De novo fatty-acid synthesis and related pathways as molecular targets for cancer therapy.

Enhanced lipid biosynthesis is a characteristic feature of cancer. Deregulated lipogenesis plays an important role in tumour cell survival. These observations suggest that enzymes in the lipid synthesis pathway would be rational therapeutic targets for cancer. To this end, we review the enzymes in de novo fatty-acid synthesis and related pathways.

Chemosensitisation by manganese superoxide dismutase inhibition is caspase-9 dependent and involves extracellular signal-regulated kinase 1/2.

Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

Activation of actin-cleavable interleukin 1beta-converting enzyme (ICE) family protease CPP-32 during chemotherapeutic agent-induced apoptosis in ovarian carcinoma cells.

We have previously reported that actin cleavage activity (ACA) by interleukin 1beta-converting enzyme (ICE) family protease was elevated during anticancer drug-induced apoptosis in human leukemia U937 cells. In this study, the involvement of ACA in the drug-induced apoptosis in solid tumor cells was investigated. Human ovarian carcinoma OVCAR-3 cells undergo apoptotic cell death when cells are treated with chemotherapeutic agents such as cisplatin and etoposide. The induction of the actin cleavage activity accompanied the development of apoptosis. ICE/ced-3 family protease inhibitors such as Z-VAD-CH2DCB and Z-EVD-CH2DCB at 100 microg/ml prevented both the emergence of ACA and the morphological change, characteristics of apoptosis, in cisplatin-treated OVCAR-3 cells. The ACA in apoptotic OVCAR-3 cell lysate was greatly adsorbed by antibody against CPP-32, an ICE family protease. Furthermore, the immunoprecipitated CPP-32 from OVCAR-3 lysate could cleave actin to generate a 15-kDa fragment, as did the apoptotic OVCAR-3 cell lysate, indicating that CPP-32 is a major protease responsible for the ACA. The activation of CPP-32 in the drug-treated cell lysate was verified with Western blot analysis. Our present results indicate that CPP-32, an actin cleavage ICE/ced-3 family protease, could be a common mediator involved in the process of chemotherapy-induced apoptosis of cancer cells.

Caspase-mediated cleavage of cytoskeletal actin plays a positive role in the process of morphological apoptosis.

Tumors result from the imbalance between cell growth and apoptosis. One of the characteristic changes in cancers is the abnormality in cytoskeleton, which suggests some roles of cytoskeletal proteins in tumorigenesis or the maintenance of tumor cells. Previously we showed that cytoskeletal actin is the substrate of caspases, the proteases responsible for apoptosis, while the role of actin cleavage in apoptosis remained unknown. To examine the cleavage of actin in vivo, we extensively performed immunoblot analysis using actin fragment-specific antibody. Here, we showed that, in some solid tumor cells, induction of apoptosis was accompanied by caspase-dependent actin-cleavage to 15 and 31 kDa fragments in vivo. To elucidate the role of actin-cleavage further, we introduced actin cleaved-fragments. We found that ectopic expression of an actin 15 kDa fragment induces morphological changes resembling those of apoptotic cells. The expression of the actin fragment induced a dramatic change of cellular actin localization, as visualized by enhanced green fluorescent protein (EGFP)-tagged actin, while the actin fragment expression did not cause caspase activation nor the cleavage of a marker substrate protein, poly (ADP-ribose) polymerase. These results indicate that actin cleavage could play a positive role in the morphological changes of apoptosis downstream of caspase activation.

Interleukin-7 inhibits apoptosis of mouse malignant T-lymphoma cells by both suppressing the CPP32-like protease activation and inducing the Bcl-2 expression.

Mouse malignant T-lymphoma CS-21 cells grow in vitro in the presence of CA-12 lymph node stromal cells, but they undergo apoptotic cell death when separated from CA-12 stromal cells. In the course of examining the nursing effects of CA-12 stromal cells, we found that these cells provided some soluble factors that suppressed CS-21 cell apoptosis. We recently found that cysteine was an antiapoptotic soluble factor. In this report, we identify interleukin-7 (IL-7) as another antiapoptotic soluble factor secreted by CA-12 stromal cells. Although the activity of CPP32-like protease was increased in induction of CS-21 cell apoptosis, the addition of IL-7 suppressed the activity. The expression of Bcl-2 protein was down-regulated when CS-21 cells were cultured alone, but the addition of IL-7 recovered the expression of Bcl-2. These results indicate that CA-12 stromal cells inhibit CS-21 cell apoptosis by producing IL-7, which leads to the suppression of CPP32-like protease activation and the expression of Bcl-2 protein.

Activation of c-Abl tyrosine kinase requires caspase activation and is not involved in JNK/SAPK activation during apoptosis of human monocytic leukemia U937 cells.

Genotoxic stress triggers the activation of several sensor molecules, such as p53, JNK1/SAPK and c-Abl, and occasionally promotes the cells to apoptosis. We previously reported that JNK1/SAPK regulates genotoxic stress-induced apoptosis in p53-negative U937 cells by activating caspases. c-Abl is expected to act upstream of JNK1/SAPK activation upon treatment with genotoxic stressors, but its involvement in apoptosis development is still unclear. We herein investigated the kinase activities of c-Abl and JNK1/SAPK during apoptosis elicited by genotoxic anticancer drugs and tumor necrosis factor (TNF) in U937 cells and their apoptosis-resistant variant UK711 cells. We found that the activation of JNK1/SAPK and c-Abl correlated well with apoptosis development in these cell lines. Unexpectedly, however, the JNK1/SAPK activation preceded the c-Abl activation. Moreover, the caspase inhibitor Z-Asp suppressed c-Abl activation and the onset of apoptosis but not the JNK1/SAPK activation. Interestingly, c-Abl tyrosine kinase inhibition by CGP 57148 reduced apoptosis without interfering with JNK1/SAPK activation. These results indicate that c-Abl acts not upstream of JNK1/ SAPK but downstream of caspases during the development of p53-independent apoptosis and is possibly involved in accelerating execution of the cell death pathway.

Chromosome 22 complements apoptosis in Fas-and TNF-resistant mutant UK110 cells.

Fas and p55 tumor necrosis factor receptor (TNFR) transfer an apoptosis signal when they are crosstinked with their ligands or agonistic antibodies. However, the signal transduction mechanism of apoptosis via Fas and p55 TNFR has not yet been elucidated. We previously described a recessive mutant UK110 from the human monocytic leukemia U937 cell line, that showed resistance against Fas- and p55 TNFR-mediated apoptosis. By cytogenetic analysis and microcell-fusion method, we demonstrate here that introduction of chromosome 22 can specifically restore the sensitivity to Fas- and TNF-mediated apoptosis in UK110 cells. Moreover, introduction of chromosome 22 into UK110 can complement the processing of interleukin-1 beta converting enzyme (ICE)-like proteases, such as CPP32/Yama/Apopain and ICH-1L, after treatment with anti-Fas and anti-p55 TNFR antibodies. These results suggest that the product of a gene located on chromosome 22 participates in the Fas-and p55 TNFR-mediated apoptosis at a point upstream of ICE-like proteases.

Actin cleavage by CPP-32/apopain during the development of apoptosis.

Interleukin-1beta-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15 kDa and 31 kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15 kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

ASK1 mediates apoptotic cell death induced by genotoxic stress.

ASKI mediates apoptotic cell death induced by genotoxic stress Genotoxic stress-induced apoptosis is mediated by caspase family proteases as triggered by other stimuli. In this study, we found that the DNA-damaging agent cisplatin (cDDP) activated MAP kinase kinase kinase ASK1 and subsequent downstream subgroups of MAP kinase kinase, SEK1 (or MKK4) and MKK3/MKK6, which in turn activated c-Jun N-terminal kinase 1/stress-activated protein kinase (JNK1/SAPK) and p38 MAP kinase prior to caspase family protease activation and the onset of apoptosis in human ovarian carcinoma (OVCAR-3) and human kidney (293T) cells. As reported previously, benzyloxy carbonyl-Asp-CH2OC(O)-2, 6-dichlorobenzene (Z-Asp), a preferential inhibitor of caspase family proteases, blocked the apoptosis of OVCAR-3 cells induced by the genotoxic stress cDDP. Z-Asp, however, did not inhibit ASKI activation and the subsequent kinase cascades. Overexpression of kinase-negative ASK1 (K709R), which inhibited ASK1 activation and the downstream MKK3-p38 and MKK4-JNK1 pathways, also suppressed the caspase protease activation and apoptosis induced by cDDP. These results indicate that the ASK1 pathway is involved in genotoxic stress-induced apoptosis and mediates apoptosis at a step upstream of caspase protease activation.

Induction of in vitro nuclear apoptosis activity coincides with the production of 50 kDa cytosolic protein.

Human monocytic leukemia U937 cells undergo apoptosis when cells are treated with the anticancer drug etoposide. To study the mechanism of drug-induced apoptosis, we used an in vitro apoptosis system with cytosol from etoposide-treated U937 cells. The cytosol from apoptotic U937 cells showed activity to induce morphologic changes and oligonucleosomal DNA fragmentation in isolated nuclei in vitro; both are typical features of apoptosis. We generated monoclonal antibodies to the proteins in the etoposide-treated U937 cytosol. We found that a 50 kDa protein, recognized by SN-1 monoclonal antibody, appeared in the cytosol of U937 cells, in accordance with its cell-free apoptosis activity. Z-Asp, an inhibitor of interleukin-1beta converting enzyme (ICE) family proteases, inhibited the appearance of the 50 kDa protein and the emergence of the cell-free apoptosis activity in the etoposide-treated U937 cytosol. These results indicate that the 50 kDa protein is produced by the activation of ICE family protease during apoptosis and suggest some roles of the protein in the development of apoptosis.

Selective activation of apoptosis program by S-p-bromobenzylglutathione cyclopentyl diester in glyoxalase I-overexpressing human lung cancer cells.

PURPOSE: Glyoxalase I (GLO1) is an enzyme that plays a role in the detoxification of methylglyoxal, a side-product of glycolysis. We previously reported that GLO1 was a resistant factor to antitumor agent-induced apoptosis, and that S-p-bromobenzylglutathione cyclopentyl diester (BBGC), an effective inhibitor of GLO1, selectively sensitized to etoposide the drug-resistant human leukemia cells that overexpressed GLO1. In this study, we quantitatively measured GLO1 enzyme activity in various human solid tumor cells, and the antiproliferative effect of the GLO1 inhibitor was examined. EXPERIMENTAL DESIGN: BBGC-induced apoptosis was assessed by flow cytometry. To evaluate antitumor activity of BBGC in vivo, we developed human cancer xenografts in nude mice. RESULTS: We found that GLO1 enzyme activity was higher in all of the 38 human cancer cell lines that we examined than in the normal tissue samples. Moreover, GLO1 activity was frequently elevated in human lung carcinoma cells. Positive correlation between cellular GLO1 activity and BBGC sensitivity was observed in the lung cancer cell lines. Human lung cancer NCI-H522 and DMS114 cells, expressing higher GLO1 activity, underwent apoptosis when treated with BBGC, whereas A549 cells, expressing lower activity, did not. BBGC induced the activation of the stress-activated protein kinases c-Jun NH(2)-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK), which led to caspase activation in GLO1-overexpressing tumor cells. BBGC significantly inhibited the growth of xenografted DMS114 and human prostate cancer DU-145. CONCLUSIONS: Our present results indicate that GLO1 is a tumor-specific target enzyme especially in human lung carcinoma cells and that the GLO1 inhibitor is a potent chemotherapeutic agent to repress GLO1-overexpressing human tumors.

Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase-3, such as DEVD-MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase-3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase-3-like activity within HeLa cells treated with etoposide. The caspase-3-like activity was increased in the cytosol at first, then expanded to the whole cell.

The effect of hydroxyapatite coating on bony ingrowth into grooved titanium implants.

This study was undertaken to investigate the relative importance of a hydroxyapatite (HA) coating and the macrotexture of titanium implants to the quality of bony ingrowth and fixation. Various types of titanium cylinders were implanted into the cancellous bone of the intercondylar region of the distal femur of the dog. The animals were sacrificed at intervals post-implantation and the implants were evaluated histologically for amount of bony ingrowth and mechanically by the means of a push-out test. Our results demonstrated that when grooved titanium implants are used, the addition of HA coating significantly improved the biologic fixation. In addition, a groove depth of 1 mm was found to give significantly better fixation than 2 mm. When compared to implants with traditional, beads-coated porous surfaces, HA-coated grooved titanium implants were found to show better fixation at 4 weeks after implantation, but, significantly inferior fixation at 12 weeks after implantation. We concluded that while a groove depth of 1 mm was optimal in HA-coated, grooved titanium implants, they remain inferior to beads-coated titanium implants with respect to longer-term fixation. More research needs to be addressed at improving the macrotexture environment of grooved implants to enhance long-term bony ingrowth.

Remodelling of bone around hydroxyapatite and titanium in experimental osteoporosis.

The affinity of the tibia bone for cylinders of hydroxyapatite (HA) and Ti-6Al-4V was investigated in three experimental animal models: intact rat tibiae, ovariectomized rat tibiae and ovariectomized plus neurectomized rat tibiae. The affinity index (the length of bone directly apposed to the implant/the total length of the bone-implant interface x 100%) was calculated. In intact and ovariectomized tibiae, no significant difference existed between the affinity index of HA and Ti-6Al-4V. In ovariectomized plus neurectomized tibiae, the affinity index of HA was greater than for Ti-6Al-4V from 8 to 24 wk after implantation (8 wk, P < 0.05; 12 wk, P < 0.005; 24 wk, P < 0.05). The affinity index of Ti-6Al-4V in normal tibiae was greater than that in ovariectomized or ovariectomized plus neurectomized tibiae. There was no significant difference of the affinity index of HA between normal, ovariectomized or ovariectomized plus neurectomized tibiae. Osteoporotic bone demonstrated a greater affinity to HA than Ti-6Al-4V in an experimental animal model.

Bone-implant interface mechanics of in vivo bio-inert ceramics.

We have previously demonstrated that there was no significant difference between the affinity of bone to bio-inert ceramics and stainless steel in a histological study. In this study, the bone-implant interface shear strength of alumina ceramics (Al2O3), zirconia ceramics (ZrO2), stainless steel (SUS316L) and sintered hydroxyapatite (HA) were compared in 19 dogs using a transcortical push-out model of the femur 4 and 12 wk after implantation. The interface shear strength of HA was significantly greater than that of alumina ceramics, zirconia ceramics and stainless steel (P < 0.001). There was no significant difference between bio-inert ceramics and stainless steel.

T cell receptor-extracellular constant regions as hetero-cross-linkers for immunoglobulin variable regions.

The T cell receptor alpha and beta chains are covalently linked via a disulfide bond in their extracellular constant regions. To use these domains as specific hetero-cross-linkers of two different polypeptides, we created genetic constructs encoding a chimeric antibody Fab fragment in which mouse immunoglobulin constant regions from a phosphorylcholine-specific antibody were substituted for human alpha beta-T cell receptor (TCR) extracellular constant regions (for solubilization, the transmembrane- and cytoplasmic-regions of the receptor were deleted). These constructs, i.e., chimeric heavy (VHC beta C kappa) and light (VLC alpha) chains, were cotransfected into murine SP2/0 myeloma cells for expression. Cells transfected with the genes expressed mRNAs for chimeric heavy and light chains. Without CD3 molecules, the two chimeric chains specifically associated via a disulfide bond to form a chimeric Fab fragment in the cells. These data indicate that the TCR C alpha- and C beta-regions might be used as potent specific hetero-cross-linkers for protein engineering.

Activation of caspase protease during apoptosis in ovarian cancer cells.

As a genetically controlled program, apoptosis has important roles in a variety of biological processes. The realization that chemotherapy can also induce apoptosis in some cancer cells both in vitro and in vivo indicates apoptosis may play a very important role in cancer and cancer therapy (1,2). Many of the molecules that participate in the apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the "caspases" (3).

Dysphagia complications in ankylosing spinal hyperostosis and ossification of the posterior longitudinal ligament. Roentgenographic findings of the developmental process of cervical osteophytes causing dysphagia.

This is the first report of the process of formulation of a cervical osteophyte causing dysphagia. The patient had ankylosing spinal hyperostosis and OPLL and was followed radiographically for a long time before the onset of dysphagia. The radiological observation suggested that dysphagia was produced when the immobile part of the esophagus was compressed by the anterior projecting cervical osteophyte. The immobility of the esophagus is an important factor in determining whether dysphagia occurs. Another possible contributing factor to dysphagia in this patient was the ossification of the cervical anterior and posterior longitudinal ligaments. The OPLL affected intervertebral segmental motion and induced the formation of anterior projecting cervical osteophytes.

Conservative treatment for cervical spondylotic myelopathy. prediction of treatment effects by multivariate analysis.

BACKGROUND CONTEXT: Many studies have suggested only slight effects of conservative treatment on cervical spondylotic myelopathy (CSM), whereas a few reports describe conservative treatment as being effective. This suggested the influence of various factors on treatment outcomes. PURPOSE: We investigated symptomatic changes after conservative treatment in patients based on a clear understanding of the effects and limitations of conservative treatment. STUDY DESIGN: We have encountered cases that showed symptomatic improvement with conservative treatment and became interested in the effectiveness of conservative treatment for CSM and whether other factors affect the results of conservative treatment. PATIENT SAMPLE: We have analyzed the results of conservative treatment for CSM in 69 cases, derived from a population of 101 CSM cases. OUTCOME MEASURES: Symptoms at the time of the first examination were compared with those at the final examination, and the patients were classified into three groups showing improvement, no change or exacerbation. METHODS: Improvement or exacerbation of the symptoms was used as dependent variables and the collected factors as independent variables, and logistic regression was performed on these variables. RESULTS: Multivariate analysis showed significant correlation between clinical outcome and the disease duration and the presence of rigorous conservative treatment. CONCLUSIONS: Conservative treatment for CSM is considered to be effective if it is performed intensively in selected patients. In treating CSM, the therapeutic approach must be selected first in consideration of the patient's disease duration. Conservative treatment must be carried out intensively after sufficient explanation to the patients. Timely surgical intervention is considered to be important if the symptoms show no change or exacerbation with conservative treatment.

Efficacy of aquatic exercises for patients with low-back pain.

We have studied 35 patients (25 female and 10 male) with low-back pain who were managed with aquatic exercises after an appropriate period of treatment for their condition in the medical institution. The exercises employed consisted of strengthening exercises for the abdominal, gluteal, and leg muscles, stretching of the back, hip, hamstrings, and calf muscles, walking in water, and swimming. All the patients had been participating in the exercise program for more than 6 months. The frequency of performing exercises was once a week for 7 patients, twice a week for 19, and 3 or more times a week for the remaining patients. The method used in this study was a survey questionnaire which was composed of questions about the patient's physical and psychological condition. Those patients who had performed exercises twice or more in a week showed a more significant improvement in the physical score than those who performed exercises only once a week. More than 90% of the patients felt they had improved after 6 months of participation in the program. The improvement in physical score was independent of the initial ability in swimming. The results obtained suggested that exercises in water may be one of the most useful modes of exercise for a patient with low-back pain.