Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro

Aim:

To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro.

Methods:

The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting.

Results:

Treatment of U251 and U87 cells with anisomycin (0.01–8 μmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC₅₀ values at 48 h were 0.233±0.021 and 0.192±0.018 μmol/L, respectively). Anisomycin (4 μmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 μmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 μmol/L) nor the JNK inhibitor SP600125 (10 μmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 μmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death.

Conclusion:

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.     

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation

Aim:

Probucol, an anti-hyperlipidemic drug, has been reported to exert antitumor activities at various stages of tumor initiation, promotion and progression. In this study we examined whether the drug affected glioma cell growth in vitro and the underlying mechanisms.

Methods:

Human glioma U87 and glioblastoma SF295 cell lines were used. Cell proliferation was accessed using the cell proliferation assay and BrdU incorporation. The phosphorylation of AMPK, liver kinase B1 (LKB1) and p27^Kip1 was detected by Western blot. The activity of 26S proteasome was assessed with an in situ fluorescent substrate. siRNAs were used to suppress the expression of the relevant signaling proteins.

Results:

Treatment of U87 glioma cells with probucol (10–100 μmol/L) suppressed the cell proliferation in dose- and time dependent manners. Meanwhile, probucol markedly increased the ROS production, phosphorylation of AMPK at Thr172 and LKB1 at Ser428 in the cells. Furthermore, probucol significantly decreased 26S proteasome activity and increased p27^Kip1 protein level in the cells in an AMPK-dependent manner. Probucol-induced suppression of U87 cell proliferation could be reversed by pretreatment with tempol (a superoxide dismutase mimetic), MG132 (proteasome inhibitor) or compound C (AMPK inhibitor), or by gene silencing of LKB1, AMPK or p27^Kip1. Similar results were observed in probucol-treated SF295 cells.

Conclusion:

Probucol suppresses human glioma cell proliferation in vitro via ROS production and LKB1-AMPK activation, which reduces 26S proteasome-dependent degradation of p27^Kip1.     

Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation

Aim:

Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms.

Methods:

Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy.

Results:

Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells.

Conclusion:

Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.     

Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms

Aim:

To investigate the anticancer effect of crocetin, a major ingredient in saffron, and its underlying mechanisms.

Methods:

Cervical cancer cell line HeLa, non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine. Cell proliferation was examined using MTT assay. Cell cycle distribution and sub-G₁ fraction were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry. Cell death was measured based on the release of lactate dehydrogenase (LDH). The expression levels of p53 and p21^WAF1/Cip1 as well as caspase activation were examined using Western blot analysis.

Results:

Treatment of the 3 types of cancer cells with crocetin (60-240 μmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (240 μmol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21^WAF1/Cip1 induction. Crocetin (120-240 μmol/L) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner. In the 3 types of cancer cells, crocetin (60 μmol/L) significantly enhanced the cytotoxicity induced by vincristine (1 μmol/L). Furthermore, this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR.

Conclusion:

Ccrocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug or as a chemosensitizer for vincristine.     

Critical role of the JNK-p53-GADD45α apoptotic cascade in mediating oxidative cytotoxicity in hippocampal neurons

BACKGROUND AND PURPOSE

Glutamate-induced oxidative stress plays a critical role in the induction of neuronal cell death in a number of disease states. We sought to determine the role of the c-Jun NH₂-terminal kinase (JNK)-p53-growth arrest and DNA damage-inducible gene (GADD) 45α apoptotic cascade in mediating glutamate-induced oxidative cytotoxicity in hippocampal neuronal cells.

EXPERIMENTAL APPROACH

HT22 cells, a mouse hippocampal neuronal cell line, were treated with glutamate to induce oxidative stress in vitro. Kainic acid-induced oxidative damage to the hippocampus in rats was used as an in vivo model. The signalling molecules along the JNK-p53-GADD45α cascade were probed with various means to determine their contributions to oxidative neurotoxicity.

KEY RESULTS

Treatment of HT22 cells with glutamate increased the mRNA and protein levels of GADD45α, and these increases were suppressed by p53 knock-down. Knock-down of either p53 or GADD45α also prevented glutamate-induced cell death. Glutamate-induced p53 activation was preceded by accumulation of reactive oxygen species, and co-treatment with N-acetyl-cysteine prevented glutamate-induced p53 activation and GADD45α expression. Knock-down of MKK4 or JNK, or the presence of SP600125 (a JNK inhibitor), each inhibited glutamate-induced p53 activation and GADD45α expression. In addition, we also confirmed the involvement of GADD45α in mediating kainic acid-induced hippocampal oxidative neurotoxicity in vivo.

CONCLUSIONS AND IMPLICATIONS

Activation of the JNK-p53-GADD45α cascade played a critical role in mediating oxidative cytotoxicity in hippocampal neurons. Pharmacological inhibition of this signalling cascade may provide an effective strategy for neuroprotection.     

Glycogen synthase kinase-3β regulates astrocytic differentiation of U87-MG human glioblastoma cells

Aim:

To evaluate the role of glycogen synthase kinase-3β (GSK-3β) in the induced differentiation of human glioblastoma cells.

Methods:

Cell proliferation was determined by bromodeoxyuridine (BrdU) incorporation assay. The protein level of p-GSK-3β, GSK-3β, glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) were determined using Western blots. The overexpression of mutant GSK-3β was analyzed by immunocytochemistry.

Results:

The biotoxin cholera toxin is capable of inducing differentiation of U87-MG human glioblastoma cells, which is characterized by morphological changes to astrocytic phenotype, increase in differentiation marker protein GFAP and decrease in proliferation. GSK-3β activation is induced during this differentiation. Small interfering RNA against GSK-3β suppresses the induced-differentiation in U87-MG cells. Conversely, overexpression of a constitutively active form of human GSK-3β (pcDNA3-GSK-3β-S9A) mutant leads to differentiation of U87-MG cells.

Conclusion:

Our findings suggest that GSK-3β plays an important role in astrocytic differentiation of human glioblastoma cells and may be a novel therapeutic target in the malignant tumor.     

Synergistic suppression of noscapine and conventional chemotherapeutics on human glioblastoma cell growth

Aim:

Noscapine (NOS) is a non-narcotic opium alkaloid with anti-tumor activity. The aim of this study was to investigate the effects of the combination of NOS with conventional chemotherapeutics temozolamide (TMZ), bis-chloroethylnitrosourea (BCNU), or cisplatin (CIS)on human glioblastoma cells.

Methods:

U87MG human glioblastoma cells were examined. Cell proliferation was quantified using MTT assay. Western blotting and flow cytometry were used to examine apoptosis and the expression of active caspase-3 and cleaved PARP. Mouse tumor xenograft model bearing U87MG cells was treated with TMZ (2 mg·kg⁻¹·d⁻¹, ip) or CIS (2 mg/kg, ip 3 times a week) alone or in combination with NOS (200 mg·kg⁻¹·d⁻¹, ig) for 3 weeks. Immunohistochemistry was used to investigate the expression of active caspase-3 and Ki67 following treatment in vivo. The safety of the combined treatments was evaluated based on the body weight and histological studies of the animal''s organs.

Results:

NOS (10 or 20 mol/L) markedly increased the anti-proliferation effects of TMZ, BCNU, and CIS on U87MG cells in vitro. The calculated combination index (CI) values of NOS-CIS, NOS-TMZ, and NOS-BCNU (20 μmol/L) were 0.45, 0.51, and 0.57, respectively, demonstrating synergistic inhibition of the drug combinations. In tumor xenograft models, combined treatment with NOS robustly augmented the anti-cancer actions of TMZ and CIS, and showed no detectable toxicity. The combined treatments significantly enhanced the apoptosis, the activated caspase-3 and PARP levels in U87MG cells in vitro, and reduced Ki67 staining and increased the activated caspase-3 level in the shrinking xenografts in vivo.

Conclusion:

NOS synergistically potentiated the efficacy of FDA-approved anti-cancer drugs against human glioblastoma cells, thereby allowing them to be used at lower doses and hence minimizing their toxic side effects.     

Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

Aim:

To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms.

Methods:

Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye.

Results:

Physalin B inhibited the viability of HCT116 cells with an IC₅₀ value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis.

Conclusion:

Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro.     

Ginsenoside Rg1 enhances the resistance of hematopoietic stem/progenitor cells to radiation- induced aging in mice

Aim： To investigate the effects of ginsenoside Rg1 on the radiation-induced aging of hematopoietic stem/progenitor cells （HSC/HPCs） in mice and the underlying mechanisms.
Methods： Male C57BL/6 mice were treated with ginsenoside Rg1 （20 mg·kg-1·d-1, ip） or normal saline （NS） for 7 d, followed by exposure to 6.5 Gy X-ray total body irradiation. A sham-irradiated group was treated with NS but without irradiation. Sca-1^＋ HSC/HPCs were isolated and purified from their bone marrow using MACS. DNA damage was detected on d 1. The changes of anti-oxidative activities, senescence-related markers senescence-associated β-galactosidase （SA-β-gal） and mixed colony-forming unit （CFU-mix）, P16INK4a and P21Cip1/Waf1 expression on d 7, and cell cycle were examined on d 1, d 3, and d 7.

Results： The irradiation caused dramatic reduction in the number of Sca-1^＋ HSC/HPCs on d 1 and the number barely recovered until d 7 compared to the sham-irradiated group. The irradiation significantly decreased SOD activity, increased MDA contents and caused DNA damage in Sca-1^＋ HSC/HPCs. Moreover, the irradiation significantly increased SA-β-gal staining, reduced CFU-mix forming, increased the expression of P16INK4a and P21Cip1/Waf1 in the core positions of the cellular senescence signaling pathways and caused G1 phase arrest of Sca-1^＋ HSC/HPCs. Administration of ginsenoside Rg1 caused small, but significant recovery in the number of Sca-1^＋ HSC/HPCs on d 3 and d 7. Furthermore, ginsenoside Rg1 significantly attenuated all the irradiation-induced changes in Sca-1^＋ HSC/HPCs, including oxidative stress reaction, DNA damage, senescence-related markers and cellular senescence signaling pathways and cell cycle, etc.

Conclusion： Administration of ginsenoside Rg1 enhances the resistance of HSC/HPCs to ionizing radiation-induced senescence in mice by inhibiting the oxidative stress reaction, reducing DNA damage, and regulating the cell cycle.     

Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage

Aim:

Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro.

Methods:

Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy.

Results:

Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G₂/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

Conclusion:

Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.     

γ-Secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway

Aim： Trans-4-[4-（3-adamantan-1-yl-ureido）-cyclohexyloxy]-benzoic acid （t-AUCB） is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-l-alanyl]-S-phenylglycine t-butyl ester （DAPT） could sensitize glioma cells to t-AUCB-induced apoptosis.
Methods： Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots.
Results： Pretreatment with DAPT （2 μmol/L） substantially potentiated the growth inhibition caused by t-AUCB （200 μmol/L） in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells.
Conclusion： The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.     

Bisphenol A activates Maxi-K (KCa1.1) channels in coronary smooth muscle

Background and purpose:

Bisphenol A (BPA) is used to manufacture plastics, including containers for food into which it may leach. High levels of exposure to this oestrogenic endocrine disruptor are associated with diabetes and heart disease. Oestrogen and oestrogen receptor modulators increase the activity of large conductance Ca²⁺/voltage-sensitive K⁺ (Maxi-K; K_Ca1.1) channels, but the effects of BPA on Maxi-K channels are unknown. We tested the hypothesis that BPA activates Maxi-K channels through a mechanism that depends upon the regulatory β1 subunit.

Experimental approach:

Patch-clamp recordings of Maxi-K channels were made in human and canine coronary smooth muscle cells as well as in AD-293 cells expressing pore-forming α or α plus β1 subunits.

Key results:

BPA (10 µM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 µM), a Maxi-K blocker. BPA increased Maxi-K activity in inside-out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD-293 cells with Maxi-K channels composed of α subunits alone, 10 µM BPA did not affect channel activity. When channels in AD-293 cells contained β1 subunits, 10 µM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 µM) increased Maxi-K current independent of the β1 subunit.

Conclusions and implications:

Our data indicate that BPA increased the activity of Maxi-K channels and may represent a basis for some potential toxicological effects.     

Modulation of the pentose phosphate pathway alters phase I metabolism of testosterone and dextromethorphan in HepG2 cells

Aim:

The pentose phosphate pathway (PPP) is involved in the activity of glucose-6-phosphate dehydrogenase (G6PD) and generation of NADPH, which plays a key role in drug metabolism. The aim of this study was to investigate the effects of modulation of the PPP on drug metabolism capacity in vitro.

Methods:

A pair of hepatic cell lines, ie, the cancerous HepG2 cells and normal L02 cells, was used. The expression of CYP450 enzymes, p53 and G6PD in the cells were analyzed. The metabolism of testosterone (TEST, 10 μmol/L) and dextromethorphan (DEM, 1 μmol/L), the two typical substrates for CYP3A4 and CYP2D6, in the cells was examined in the presence of different agents.

Results:

Both the expression and metabolic activities of CYP3A4 and CYP2D6 were considerably higher in HepG2 cells than in L02 cells. The metabolism of TEST and DEM in HepG2 cells was dose-dependently inhibited by the specific CYP3A4 inhibitor ketoconazole and CYP2D6 inhibitor quinidine. Addition of the p53 inhibitor cyclic PFT-α (5, 25 μmol/L) in HepG2 cells dose-dependently enhanced the metabolism of DEM and TEST, whereas addition of the p53 activator NSC 66811 (3, 10, 25 μmol/L) dose-dependently inhibited the metabolism. Furthermore, addition of the G6PD inhibitor 6-aminonicotinamide (5, 15 μmol/L) in HepG2 cells dose-dependently inhibited the metabolism of DEM and TEST, whereas addition of the PPP activity stimulator menadione (1, 5, 15 μmol/L) dose-dependently enhanced the metabolism.

Conclusion:

Modulation of p53 and the PPP alters the metabolism of DEM and TEST, suggesting that the metabolic flux pattern of PPP may be closely involved in drug metabolism and the individual variance.     

Control of enteric neuromuscular functions by purinergic A3 receptors in normal rat distal colon and experimental bowel inflammation

Background and purpose:

Elephantopus scaber L. (Asteraceae) is a traditional herbal medicine with anti-cancer effects. We evaluated the in vitro and in vivo efficacy of a major sesquiterpene lactone constituent of E. scaber, deoxyelephantopin (DET), against mammary adenocarcinoma and the underlying molecular mechanism.

Experimental approach:

A variety of cellular assays, immunoblotting and immunohistochemistry, as well as both orthotopic and metastatic TS/A tumour models in BALB/c mice, were used. Test mice were pretreated and post-treated with DET or paclitaxel and mammary tumour growth evaluated.

Key results:

DET (≤2 µg·mL⁻¹) significantly inhibited colony formation, cell proliferation, migration and invasion of TS/A cells and induced G₂/M arrest and apoptosis in TS/A cells. c-Jun N-terminal kinase-mediated p21^Waf1/Cip1 expression and caspase activation cascades were up-regulated by DET, effects suppressed by N-acetyl-L-cysteine. Moreover, tumour necrosis factor α-induced matrix metalloproteinase-9 enzyme activity and expression and nuclear factor-kappa B activation were abolished by DET. Pretreatment with DET was more effective than paclitaxel, for profound suppression of orthotopic tumour growth (99% vs. 68% reduction in tumour size) and lung metastasis of TS/A cells (82% vs. 63% reduction in metastatic pulmonary foci) and prolonged median survival time (56 vs. 37 days, P < 0.01) in mice. The levels of cyclooxygenase-2 and vascular endothelial growth factor in metastatic lung tissues of TS/A-bearing mice were attenuated by DET.

Conclusions and implications:

Our data provide evidence for the suppression of mammary adenocarcinoma by DET with several mechanisms and suggest that DET has potential as a chemopreventive agent for breast cancer.     

Lobolide,a diterpene,blockades the NF-κB pathway and p38 and ERK MAPK activity in macrophages in vitro

Aim:

Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway.

Methods:

A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1β production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis.

Results:

In HEK 293/NF-κB-Luc stable cells, lobolide (0.19–50 μmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC₅₀ value of 4.2±0.3 μmol/L. Treatment with lobolide (2.5–10 μmol/L) significantly suppressed LPS-induced production of TNFα and IL-1β in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity.

Conclusion:

Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1β release, suggesting that the compound might be an anti-inflammatory compound.     

G226, a new epipolythiodioxopiperazine derivative,triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation

Aim:

G226 is a novel derivative of epipolythiodioxopiperazines with potent inhibitory activity against cancer cells. Here, we sought to identify potential targets involved in the anti-cancer activity of G226.

Methods:

Cell proliferation assay was conducted in a panel of 12 human cancer cell lines. The activities of topoisomerase I (Topo I) and Topo II were studied using supercoiled pBR322 DNA relaxation and kDNA decatenation assays. ROS production was assessed with probes DCFH-DA and H&E. Western blot analysis and flow cytometry were used to examine DNA damage, apoptosis and cell cycle changes.

Results:

G226 displayed potent cytotoxicity in the 12 human cancer cell lines with a mean IC₅₀ value of 92.7 nmol/L. This compound (1–100 μmol/L) selectively inhibited the activity of Topo II, and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.125–2 μmol/L) dose-dependently elevated the intracellular levels of H₂O₂ and in the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity.

Conclusion:

G226-mediated ROS production contributes to the anti-cancer activity of this compound.     

Noradrenaline stimulates cell proliferation by suppressing potassium channels via Gi/o-protein-coupled α1B-adrenoceptors in human osteoblasts

Background and Purpose

Recent studies demonstrated that the sympathetic nervous system regulates bone metabolism via β₂-adrenoceptors. Although α-adrenoceptors are also expressed in osteogenic cells, their functions in bone metabolism have been less studied. We previously demonstrated that noradrenaline suppressed potassium currents via α_1B-adrenoceptors in the human osteoblast SaM-1 cell line. The aim of this study was to investigate the signal transduction pathway and the physiological role of noradrenaline in human osteoblasts in more detail.

Experimental Approach

To investigate signal transduction through α_1B-adrenoceptors, we used whole-cell patch clamp recording and Ca fluorescence imaging. Potassium channels regulate membrane potential and cell proliferation activity in non-excitable cells, so we evaluated cell proliferation activity by BrdU incorporation and WST assay.

Key Results

In SaM-1 cells, bath-applied noradrenaline elevated intracellular Ca²⁺ concentration and this effect was abolished by both chloroethylclonidine, an α_1B-adrenoceptor antagonist, and {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122, a PLC inhibitor. However, the inhibitory effect of noradrenaline on whole-cell current was unaffected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122. In contrast, in cells pretreated with either Pertussis toxin, a G_i/o-protein-coupled receptor inhibitor, or gallein, a Gβγ-protein inhibitor, the inhibitory effect of noradrenaline on whole-cell current was significantly suppressed. Noradrenaline-induced enhancement of cell proliferation was inhibited by CsCl, a non-selective potassium channel blocker, gallein and H89, a PKA inhibitor, but not by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122.

Conclusions and Implications

Noradrenaline facilitated cell proliferation by regulation of potassium currents in human osteoblasts via G_i/o-protein-coupled α_1B-adrenoceptors, not via coupling to Gq-proteins.