itself produce a fluorescence signal, it produces a lag in the signal for deac-aminoADP dissociation. The lag provides direct evidence that the principal pathway of product dissociation from actomyosinV-ADP-Pi is an ordered mechanism in which phosphate precedes ADP. Although the mechanism of hydrolysis of deac-aminoATP by (acto)myosinV-S1 is qualitatively similar to the ATP hydrolysis mechanism, there are significant differences in some of the rate constants. Deac-aminoATP binds 3-fold faster to myosinV-S1, and the rate of deac-aminoADP dissociation from actomyosinV-S1 is 20-fold slower. Deac-aminoATP supports motility by myosinV-HMM on actin at a rate consistent with the slower rate of deac-aminoADP dissociation.

Adenosine Diphosphate↗

PubMed2006

The cargo-binding domain regulates structure and activity of myosin 5.

Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments. Its tail ends in paired globular tail domains (GTDs) thought to bind cargo. At nanomolar calcium levels, actin-activated ATPase is low and the molecule is folded. Micromolar calcium concentrations activate ATPase and the molecule unfolds. Here we describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows that the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (approximately Pro 117-Pro 137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin, a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium levels heavy meromyosin moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains, producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.

Actins↗

PubMed2006

Dimerized Drosophila myosin VIIa: a processive motor.

The molecular mechanism of processive movement of single myosin molecules from classes V and VI along their actin tracks has recently attracted extraordinary attention. Another member of the myosin superfamily, myosin VII, plays vital roles in the sensory function of Drosophila and mammals. We studied the molecular mechanism of Drosophila myosin VIIa, using transient kinetics and single-molecule motility assays. Myosin VIIa moves along actin filaments as a processive, double-headed single molecule when dimerized by the inclusion of a leucine zipper at the C terminus of the coiled-coil domain. Its motility is approximately 8-10 times slower than that of myosin V, and its step size is 30 nm, which is consistent with the presence of five IQ motifs in its neck region. The kinetic basis for the processive motility of myosin VIIa is the relative magnitude of the release rate constants of phosphate (fast) and ADP (slow) as in myosins V and VI. The ATPase pathway is rate-limited by a reversible interconversion between two distinct ADP-bound actomyosin states, which results in high steady-state occupancy of a strongly actin-bound myosin species. The distinctive features of myosin VIIa (long run lengths, slow motility) will be very useful in video-based single-molecule applications. In cells, this kinetic behavior would allow myosin VIIa to exert and hold tension on actin filaments and, if dimerized, to function as a processive cargo transporter.

Animals↗

PubMed2006

Delayed postoperative spinal epidural hematoma causing tetraplegia. Case report.

The authors describe a case of postoperative spinal epidural hematoma (PSEH) that developed in a patient 9 days after he underwent laminoplasty. A PSEH is a rare but critical complication of spinal surgery that usually occurs within a few days of the procedure. The authors draw attention to the possibility of delayed PSEH and its triggering mechanism. In this case, a 59-year-old man with no history of bleeding disorder underwent cervical laminoplasty for mild myelopathy. On the 7th postoperative day computed tomography demonstrated no abnormal findings in the operative field. On the 9th postoperative day, while straining to defecate, the patient suddenly felt neck and shoulder pain, and tetraplegia rapidly developed. Magnetic resonance imaging demonstrated a huge epidural hematoma. The clot was evacuated during emergency revision surgery, during which the arterial bleeding from a split muscle wall was confirmed. The postoperative course after the revision surgery was uneventful and the patient had none of the previous symptoms 1 year later. A PSEH causing paralysis can occur even more than a week after surgery. The possibility of a delayed-onset PSEH should be kept in mind, and prompt diagnosis should be made when a patient presents with paresis or paralysis after an operation. The authors recommend advising patients that for a while after surgery they avoid strenuous activity.

Cervical Vertebrae↗

PubMed2005

The clinical risk of vertebral artery injury from cervical pedicle screws inserted in degenerative vertebrae.

STUDY DESIGN: Postoperative outcomes of cervical pedicle screw (CPS) placement were evaluated. The screws were inserted in degenerative vertebrae using anatomic landmarks. OBJECTIVE: To evaluate the risks of this procedure caused by misplacements and subsequent complications. SUMMARY OF BACKGROUND DATA: The CPS gives superior vertebral fixation but risks causing vertebral artery and spinal cord injury. However, few clinical reports have been published concerning CPS misplacement and subsequent complications. Therefore, the risk of this procedure is still unclear. METHODS: There were 18 consecutive patients, who submitted to CPS fixation of degenerative vertebrae C2-C6, evaluated using computerized tomography (CT). In 5 of 9 patients in whom the screw critically violated the transverse foramen, viability of the vertebral artery was evaluated using multi-detector row CT angiography. RESULTS: There were no intraoperative vertebral artery or spinal cord injuries and no serious postoperative complications. A total of 86 screws were inserted, and 25 (29%) breached the pedicle cortex. Of them, 84% deviated laterally and violated the transverse foramen. There were 13 screws (15%) that deviated more than 2 mm. However, multi-detector row CT angiography showed the continuity of the vertebral artery in all cases. CONCLUSIONS: The deviation rate of CPS placement using anatomic landmarks was higher than anticipated, although no serious complications were encountered. Refinement of the technique (e.g., using a computer-assisted navigation system) is strongly recommended.

Adult↗

PubMed2005

Step-size is determined by neck length in myosin V.

The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.

Actin Cytoskeleton↗

PubMed2005

Effect of Smad7 expression on metastasis of mouse mammary carcinoma JygMC(A) cells.

BACKGROUND: Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. We used adenovirus-mediated gene transfer of these natural inhibitors in a mouse model of breast cancer to examine the roles of TGF-beta superfamily signaling in tumor growth and metastasis. METHODS: We systemically administered, by intravenous injection, adenoviruses (AdCMV) containing the mouse cDNAs for Smad7, Smad6, c-Ski, the c-Ski mutant c-Ski (ARPG), or LacZ (control) to nude mice (>19 mice/group) bearing tumors derived from mouse mammary carcinoma JygMC(A) cells, which spontaneously metastasize to lung and liver, and examined their effects on survival and metastasis. High-throughput western blotting analysis was used to examine the expression levels for 47 signal transduction proteins in JygMC(A) cells and primary tumors. We also investigated the proliferation, migration, and invasion of JygMC(A) cells that stably overexpressed Smad6 or Smad7. Nonparametric comparisons were done by Kruskal-Wallis H statistic and Wilcoxon's rank sum tests. Parametric comparisons were done by one-way analysis of variance or two-sided unpaired Student's t tests. All statistical tests were two-sided. RESULTS: Control mice bearing tumors derived from JygMC(A) cells showed many metastases to the lung and liver; all animals died by 50 days after cell inoculation. By contrast, mice treated with AdCMV-Smad7 or AdCMV-c-Ski demonstrated a dramatic decrease in metastasis and statistically significantly longer survival than control mice (Smad7 versus LacZ: medium survival = 55 days versus 41 days, difference = 14 days [95% confidence interval {CI} = 6 days to 22 days], P < .001), whereas mice treated with AdCMV-Smad6 or AdCMV-c-Ski (ARPG) did not. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and tight junctions, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells. CONCLUSION: Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers.

Adenoviridae↗

PubMed2005

Applications of 4,4'-(Me3Si)2-BINAP in transition-metal-catalyzed asymmetric carbon-carbon bond-forming reactions.

[structure: see text] A recently developed BINAP derivative with trimethylsilyl substituents on the 4- and 4'-positions of the binaphthyl skeleton, 2,2'-bis(diphenylphosphino)-4,4'-bis(trimethylsilyl)-1,1'-binaphthyl (tms-BINAP), was used in a variety of transition-metal-catalyzed asymmetric carbon-carbon bond-forming reactions. In pi-allylpalladium-mediated reactions, tms-BINAP gave better enantioselectivity than the unsubstituted BINAP, and the origin of the improved enantioselectivity was gained from an X-ray structural study of [Pd(eta(3)-C(3)H(5))((R)-tms-BINAP)]ClO(4).

Journal Article↗

PubMed2005

Crystallization of halorhodopsin from Halobacterium sp. shark.

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.

Crystallization↗

PubMed2005

A safe screw trajectory for atlantoaxial transarticular fixation achieved using an aiming device.

STUDY DESIGN: A retrospective evaluation and characterization of the trajectory of atlantoaxial transarticular screws inserted using an aiming device. OBJECTIVES: To confirm that the screws were inserted through the safest trajectory, which is through the most dorsal and medial part of the isthmus of C2, and to characterize the trajectory on lateral radiograms by comparison with historical controls. SUMMARY OF BACKGROUND DATA: Posterior atlantoaxial transarticular screw fixation entails the potential risk of vertebral artery (VA) injury, which may be lethal. Although much literature recommends that the screws should be inserted aiming at the anterior arch of C1, the authors considered that the safest screw path is via the most dorsal and medial part of the isthmus regardless of the C1 anterior arch, and have used an original aiming device to achieve this trajectory. METHODS: Forty-three consecutive patients who submitted to atlantoaxial transarticular screw fixation using the aiming device were evaluated for screw position using computed tomography (CT) and lateral radiogram. The medialization index (the distance between the screw and the cortex of the spinal canal of C2 on axial CT) and the dorsalization index (the thickness of the bone remaining dorsal to the screw at the isthmus of C2 on sagittal reconstruction CT) were measured. Further, three parameters on the lateral radiograms of these patients were compared with those in the literature and those of our previous cases performed without the aiming device. RESULTS: Neither VA injury nor violation of the spinal canal was encountered, although 12 high-riding VAs were included in this series. The mean medialization index was 0.21 mm, and the indexes of 86.3% of the screws were zero. The mean dorsalization index was 0.36 mm, and the indexes of 76.8% of the screws were zero. These results demonstrated that most of the screws were inserted as aimed, proving the usefulness of the aiming device. The trajectory of these screws on lateral radiograms was characterized by significantly less bone thickness dorsal to the screw at the isthmus compared with the two control groups. As a result, more screws were pointed above the anterior arch of C1. CONCLUSIONS: The atlantoaxial transarticular screw was inserted safely as aimed by using the aiming device. The trajectory was characterized by less bone thickness dorsal to the screw on lateral radiogram, which should be a new intraoperative landmark for screw insertion, in place of the anterior arch of C1.

Adult↗

PubMed2005

Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light.

Blebbistatin is a small molecule inhibitor discovered in a screen for inhibitors of nonmuscle myosin IIA. Blebbistatin inhibits the actin-activated MgATPase activity and in vitro motility of class II myosins. In cells, it has been shown to inhibit contraction of the cytokinetic ring. Blebbistatin has some photochemical properties that may affect its behavior in cells. In particular, we have found that exposure to light at wavelengths below 488 nm rapidly inactivates the inhibitory action of blebbistatin using the in vitro motility of myosin as an assay. In addition, the inhibition of cytokinetic ring contraction can be reversed by exposure of the cells to blue light. This property may be useful in locally reversing the action of blebbistatin treatment in a cell. However, caution should be exercised as free radicals may be produced upon irradiation of blebbistatin that could result in cell damage.

Actins↗

PubMed2005

A rare case of penile metastasis of testicular cancer presented with priapism.

Priapism is thought as a condition of penile erection that persists beyond or is unrelated to sexual stimulation. Commonly two different entities of priapism are known, one is low-flow priapism and the other is high-flow priapism. It is important to distinguish these two conditions for the subsequent different treatments. We report a rare case of an indistinguishable priapism caused by penile metastasis of testicular cancer.

Aged↗

PubMed2004

Transpedicular screw placement evaluated by axial computed tomography of the cervical pedicle.

STUDY DESIGN: We evaluated the trajectory of transpedicular screws in the cervical spine using axial computed tomography (CT). OBJECTIVES: To provide a safe transpedicular screw trajectory by measuring the dimensions of the cervical pedicle and evaluating the entrance points and the insertion angles of transpedicular screws. SUMMARY OF BACKGROUND DATA: The morphology of the cervical pedicle has been studied, but few in vivo CT-based studies of pedicle dimensions and transpedicular screw placement in the cervical spine have been reported. METHODS: The dimensions of the pedicles (C3-C7) were determined in 30 patients with cervical spinal lesions from CT images. The space available for transpedicular screws (SAS) was defined as the distance between two parallel lines tangential to the spinal canal and the transverse foramen, respectively. SAS was evaluated at 25 degrees and 50 degrees insertion angles. RESULTS: SAS at a 25 degrees insertion angle (SAS-25) ranged from 4.7 to 5.4 mm. SAS at 50 degrees (SAS-50) ranged from 6.1 to 6.6 mm. SAS-25 and SAS-50 were significantly different. Four-millimeter-diameter screws would fit in all 120 C3-C6 vertebrae studied at 50 degrees, but 20 (17%) would not fit at 25 degrees. CONCLUSIONS: Axial CT measurements should facilitate transpedicular screw fixation in the cervical spine. We believe that the screw insertion angle should be close to 50 degrees, which is the mean pedicle transverse angle from C3-C6. The entry point of the pedicle screw should be located as laterally as possible in the posterior surface of the lateral mass.

Adult↗

PubMed2004

Detection of a dural defect by cinematic magnetic resonance imaging and its selective closure as a treatment for a spinal extradural arachnoid cyst.

STUDY DESIGN: We document a giant spinal extradural arachnoid cyst treated by selective closure of the dural defect. It was diagnosed using cinematic magnetic resonance imaging (cine-MRI). OBJECTIVE: To demonstrate the effectiveness of selective closure of the dural defect as a treatment for a spinal extradural arachnoid cyst. SUMMARY OF BACKGROUND DATA: The standard treatment for a spinal extradural arachnoid cyst is total resection of the cyst wall, if possible, and the closure of the communication site, if any, between the cyst and the subarachnoid space, after an extensive laminectomy. To our knowledge, selective closure of the dural defect through minimal laminotomy with little cyst resection has not been reported. METHODS: A 29-year-old woman presented with right leg muscle weakness and was diagnosed with an extradural arachnoid cyst ranging from spinal regions T11 to L3 using MRI. Myelography demonstrated that the cyst communicated with the subarachnoid space. Cine-MRI showed a pulsating flow voiding on the left side of level L1, suggesting the location of the communication site. Fenestration of the T12-L1 region was performed, preserving the spinous processes and the facet joints. A small dural rent was found on the left side of level L1. This was closed using small clips. RESULTS: MRI 12 days later demonstrated that the cyst had shrunk dramatically. It had disappeared completely by 4 months. The patient's muscle weakness improved gradually, and she was almost complaint-free 6 months after the operation. CONCLUSIONS: Selective closure of the dural defect based on cine-MRI will be useful for treating extradural arachnoid cysts.

Adult↗

PubMed2004

S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase.

An amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas azotoformans IAM 1603 and characterized. The enzyme acted S-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined. The gene encoding the S-stereoselective piperazine-2-tert-butylcarboxamide amidase was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 2.1 kb of genomic DNA revealed the presence of two ORFs, one of which (laaA) encodes the amidase. This enzyme, LaaA is composed of 310 amino acid residues (molecular mass 34 514 Da), and the deduced amino acid sequence exhibits significant similarity to hypothetical and functionally characterized proline iminopeptidases from several bacteria. The laaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant LaaA enzyme in cell-free extracts of E. coli was 13.1 units.mg(-1) with l-prolinamide as substrate. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and two column chromatography steps. On gel-filtration chromatography, the enzyme appeared to be a monomer with a molecular mass of 32 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylhydrazine, Zn2+, Ag+, Cd2+ or Hg2+. LaaA had hydrolyzing activity toward L-amino acid amides such as L-prolinamide, L-proline-p-nitroanilide, L-alaninamide and L-methioninamide, but did not act on the peptide substrates for the proline iminopeptidases despite their sequence similarity to LaaA. The enzyme also acted S-stereoselectively on (R,S)-piperidine-2-carboxamide, (R,S)-piperazine-2-carboxamide and (R,S)-piperazine-2-tert-butylcarboxamide. Based on its specificity towards L-amino acid amides, the enzyme was named L-amino acid amidase. E. coli transformants overexpressing the laaA gene could be used for the S-stereoselective hydrolysis of (R,S)-piperazine-2-tert-butylcarboxamide.

Amides↗

PubMed2004

A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide.

A novel amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas sp. MCI3434 and characterized. The enzyme acted R-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (R)-piperazine-2-carboxylic acid, and was tentatively named R-amidase. The N-terminal amino acid sequence of the enzyme showed high sequence identity with that deduced from a gene named PA3598 encoding a hypothetical hydrolase in Pseudomonas aeruginosa PAO1. The gene encoding R-amidase was cloned from the genomic DNA of Pseudomonas sp. MCI3434 and sequenced. Analysis of 1332 bp of the genomic DNA revealed the presence of one open reading frame (ramA) which encodes the R-amidase. This enzyme, RamA, is composed of 274 amino acid residues (molecular mass, 30 128 Da), and the deduced amino acid sequence exhibits homology to a carbon-nitrogen hydrolase protein (PP3846) from Pseudomonas putida strain KT2440 (72.6% identity) and PA3598 protein from P. aeruginosa strain PAO1 (65.6% identity) and may be classified into a new subfamily in the carbon-nitrogen hydrolase family consisting of aliphatic amidase, beta-ureidopropionase, carbamylase, nitrilase, and so on. The amount of R-amidase in the supernatant of the sonicated cell-free extract of an Escherichia coli transformant overexpressing the ramA gene was about 30 000 times higher than that of Pseudomonas sp. MCI3434. The intact cells of the E. coli transformant could be used for the R-stereoselective hydrolysis of racemic piperazine-2-tert-butylcarboxamide. The recombinant enzyme was purified to electrophoretic homogeneity from cell-free extract of the E. coli transformant overexpressing the ramA gene. On gel-filtration chromatography, the enzyme appeared to be a monomer. It had maximal activity at 45 degrees C and pH 8.0, and was completely inactivated in the presence of p-chloromercuribenzoate, N-ethylmaleimide, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Hg2+, or Pb2+. RamA had hydrolyzing activity toward the carboxamide compounds, in which amino or imino group is connected to beta- or gamma-carbon, such as beta-alaninamide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutaminamide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme, however, did not act on the other amide substrates for the aliphatic amidase despite its sequence similarity to RamA.

Amidohydrolases↗

PubMed2004

Nanometer localization of single green fluorescent proteins: evidence that myosin V walks hand-over-hand via telemark configuration.

Myosin V is a homodimeric motor protein involved in trafficking of vesicles in the cell. It walks bipedally along actin filaments, moving cargo approximately 37 nm per step. We have measured the step size of individual myosin heads by fusing an enhanced green fluorescent protein (eGFP) to the N-terminus of one head of the myosin dimer and following the motion with nanometer precision and subsecond resolution. We find the average step size to be 74.1 nm with 9.4 nm (SD) and 0.3 nm (SE). Our measurements demonstrate nanometer localization of single eGFPs, confirm the hand-over-hand model of myosin V procession, and when combined with previous data, suggest that there is a kink in the leading lever arm in the waiting state of myosin V. This kink, or "telemark skier" configuration, may cause strain, which, when released, leads to the powerstroke of myosin, throwing the rear head forward and leading to unidirectional motion.

Adenosine Triphosphate↗

PubMed2003

Gamma-aminobutyric acid as a promoting factor of cancer metastasis; induction of matrix metalloproteinase production is potentially its underlying mechanism.

We investigated expression of gamma-aminobutyric acid (GABA), glutamate decarboxylase, and matrix metalloproteinase (MMP) in the prostates of patients with cancer or benign prostatic hypertrophy by immunohistochemical study. Marked expression of GABA, glutamate decarboxylase 67, and MMPs was observed in the prostates of cancer patients with metastasis (n = 72) and lymph node metastasis, although only sparse expression was noted in those of cancer patients without metastasis (n = 76) or patients with benign prostatic hypertrophy (n = 152). We then investigated the influence of GABA stimulation on in vitro MMP production and the invasive ability of cancer cells using human prostate cancer cell line C4-2. The production of MMPs increased significantly in cancer cells after a 24-h incubation with GABA. Cell invasion assay using a BioCoat Matrigel Invasion Chamber kit revealed that GABA stimulation significantly promoted the invasive ability of cancer cells and that addition of MMP inhibitor GM6001 significantly decreased GABA-induced migration. This may indicate the involvement of MMP activity in GABA-induced cancer cell invasion. We further analyzed the transmission pathway by performing GABA receptor modulation. The GABA(B) receptor agonist baclofen significantly increased MMP production as well as invasive ability. Moreover, blockade of the GABA(B) receptor pathway using GABA(B) receptor antagonist CGP 35348 significantly inhibited GABA-induced MMP production and invasive ability in cancer cells, whereas GABA(A) receptor modulation did not influence MMP production or the invasive ability of cancer cells. Thus, increased expression of GABA may be implicated in cancer metastasis by promoting MMP production in cancer cells, and the GABA(B) receptor pathway may be involved in the process.

Aged↗