Comparison of blood loss according to use of aspirin in lumbar fusion patients.

OBJECTIVE: To investigate and compare blood losses intra and postoperatively between lumbar fusion patients with and without antiplatelet use. METHODS: A total of 106 patients who had undergone at least 2 or more segments of lumbar fusion surgery were selected for the study. They were divided into three groups. Group 1 was not on medication before the surgery. Groups 2 and 3 had taken aspirin prior to the surgery. Group 2 discontinued the medication 1 week before the operation, but group 3 continued the use. In addition, non-steroid anti-inflammatory drug (NSAIDs) use in all patients was questioned. Amount of blood losses and platelet function were evaluated. RESULTS: When usage of NSAID was not controlled, intraoperative, postoperative, and total blood losses were found to have no statistical significance among the groups. However, when NSAID usage was taken into account, there were significantly higher blood losses in groups 2 and 3 compared with group 1. The use of NSAID resulted in significantly higher blood loss in group 1, but not in groups 2 or 3. The platelet function test results disclosed statistical differences between groups 1 and 2 and groups 1 and 3. CONCLUSION: Aspirin significantly increases the risk of bleeding in patients undergoing lumbar fusion at two or more levels. This risk is present even in patients who discontinued aspirin 1 week prior to surgery. In patients with high risk of complications resulting from aspirin discontinuation, the use should be allowed in lumbar fusion surgery. However, strong attention must be paid to avoid excessive bleeding. Because NSAID use also increases surgical blood loss, proper interval from discontinuation to surgery must be granted to minimize the risk.

High levels of the Mps1 checkpoint protein are protective of aneuploidy in breast cancer cells.

Most human cancers are aneuploid and have chromosomal instability, which contrasts to the inability of human cells to normally tolerate aneuploidy. Noting that aneuploidy in human breast cancer correlates with increased expression levels of the Mps1 checkpoint gene, we investigated whether these high levels of Mps1 contribute to the ability of breast cancer cells to tolerate this aneuploidy. Reducing Mps1 levels in cultured human breast cancer cells by RNAi resulted in aberrant mitoses, induction of apoptosis, and decreased ability of human breast cancer cells to grow as xenografts in nude mice. Remarkably, breast cancer cells that survive reductions in levels of Mps1 have relatively less aneuploidy, as measured by copies of specific chromosomes, compared with cells that have constitutively high levels of Mps1. Thus, high levels of Mps1 in breast cancer cells likely contribute to these cells tolerating aneuploidy.

Nuclear factor-kappaB (NF-kappaB) mediates a protective response in cancer cells treated with inhibitors of fatty acid synthase.

The efficacy of drugs used to treat cancer can be significantly attenuated by adaptive responses of neoplastic cells to drug-induced stress. To determine how cancer cells respond to inhibition of the enzyme fatty acid synthase (FAS), we focused on NF-κB-mediated pathways, which can be activated by various cellular stresses. Treating lung cancer cells with C93, a pharmacological inhibitor of FAS, results in changes indicative of a rapid initiation of NF-κB signaling, including translocation of RelA/p65 NF-κB to the nucleus, activation of a transfected NF-κB-luciferase reporter, and increased expression of NF-κB-dependent transcripts, IL-6, IL-8, and COX-2. Verifying that these responses to C93 are specifically related to inhibition of FAS, we confirmed that levels of these same transcripts increase in response to siRNA targeting FAS. Inhibiting this NF-κB response (either by transfecting a mutant IκBα or treating with bortezomib) resulted in increased cell killing by C93, indicating that the NF-κB response is protective in this setting. Because inhibiting FAS leads to accumulation of intermediate metabolites of fatty acid biosynthesis, we then questioned whether protein kinase C (PKC) is involved in this response to metabolic stress. Immunofluorescence microscopy revealed that C93 treatment results in cellular translocation of PKCα and PKCß isoforms and increased PKCα-dependent phosphorylation of the IκBα subunit of NF-κB. Furthermore, inhibiting PKC activity with RO-31-8220 or PKCα isoform-specific siRNA attenuates C93-induced IκBα phosphorylation and NF-κB activation and also potentiates C93-induced cell killing. These results suggest a link between PKC and NF-κB in protecting cancer cells from metabolic stress induced by inhibiting FAS.

Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability.

PURPOSE: Most breast cancers have chromosomal instability that seems related to defective mitotic spindle checkpoints. Because the molecular basis of this defect is unknown, we evaluated breast cancer cell lines and tissues for possible defects involving the major mitotic checkpoint genes responsible for maintaining chromosomal stability. EXPERIMENTAL DESIGN: We analyzed sequences and expression levels (RNA and protein) of eight major spindle checkpoint genes (MAD1L1, MAD2L1, MAD2L2, BUB1, BUB1B, BUB3, CDC20, and TTK) in a panel of 12 breast cancer cell lines, most with established genetic instability and defective spindle damage checkpoint response. mRNA levels of these genes were also measured in primary tumor samples, and immunohistochemical staining was used to evaluate BUB1B protein levels in a panel of 270 additional cases of breast cancer. RESULTS: No functionally significant sequence variations were found for any of the eight genes in the breast cancer cell lines with chromosomal instability. More surprisingly, the mRNA and protein levels for these checkpoint genes are significantly higher in the genetically unstable breast cancer cell lines and in high-grade primary breast cancer tissues than in the stable (and checkpoint proficient) MCF-10A and normal mammary epithelial cells, or in normal breast tissues. In fact, overexpression of the BUB1B protein is a marker that recognizes nearly 80% of breast cancers in paraffin-embedded tissues. CONCLUSIONS: Defective mitotic spindle checkpoints in breast cancer are most likely not caused by low expression or mutations of these eight checkpoint genes. High levels of these particular transcripts could represent a cellular compensation for defects in other molecular components of the mitotic spindle damage checkpoint, and increased expression of these genes might be markers of breast cancers with chromosomal instability.

Downregulation of protein kinase CKII is associated with cellular senescence.

Protein kinase CKII (CKII) plays a critical role in cell growth and proliferation. In this study, we examine how CKII activity is regulated during cellular senescence. Our results demonstrate that CKII activity apparently decreases during both replicative and H2O2-induced senescence in human diploid fibroblast IMR-90 cells. The mRNA and protein levels of CKIIalpha decreases significantly during replicative and H2O2-induced senescence, while only slight reduction in those of CKIIbeta is observed during replicative senescence. Treatment of IMR-90 cells with CKII inhibitors 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole and apigenin led cells to acquire a senescent phenotype as judged by the senescence-associated beta-galactosidase marker and overexpression of p53 and p21(Waf-1). Knockdown of CKIIalpha in IMR-90 cells by RNA interference also dramatically induced the senescent phenotype. In parallel, CKII activity was transcriptional downregulated in rat liver and testis with advancing age. Taken together, these results suggest that downregulation of CKII activity is tightly associated not only with cellular senescence but also with organism aging.

Dykellic acid inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting nuclear factor kappa B transcriptional activity.

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with expression of endopeptidases known as matrix metalloproteinases (MMPs). Expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate. We found that dykellic acid, a fungal metabolite, significantly inhibits the phorbol myristate acetate-induced increase in MMP-9 expression and activity. These effects of dykellic acid are time- and dose-dependent, and correlate with decreased MMP-9 promoter activity and mRNA expression. Whereas this compound does not affect DNA binding activity of nuclear factor kappa B (NF kappa B), dykellic acid does inhibit transactivation of NF kappa B. These data demonstrate a role for NF kappa B in the regulation of MMP-9 expression and the ability of dykellic acid to suppress this action of NF kappa B.

Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta signal transduction.

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase in MMP-9 expression and activity. These effects of resveratrol are dose dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection utilizing MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via an activator protein-1 and nuclear factor-kappaB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-delta may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.

Failure to activate caspase 3 in phorbol ester-resistant leukemia cells is associated with resistance to apoptotic cell death.

The protein kinase C (PKC)-specific inhibitor, Ro-31-8220, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the molecular pathways that lead to apoptosis after treatment of cells with the PKC-specific inhibitor RO-31-8220. For this, we used the U937 human leukemia cell line and a phorbolmyristate acetate (PMA)-resistant derivative cell line, R-U937. Ro-31-8220 treatment of U937 cells leads to apoptosis, which is accompanied by activation of caspase 3 (as measured by decreased levels of the 32kDa inactive form and increased proteolytic cleavage of phospholipase C (PLC)-gamma1). The broad-range caspase inhibitor z-VAD-fmk inhibits this induction of apoptosis, supporting a direct link between caspase activation and Ro-31-8220 induction of apoptosis. This activation of apoptosis is also accompanied by release of cytochrome c, but not by altered expression of Bcl-2 family protein or IAP family proteins. In R-U937 cells, Ro-31-8220 fails to cause release of cytochrome c, activation of caspase 3, or apoptosis. Activation of Akt occurs to a greater extent in the R-U937 cells than the U937 cells and thus might be related to protection from Ro-31-8220-induced apoptosis.

Coix seed extract, a commonly used treatment for cancer in China, inhibits NFkappaB and protein kinase C signaling.

A pharmaceutical grade extract of Coix lachryma-jobi seeds is currently the most commonly used treatment for cancer in China. Although clinical data support the use of this preparation of a Traditional Chinese Medicine for cancer treatment, biological basis for the activity of this preparation has not been previously established. To address this issue, we first evaluated the anti-neoplastic activity of a Coix extract emulsion in xenografts of MDA-MB-231 breast cancer cells and found that the extract significantly inhibits growth of MDA-MB-231 xenografts in athymic nude mice. Using oligonucleotide microarrays, we determined that Coix seed extract also significantly affects gene expression in these cells, including downregulation of genes (such as COX-2 and matrixmetalloproteinases) that are considered to be important in neoplasia. The specific gene expression changes noted after Coix seed extract treatment are characteristic of inhibition of NFkappaB-dependent transcription, leading us to evaluate how the treatment affects that pathway. An NFkappaB-dependent reporter assay demonstrated dose-dependant inhibition of NFkappaB signaling by treatment of cultures with the extract, and immunofluorescent microscopy found that these effects are associated with reduced translocation of the Rel-A/p65 subunit of NFkappaB to the nucleus. Coix extract also inhibits activity of protein kinase C, a major mediator of signal transduction and activator of NFkappaB. Thus, this Traditional Chinese Medicine-based cancer treatment affects cellular pathways of recognized importance in neoplasia.

Sodium orthovanadate potentiates EGCG-induced apoptosis that is dependent on the ERK pathway.

Epigallocatechin-3-gallate (EGCG) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis. In the present study, we determined the effect of vanadate, a potent inhibitor of tyrosine phosphatase, on EGCG-induced apoptosis. Investigation of the mechanism of EGCG or vanadate-induced apoptosis revealed induction of caspase 3 activity and cleavage of phospholipase-gamma1 (PLC-gamma1). Furthermore, vanadate potentiated EGCG-induced apoptosis by mitogen-activated protein kinase (MAPK) signaling pathway. Treatment with EGCG plus vanadate for 24h produced morphological features of apoptosis and DNA fragmentation in U937 cells. This was associated with cytochrome c release, caspase 3 activation, and PLC-gamma1 degradation. EGCG plus vanadate activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in EGCG plus vanadate-induced apoptosis in U937 cells. Elevated ERK activity that contributed to apoptosis by EGCG plus vanadate was supported by PD98059 and U0126, chemical inhibitor of MEK/ERK signaling pathway, prevented apoptosis. Taken together, our finding suggests that ERK activation plays an active role in mediating EGCG plus vanadate-induced apoptosis of U937 cells and functions upstream of caspase activation to initiate the apoptotic signal.

Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of Akt.

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human renal Caki cells. Treatment of Caki cells with 50 microM curcumin resulted in the activation of caspase 3, cleavage of phospholipase C-gamma1 and DNA fragmentation. Curcumin-induced apoptosis is mediated through the activation of caspase, which is specifically inhibited by the caspase inhibitor, benzyloxycarbony-Val-Ala-Asp-fluoromethyl ketone. Curcumin causes dose-dependent apoptosis and DNA fragmentation of Caki cells, which is preceded by the sequential dephosphorylation of Akt, down-regulation of the anti-apoptotic Bcl-2, Bcl-XL and IAP proteins, release of cytochrome c and activation of caspase 3. Cyclosporin A, as well as caspase inhibitor, specifically inhibit curcumin-induced apoptosis in Caki cells. Pre-treatment with N-acetyl-cysteine, markedly prevented dephosphorylation of Akt, and cytochrome c release, and cell death, suggesting a role for reactive oxygen species in this process. The data indicate that curcumin can cause cell damage by inactivating the Akt-related cell survival pathway and release of cytochrome c, providing a new mechanism for curcumin-induced cytotoxicity.

Differential expression of transforming growth factor-beta in the interstitial tissue of testis during aging.

Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.