Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Detecting mRNA Predictors of Acetaminophen-Induced Hepatotoxicity in Mouse Blood Using Quantitative Real-Time PCR.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. Drug-induced liver injury from agents such as APAP is known to vary between individuals within a species. To avoid liver injury and ensure the proper use of pharmaceutical products, it is important to be able to predict such risks using genetic information. This study evaluated the use of quantitative real-time polymerase chain reaction (RT-qPCR) to identify mRNAs (carried in the blood of male ddY mice) capable of predicting susceptibility to APAP-induced hepatotoxicity. Screening was performed on samples obtained at 18 h after treatment from mice that had been orally treated with 500 mg/kg APAP. APAP-induced hepatotoxicity was seen in 60% of the mice, and the mortality rate was 12%. Blood APAP concentration did not differ significantly between mice with and without APAP-induced hepatotoxicity. We compared blood mRNA expression levels between mice with (positive, serious or lethal injury) and without hepatotoxicity in the APAP-treated group. The transcript levels of interleukin-encoding loci Il1ß, Il10, and tumor necrosis factor (Tnf) were increased in the lethal injury group. Transcripts of the loci encoding transthyretin (Ttr) and metallothionein 1 (Mt1) showed increases in the liver injury group, while those of the glutathione peroxidase 3-encoding locus (Gpx3) were decreased. APAP hepatotoxicity was potentiated in fasted animals, although fasting did not appear to affect the level of expression of these genes. These results indicate that mRNA expression of Il1ß, Il10, Tnf, Ttr, Mt1, and Gpx3 in mouse blood may provide useful surrogate markers of APAP-induced hepatotoxicity.

Angiotensin II produces nociceptive behavior through spinal AT1 receptor-mediated p38 mitogen-activated protein kinase activation in mice.

BACKGROUND: It has been demonstrated that angiotensin II (Ang II) participates in either the inhibition or the facilitation of nociceptive transmission depending on the brain area. Neuronal Ang II is locally synthesized not only in the brain, but also in the spinal cord. Though the spinal cord is an important area for the modulation of nociception, the role of spinal Ang II in nociceptive transmission remains unclear. Therefore, in order to elucidate the role of Ang II in nociceptive transmission in the spinal cord, we examined the effect of intrathecal (i.t.) administration of Ang II into mice. RESULTS: I.t. administration of Ang II produced a behavioral response in mice mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by Ang II (3 pmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.1-0.3 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also inhibited dose-dependently by i.t. co-administration of losartan (0.3-3 nmol), an Ang II type 1 (AT1) receptor antagonist, and SB203580 (0.1-1 nmol), a p38 MAPK inhibitor. However, the Ang II type 2 (AT2) receptor antagonist PD123319, the upstream inhibitor of ERK1/2 phosphorylation U0126, and the JNK inhibitor SP600125 had no effect on Ang II-induced nociceptive behavior. Western blot analysis showed that the i.t. injection of Ang II induced phosphorylation of p38 MAPK in the lumbar dorsal spinal cord, which was inhibited by losartan, without affecting ERK1/2 and JNK. Furthermore, we found that AT1 receptor expression was relatively high in the lumbar superficial dorsal horn. CONCLUSIONS: Our data show that i.t. administration of Ang II induces nociceptive behavior accompanied by the activation of p38 MAPK signaling mediated through AT1 receptors. This observation indicates that Ang II may act as a neurotransmitter and/or neuromodulator in the spinal transmission of nociceptive information.

Does the balance strategy during walking in elderly persons show an association with fall risk assessment?

The primary objective of this study was to clarify whether balance evaluation during walking in elderly people was related to fall risk assessment; the second objective was to clarify the difference in balance strategy between young and elderly people based on the balance evaluation through a gait cycle. Thirty healthy young adults and 25 healthy elderly adults participated. All participants performed walking at their preferred speed and at a fast speed. Based on the margin of stability (MoS), balance during a gait cycle was divided into medial/lateral and anterior/posterior direction (ML/AP-MoS). Positive/negative integral values of ML-MoS were defined as ML-MoSPOS/ML-MoSNEG, and the average of AP-MoS over the gait cycle was defined as AP-MoSmean. The fast/preferred ratio of AP-MoSmean/ML-MoSPOS (AP-MoSmean (Fast/Preferred)/ML-MoSPOS (Fast/Preferred)) and the fast-preferred difference of ML-MoSNEG (ML-MoSNEG (Fast-Preferred)) were compared between groups. ML/AP-MoS at the preferred/fast gait was also compared between 12 gait events and groups. The Japanese version of the Mini-Balance Evaluation Systems Test (J-Mini-BESTest), the Japanese version of the Activities-specific Balance Confidence Scale (J-ABC scale), and the number of falls in the past year were obtained from all subjects. ML-MoSPOS (Fast/Preferred), ML-MoSNEG (Fast-Preferred), and AP-MoSmean (Fast/Preferred) were significantly correlated with J-Mini-BESTest. Gait balance evaluation based on MoS may reflect an individual's balance function. In fast gait, ML-MoS at foot flat and toe off and AP-MoS at just before heel strike were highly likely to be gait events to identify elderly adults with balance disorders.

Antimicrobial cathelicidin peptide CAP11 suppresses HMGB1 release from lipopolysaccharide-stimulated mononuclear phagocytes via the prevention of necrotic cell death.

High mobility group box-1 (HMGB1) is extracellularly released from mononuclear phagocytes by lipopolysaccharide (LPS)-stimulation accompanied with cell death, and plays an important role in septic/endotoxin shock as a late phase mediator. Notably, CAP11 (cationic antibacterial polypeptide of 11-kDa), a member of cathelicidin family of antimicrobial peptides, has a potential to bind with LPS and neutralize the biological activity of LPS. In this context, we previously revealed that CAP11 can suppress the elevation of serum HMGB1 level in mouse endotoxin shock model and protect mice from endotoxin lethality. In the present study, to clarify the inhibitory mechanism of CAP11 on HMGB1 release, we evaluated the effect of CAP11 on the LPS-induced HMGB1 release and apoptotic/necrotic cell death using a murine macrophage cell line RAW264.7. The results indicated that LPS-stimulation induced the release of HMGB1 from RAW264.7 cells, accompanied with both apoptotic and necrotic cell death. Of interest, CAP11 markedly inhibited the binding of LPS to target RAW264.7 cells, and suppressed HMGB1 release as well as necrotic cell death; however, CAP11 could not affect the LPS-induced apoptotic cell death. These observations clearly indicate that CAP11 can efficiently abolish necrotic cell death via the inhibition of LPS-binding to target cells, thereby suppressing the release of HMGB1. Thus, CAP11 could be a therapeutic agent for septic/endotoxin shock, with a potential to regulate the release of HMGB1 from LPS-stimulated mononuclear phagocytes via the suppression of LPS-binding to target cells and prevention of necrotic cell death due to its potent LPS-binding activity.

Augmentation of the antimicrobial activities of guinea pig cathelicidin CAP11-derived peptides by amino acid substitutions.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensin and cathelicidin) that contribute to the innate host defense against invading microorganisms. Among these, guinea pig cathelicidin CAP11 (G1-I43) possesses potent antibacterial activities against Gram-positive and -negative bacteria, and also lipopolysaccharide-neutralizing activity. We previously revealed that the active region with antibacterial activity is localized at G1 to R18 of CAP11. In this study, to develop peptide derivatives with enhanced antimicrobial actions, we utilized the amphipathic 18-mer peptide (G1-R18) as a template. Anti-microbial activities of the peptides were assessed by alamarBlue assay (Escherichia coli, Staphylococcus aureus and Candida albicans) and colony formation assay (Porphyromonas gingivalis). Furthermore, the membrane-permeabilization activities were determined by using E. coli ML-35p as a target. By substituting K5, T9, R10, R12, and G17 with five L residues, the hydrophobicity of the peptide (1-18m1) was increased, and by substituting G1, and Q14 with K and R residues, respectively, the hydrophilicity (positive charge) of the peptide (1-18m2) was enhanced. Among the peptides, 1-18m2 exhibits the most potent antimicrobial and membrane-permeabilizing activities against the microorganisms examined. Thus, the antimicrobial activities of the amphipathic CAP11-derived 18-mer peptide can be augmented by modifying its hydrophobicity and hydrophilicity (positive charge), and 1-18m2 is the most potent among the peptide derivatives with therapeutic potential for Gram-positive and -negative bacterial, and fungal infections.

Investigation of balance strategy over gait cycle based on margin of stability.

This study was conducted to investigate the balance strategy of healthy young adults through a gait cycle using the margin of stability (MoS). Thirty healthy young adults participated in this study. Each performed walking five times at a preferred speed and at a fast speed. The MoS was calculated over a gait cycle by defining the base of support (BoS) changes during a gait cycle. The MoS was divided into medial/lateral and anterior/posterior components (ML MoS and AP MoS). The central values and the values at 12 gait events of the MoS were compared. Positive/negative integration of ML MoS (ML MoSPOS and ML MoSNEG, respectively) and the average ML/AP MoS over a cycle (ML/AP MoSmean) were significantly lower at a fast gait than at a preferred gait. ML/AP MoS were lower at a fast speed than at the preferred speed, except for the ML MoS immediately before left heel strike (pre left HS) and right and left heel strike (HS). ML/AP MoS were significantly lower immediately before heel strike (pre-HS) than in other gait events, regardless of walking speed. It was suggested that pre-HS is the most unstable moment in both ML/AP directions and a crucial moment in control of gait stability. The results presented above might be applicable as basic data regarding dynamic stability of healthy young adults through a gait cycle for comparisons with elderly people and patients with orthopedic disorders or neurological disorders.

Glucosamine suppresses interleukin-8 production and ICAM-1 expression by TNF-alpha-stimulated human colonic epithelial HT-29 cells.

Intestinal epithelial cells play an important role in the mucosal immune reaction in inflammatory bowel diseases via the production and expression of chemokines and adhesion molecules, such as interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1), which are involved in the neutrophil infiltration and tissue damage in the inflamed colon. Notably, glucosamine, a naturally-occurring amino monosaccharide, has been shown to exhibit an anti-inflammatory action by inhibiting neutrophil functions. In the present study, to evaluate the anti-inflammatory action of glucosamine on intestinal epithelial cells, we examined the effects of glucosamine on the activation of a human colonic epithelial cell line HT-29. The results revealed that glucosamine suppressed the IL-8 production and ICAM-1 expression by TNF-alpha-activated HT-29 cells. Furthermore, glucosamine inhibited the TNF-alpha-induced phosphorylation of p38MAPK and NF-kappaB p65, and the nuclear translocation of NF-kappaB in the cells. Thus, glucosamine demonstrates inhibitory actions on the inflammatory and signaling molecules (IL-8, ICAM-1, p38MAPK and NF-kappaB) in intestinal epithelial cells. However, glucosamine did not essentially affect the binding of TNF-alpha to its receptor on HT-29 cells. Together, these observations suggest that glucosamine may have the potential to exhibit an anti-inflammatory action on intestinal epithelial cells, by possibly interfering with the activation signaling downstream of the ligand/receptor binding.

Glucosamine, a naturally occurring amino monosaccharide, suppresses dextran sulfate sodium-induced colitis in rats.

Glucosamine, a naturally occurring amino monosaccharide, is widely used to treat osteoarthritis in humans. Furthermore, glucosamine exhibits an anti-inflammatory action by inhibiting the activation of neutrophils, chondrocytes and synoviocytes. Recently, we revealed that glucosamine suppresses cytokine-induced activation of intestinal epithelial cells in vitro. In the present study therefore, we investigated whether glucosamine exhibits the anti-inflammatory effect in vivo, using dextran sulfate sodium (DSS)-induced colitis in rats, a model of inflammatory bowel diseases (IBD). The results indicated that glucosamine improved the clinical symptoms (evaluated by disease activity index), and suppressed colonic inflammation (evaluated by colon length and weight/length ratio) and tissue injury (evaluated by histological damage score) in DSS-induced colitis. Furthermore, glucosamine inhibited the activation of intestinal epithelial cells, as evidenced by the suppressed phosphorylation of NF-kappaB in the intestinal mucosa of DSS-induced colitis. Collectively, these observations suggest that glucosamine is likely to suppress the activation of intestinal epithelial cells in vivo, thereby possibly exhibiting anti-inflammatory action in a DSS-induced rat colitis model. Thus, glucosamine could prove to be a useful agent for IBD.

Antibacterial cathelicidin peptide CAP11 suppresses the anandamide production from lipopolysaccharide-stimulated mononuclear phagocytes.

The action of antibacterial cathelicidin peptide CAP11 on the anandamide production from mononuclear phagocytes was examined. Lipopolysaccharide (LPS)-stimulation induced the anandamide production from macrophage-like RAW264.7, accompanied with the enhanced anandamide-synthesizing enzyme activity; however, the anandamide-degrading enzyme activity was not changed by LPS-stimulation. Importantly, CAP11 suppressed the LPS-induced anandamide production and the increase of anandamide-synthesizing enzyme activity. Furthermore, CAP11 abrogated the LPS-binding to CD14-positive RAW264.7. These observations indicate that CAP11 inhibits the binding of LPS to CD14-positive mononuclear phagocytes, thereby suppressing the anandamide synthesizing enzyme activity and the anandamide production from the cells.

Glutathione peroxidase 3 is a protective factor against acetaminophen­induced hepatotoxicity in vivo and in vitro.

Acetaminophen (APAP) is a widely available antipyretic and analgesic; however, overdose of the drug inflicts severe damage to the liver. It is well established that the hepatotoxicity of APAP is initiated by formation of a reactive metabolite, N­acetyl­p­benzoquinone imine (NAPQI), which can be detoxified by conjugation with reduced glutathione (GSH), a typical antioxidant. We recently found that the blood mRNA expression level of glutathione peroxidase 3 (Gpx3), which catalyzes the oxidation of GSH, is associated with the extent of APAP­induced hepatotoxicity in mice. The present study was carried out to determine the in vivo and in vitro role of GPx3 in APAP­induced hepatotoxicity. In in vivo experiments, oral administration of APAP to mice induced liver injury. Such liver injury was greater in males than in females, although no gender difference in the plasma concentration of APAP was found. Female mice had a 2­fold higher expression of Gpx3 mRNA and higher plasma GPx activity than male mice. 17ß­estradiol, a major female hormone, decreased APAP­induced hepatotoxicity and increased both the expression of blood Gpx3 mRNA and plasma GPx activity, suggesting that the cytoprotective action of this hormone is mediated by the increase in GPx3. To further clarify the role of GPx3 in APAP­induced hepatotoxicity, we evaluated the effect of a change in cellular GPx3 expression resulting from transfection of either siRNA­GPx3 or a GPx3 expression vector on NAPQI­induced cellular injury (as assessed by a tetrazolium assay) in in vitro experiments using heterogeneous cultured human cell lines (Huh­7 or K562). NAPQI­induced cell death was reduced by increased GPx3 and was enhanced by decreased GPx3. These results suggest that GPx3 is an important factor for inhibition of APAP­induced hepatotoxicity both in vivo and in vitro. To our knowledge, this is the first report to show a hepatoprotective role of cellular GPx3 against APAP­induced liver damage.

Pifithrin-alpha has a p53-independent cytoprotective effect on docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma HepG2 cells.

Pifithrin-alpha (PFT) is an inhibitor of p53 and is known to protect against a variety of p53-mediated genotoxic agents. In this report, we examined the inhibitory effects of PFT against docosahexaenoic acid (DHA)-induced cytotoxicity in the human hepatocellular carcinoma (HCC) cell line HepG2. PFT significantly abrogated DHA-induced cytotoxicity in wild-type HepG2 cells (normal expression of p53) and after p53-knockdown by siRNA, as well as in Hep3B (p53 null) and Huh7 (p53 mutant) cells. DHA-induced cytotoxicity is mediated by induction of oxidative stress, and PFT inhibited this event, but it does not exert antioxidant effects. PFT significantly suppressed the release of cytochrome c from mitochondria to cytosol, as well as changes in the mitochondrial membrane potential (ΔΨM) by DHA. Therefore, protection of mitochondria by PFT is crucial for its inhibition of DHA-induced cytotoxicity. Although it has been reported that PFT is able to block p53 function, our data suggest that PFT also has a p53-independent inhibition mechanism. This work provided insights into the mechanisms of PFT action on DHA-induced cytotoxicity in HCC.

Differential inhibition by TAK-044 of the inotropic effects of endothelin-1 and endothelin-3.

The influence of a nonselective antagonist of endothelin receptors, TAK-044 (cyclo-[d-alpha-aspartyl-3-[(4-phenylpiperazin-1-yl)carbonyl]-l-alanyl-l-alpha-aspartyl-d-2-(2-thienyl)glycyl-l-leucyl-d-tryptophyl] disodium), on the positive inotropic effect of endothelin-1 and endothelin-3 was investigated in isolated rabbit myocardium. While TAK-044 produced a concentration-dependent rightward shift of the concentration-response curve for endothelin-1 and endothelin-3, the effect of endothelin-3 was hundred times more sensitive to TAK-044 than that of endothelin-1. The combination of FR139317 ([2-(R)-[2(R)-[2(S)-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indolyl)]propionyl] amino-3-(2-pyridyl)propionic acid]) and BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-l-gamma-methylleucyl-d-1-methoxycarbonyltryptophanyl-d-norleucine) mimicked the inhibitory action of TAK-044 on the positive inotropic effect of endothelin-3 but enhanced the effect of endothelin-1. In a receptor-binding assay, TAK-044 was four times more potent in antagonizing the specific binding of endothelin-1 than that of endothelin-3. Endothelin-1 may activate receptor subtypes that trigger both positive and negative inotropic effects, the latter being more susceptible to the antagonistic action of TAK-044, which may explain in part the differential antagonistic action of TAK-044 on the inotropic effect of endothelin-1 and endothelin-3.

Combined effect of papuamine and doxorubicin in human breast cancer MCF-7 cells.

Our previous study reported that an extract of an Indonesian marine sponge, Haliclona sp., showed potent cytotoxicity and induced apoptosis. The major cytotoxic chemical compound was identified as papuamine, which caused reduction of cell survival through activation of c-Jun N-terminal kinase (JNK) in human breast cancer MCF-7 cells. Doxorubicin (DOX), a Streptomyces metabolite, is used in chemotherapy against a wide range of cancers, including breast cancer. The present study examined the combined effect of papuamine and DOX on MCF-7 cells. The effect of these reagents on cell growth was assessed by a colony formation assay. Incubation with either of the reagents alone resulted in concentration-dependent decreases in the colony formation of the MCF-7 cells. Incubation with the reagents together at sub-cytotoxic concentrations resulted in significant decreases in colony formation. The phosphorylation of JNK, the activated form of the protein, was elevated in a concentration-dependent manner upon co-incubation with papuamine and DOX. Fluorescence intensity analysis demonstrated that papuamine caused a small, but non-significant, decrease in cellular accumulation of DOX. These results indicate that the combinatory effect of papuamine and DOX is not associated with changes in the cellular accumulation of DOX, and may instead reflect additive effects on JNK activation. This study indicates that papuamine may represent a novel type of modulator for DOX chemotherapy.

Cytotoxic effects of caffeic acid undecyl ester are involved in the inhibition of telomerase activity in NALM-6 human B-cell leukemia cells.

Our previous study reported that caffeic acid undecyl ester (CAUE) has a potent cytotoxic effect and induces apoptosis in NALM-6 cells, but not in normal human lymphocytes. The majority of normal human cells have no detectable telomerase activity, however, activity is commonly detected in cancer cells. Thus, inhibiting telomerase activity and inducing apoptosis may have a selective effect on cancer cells. The aim of the present study was to investigate the inhibitory effects of telomerase activity by CAUE in a NALM-6 cell culture system. CAUE was shown to preferentially damage DNA synthesis compared with RNA or protein synthesis. In addition, telomerase activity was significantly suppressed and the activity of human telomerase reverse transcriptase (hTERT), a subunit of telomerase, was decreased following treatment with CAUE, each in a concentration-dependent manner. These results indicated that the cytotoxic effects of CAUE are mediated by the inhibition of DNA synthesis and telomerase activity. The present study is the first to identify the cytotoxic mechanisms of CAUE in leukemia cells.

Exogenous albumin inhibits sorafenib-induced cytotoxicity in human cancer cell lines.

Sorafenib is an orally administered multikinase inhibitor that exhibits anti-angiogenic and anti-tumor activity. Sorafenib is also known to bind to protein (>99.5%), suggesting protein binding may be involved in sorafenib pharmacokinetic variability. Albumin is a major drug-binding protein. In this study, we examined the effect of albumin on sorafenib-induced cytotoxicity using two in vitro culture cell lines, human hepatoma Huh-7 cells and androgen-independent prostate cancer PC-3 cells. The cells were cultured and incubated, and cytotoxicity was assessed. Results were confirmed by western blotting. The presence of exogenous albumin markedly blocked the sorafenib-induced cytotoxicity in the two cell lines. Albumin concentration, the change of pharmacological signal transduction as Raf-B, vascular endothelial growth factor (VEGF), and phosphorylation of MEK1/2 or ERK1/2 were found to be decreased by sorafenib. Co-incubation of warfarin, a representative coumarin anticoagulant and potent binding activity, with albumin enhanced the cytotoxic effects by sorafenib. These mechanisms depend on the high binding proper ties of sorafenib and exogenous albumin. Furthermore, we investigated the effects of endo genous albumin expression on sorafenib-induced cytotoxicity using the siRNA knockdown system or transfected expression vector assay. However, the cytotoxic effects by sorafenib showed little change either with the knockdown or overexpression of albumin. Our results suggest that particular care should be taken with albuminemia or the combined use of drugs with a high affinity for albumin, such as warfarin, and sorafenib in the treatment of cancer patients. Our findings may be useful to the cancer therapeutic strategy by sorafenib.

Induction of apoptosis by a potent caffeic acid derivative, caffeic acid undecyl ester, is mediated by mitochondrial damage in NALM-6 human B cell leukemia cells.

Caffeic acid esters have various biological activities, and we previously reported that undecyl caffeate (caffeic acid undecyl ester, CAUE), a new caffeic acid derivative, has strong pharmacological activity. The present study investigated the cytotoxicity of both CAUE and its parent compound, caffeic acid phenethyl ester (CAPE), and characterized the mechanisms by which they induce apoptosis in the human B cell leukemia cell line NALM-6. Treatment with CAUE reduced cell survival in NALM-6 cells but had no significant effect on the survival of normal lymphocytes. When assessing the 50% inhibitory concentration (IC(50)) for cytotoxicity, CAUE had 10-fold higher activity than CAPE in NALM-6 cells. CAUE treatment resulted in induction of apoptotic features in NALM-6 cells, including cleaved poly (ADP-ribose) polymerase and activated caspase-3. A caspase inhibitor completely blocked CAUE-induced apoptosis. CAUE treatment resulted in a concentration- and time-dependent decrease in both mitochondrial membrane potential and downregulation of Bcl-2 expression. Moreover, CAUE-induced apoptosis was enhanced in the Bcl-2 knockdown condition induced by small interfering RNA. These data suggest that CAUE-induced apoptosis was mediated via an apoptotic intrinsic pathway including mitochondrial damage and was caspase-dependent. These data also suggest that CAUE is a powerful anti-leukemic agent that acts via induction of apoptosis by mitochondrial damage and selective action in leukemia cells.

Papuamine causes autophagy following the reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 human breast cancer cells.

We previously reported that extracts of an Indonesian marine sponge Haliclona sp. showed potent cytotoxicity and the induction of apoptosis against human solid cancer cell lines. In this study, we examine the cytotoxic mechanism of the major chemical compound, papuamine, on MCF-7 human breast cancer cells. Papuamine at 5 µM did not show significant cytotoxic effects after incubation for 24 h, but autophagosome vesicular formation was apparent. At 10 µM of papuamine, significant reduction in cell survival was observed at 12 h, and increases in autophagy at this concentration were time-dependent and apparent before the appearance of cytotoxic effects. Both the release of cytochrome c to the cytosol and increase in Bax in the mitochondrial fraction were found to be concentration-dependent. Moreover, mitochondrial membrane potential shows concentration- and time-dependent decreases with exposure to papuamine. The release of cytochrome c has been shown to be accompanied by an increase in JNK activation. 3-Methyladenine (MA), a classical autophagy inhibitor showed increased JNK activation by exposure to papuamine. In conclusion, our results indicate that papuamine causes earlier onset autophagy and delayed reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 cells.

Involvement of p21waf1/cip1 expression in the cytotoxicity of the potent histone deacetylase inhibitor spiruchostatin B towards susceptible NALM-6 human B cell leukemia cells.

Spiruchostatin B (SP-B) is a potent histone deacetylase (HDAC) inhibitor that has potential for the chemotherapy of leukemia. The aim of this study was to study the susceptibility of human leukemia cell lines to SP-B. We found that NALM-6 human B cell leukemia cells are the most susceptible to SP-B. There was a low correlation between the expression of HDAC1 mRNA and HDI susceptibility of leukemia cells. NALM-6 has higher endogenous p21waf1/cip1 mRNA expression than other leukemia cells. SP-B-induced cytotoxicity was mediated by induction of histone acetylation via inhibition of HDACs, and this effect of SP-B was associated with apoptosis, which was mediated by caspase activation in NALM-6 cells. SP-B time-dependently increased the size of the sub-G1 (apoptotic) peak, and this effect correlated with SP-B induction of cellular apoptotic features such as changes in nuclear morphology. SP-B significantly increased p21waf1/cip1 expression prior to induction of apoptosis. In conclusion, NALM-6 cells, which have a higher expression of p21waf1/cip1 mRNA than other leukemia cell lines, were susceptible to SP-B-induced cytotoxicity that resulted in induction of apoptosis. Our findings may be useful when establishing a therapeutic strategy based on SP-B.

Characterization of cells resistant to the potent histone deacetylase inhibitor spiruchostatin B (SP-B) and effect of overexpressed p21waf1/cip1 on the SP-B resistance or susceptibility of human leukemia cells.

We previously showed that the B cell leukemia cell line NALM-6 had the highest susceptibility among a number of leukemia cell lines to spiruchostatin B (SP-B), a potent histone deacetylase (HDAC) inhibitor. We also showed that SP-B-induced cytotoxicity depended on induction of apoptosis that was mediated by p21waf1/cip1 expression. In the present study, we generated and characterized a stable, SP-B-resistant NALM-6 cell line (NALM-6/SP-B) by continuous exposure to SP-B, starting with a low SP-B concentration. NALM-6/SP-B cells were also more resistant to FK228, which has a similar chemical structure to SP-B, and were slightly more resistant to the P-gp substrates doxorubicin and vincristine than parental cells, but displayed similar susceptibility to other HDAC inhibitors and to paclitaxel as the parental cells. There was little change in the basal mRNA expression of HDAC1, p53, Bax, Bcl-2, Fas, caspase-3, c-Myc and MDR1 in NALM-6/SP-B compared to parental cells, but the mRNA expression of p21waf1/cip1 was decreased. The introduction of an exogenous p21waf1/cip1 expression vector restored SP-B induction of NALM-6/SP-B cell apoptosis. Moreover, overexpressed p21waf1/cip1 enhanced SP-B induction of the apoptosis of the human erythroleukemia leukemia cell line K562 which is less susceptible to SP-B than NALM-6 cells. These results suggest that downregulation of p21waf1/cip1, which is a characteristic feature of NALM-6/SP-B cells, was important for their resistance to SP-B, and that this SP-B resistance could be overcome by the introduction of exogenous p21waf1/cip1. Furthermore, introduction of p21waf1/cip1 to other leukemia cells such as K562 may enhance their susceptibility to SP-B. This is the first report of the characterization of SP-B-resistant cells and of the effect of overexpressed p21waf1/cip1 on the resistance or susceptibility of human leukemia cells to SP-B.