Regulation of Bax by c-Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis

Microtubule inhibitors, such as vinblastine, are widely used in cancer chemotherapy. Vinblastine exerts its antitumor effect by inducing apoptosis. In KB-3 cells, we have shown previously that vinblastine activates c-Jun NH₂-terminal protein kinase (JNK) and causes Bax mitochondrial translocation and activation. In this study, we sought to test the hypothesis that JNK and Bcl-xL act as positive and negative regulators, respectively, of Bax translocation. The JNK inhibitor SP600125 inhibited vinblastine-induced JNK activation and in concert inhibited Bax mitochondrial translocation, Bax oligomerization, and Bax activation. Furthermore, the JNK inhibitor blocked vinblastine-induced apoptosis. The ability of vinblastine to induce Bax translocation and the inhibitory effect of SP600125 were confirmed in cells stably expressing GFP-Bax. However, if transiently overexpressed, Bax localized to the mitochondria, and this was associated with loss of viability and subsequent cell death. If Bcl-xL was co-expressed with Bax, the cells readily tolerated Bax overexpression. Indeed, physical interaction between Bcl-xL and Bax but not Bak was demonstrated by co-immunoprecipitation. These findings provide novel insight into the role of Bax and its regulation in vinblastine-induced apoptosis.     

Binding thermodynamics at the human cannabinoid CB1 and CB2 receptors

The thermodynamic parameters ΔG°, ΔH° and ΔS° of the binding equilibrium of agonists and antagonists at cannabinoid CB₁ and CB₂ receptors were determined by means of affinity measurements at different temperatures and van’t Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB₁ and CB₂ receptors by inhibition assays of the binding of the cannabinoid receptor agonist [³H]-CP-55,940. van’t Hoff plots were linear for agonists and antagonists in the temperature range 0-30 °C. The thermodynamic parameters for CB₁ receptors fall in the ranges 17 ≤ ΔH° ≤ 59 kJ/mol and 213 ≤ ΔS° ≤ 361 kJ/mol for agonists and −52 ≤ ΔH° ≤ −26 kJ/mol and −12 ≤ ΔS° ≤ 38 kJ/mol for antagonists. The thermodynamic parameters for CB₂ receptors fall in the ranges 27 ≤ ΔH° ≤ 48 kJ/mol and 234 ≤ ΔS° ≤ 300 kJ/mol for agonists and −19 ≤ ΔH° ≤ −17 kJ/mol and 43 ≤ ΔS° ≤ 74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB₁ and CB₂ receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB₁ and CB₂ binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.     

Activation of the P2Y1 receptor induces apoptosis and inhibits proliferation of prostate cancer cells

G protein-coupled receptors, the largest cell surface receptor family, have emerged as critical players in cell death and survival. High gene expression level of the G_q-coupled P2Y₁ nucleotide receptor in PC-3 prostate cancer cells was demonstrated using real-time quantitative PCR and confirmed by Western blotting and confocal laser scanning microscopy. A selective P2Y₁ receptor agonist, the ADP analogue MRS2365, concentration-dependently induced intracellular calcium mobilization (EC₅₀ 5.28 nM), which was diminished by P2Y₁ receptor-selective antagonist MRS2500. P2Y₁ receptor activation by MRS2365 induced apoptosis in assays of Caspase-3, LDH release, and annexin-V staining. The pro-apoptotic effect of MRS2365 was blocked by MRS2500, P2Y₁ siRNA, and an inhibitor of the MAP kinase pathway PD98059. MRS2365 significantly inhibited the proliferation of PC-3 cells, examined using a MTT assay. Thus, activation of the P2Y₁ receptor induced cell death and inhibited growth of human prostatic carcinoma PC-3 cells. Activation of the P2Y₁ receptor should be a novel and promising therapeutic strategy for prostate cancer.     

Inhibition of calcium-independent phospholipase A2 activates p38 MAPK signaling pathways during cytostasis in prostate cancer cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathways activated during cytostasis induced by Ca²⁺-independent phospholipase A₂ (iPLA₂) inhibition in prostate cancer cells were investigated. iPLA₂ inhibition using siRNA, or the selective inhibitor bromoenol lactone (BEL) and it's enantiomers, decreased growth in LNCaP (p53 positive) and PC-3 (p53 negative) human prostate cancer cells. Decreased cell growth correlated to time- and concentration-dependent activation of the mitogen-activated protein kinase p38 in both cell lines. Inhibition of cytosolic iPLA₂β using S-BEL, induced significantly higher levels of P-p53, p53, p21 and P-p38 expression than inhibition of microsomal iPLA₂γ using R-BEL. Inhibition of p38 using SB202190 or SB203580 inhibited BEL-induced increases in P-p53 (ser15), p53 and p21, and altered the number of cells in G1 in LNCaP cells, and S-phase in PC-3 cells. BEL treatment also induced reactive species in PC-3 and LNCaP cells, which was partially reversed by pretreatment with N-acetyl-cysteine (NAC). NAC subsequently inhibited BEL-induced activation of p38 and p53 in LNCaP cells. In addition, treatment of cells with NAC partially reversed the effect of BEL on cell growth and preserved cell morphology. Collectively, these data demonstrate the novel findings that iPLA₂ inhibition activates p38 by inducing reactive species, and further suggest that this signaling kinase is involved in p53 activation, cell cycle arrest and cytostasis.     

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin.

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.     

Increased responsiveness to JNK1/2 mediates the enhanced H2O2-induced stimulation of Cl/HCO3 exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR

We have previously demonstrated that exogenous H₂O₂ stimulates Cl⁻/HCO₃⁻ exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H₂O₂, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H₂O₂ on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H₂O₂ had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H₂O₂ treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity. It is concluded that H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H₂O₂, which contributes to their increased sensitivity to H₂O₂.     

Pterostilbene from Vitis coignetiae protect H2O2-induced inhibition of gap junctional intercellular communication in rat liver cell line

In this study, we identified various stilbenoids derived from Vitis coignetiae and investigated the protective effect of the main component, pterostilbene, against the inhibition of gap junctional intercellular communication (GJIC) induced by H₂O₂ in WB-F344 rat liver epithelial cells. We analyzed seven kinds of stilbenoids, pterostilbene, astringin, piceid, vitisin, rhaponticin, resveratrol, and rhapontigenin, using DAD/UV HPLC. Total stilbenoid content was 127.37 ± 19.29 mg/100 g dry weight. Pretreatment with 0.5, 1.0, and 5.0 μM pterostilbene for 24 h was shown to recover GJIC blocked by 500 μM H₂O₂. Pretreatment with pterostilbene prevented the inhibition of GJIC via the down-regulation of connexin43 phosphorylation by the inactivation of ERK1/2 and p38 MAP kinase. Our results suggest that pterostilbene may be a functional chemopreventative agent and that dietary exposure of pterostilbene would be helpful for improving health.     

Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase

Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.     

Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata.

The objective of this study was to investigate the fermented culture broth of Antrodia camphorata (A. camphorata) to induce apoptosis and inhibit cyclooxygenase-2 (COX-2) in estrogen-nonresponsive (MDA-MB-231) human breast cancer cells. Treatment of the highly invasive MDA-MB-231 cells with A. camphorata (40-240 microg/ml) resulted in dose and time-dependent sequences of events marked by apoptosis, as evidenced by loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. Apoptosis in the MDA-MB-231 cells was accompanied by release of cytochrome c, activation of caspase-3, -8, and -9, and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). Although the A. camphorata-induced apoptosis was associated with a reduction in Bcl-2 protein levels, negligible Bax increase was observed. Furthermore, A. camphorata treatment inhibited COX-2 protein expression and prostaglandin E2 (PGE2) production in MDA-MB-231 cells. Analysis of the study data suggests that A. camphorata exerts growth inhibition on (highly invasive) estrogen-nonresponsive human breast cancer cells through apoptosis induction associated with COX-2 inhibition, and that it may possess anticancer properties potentially valuable for application in drug products.     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

Pyrazolotriazolopyrimidine derivatives sensitize melanoma cells to the chemotherapic drugs: taxol and vindesine

In this study, we have evaluated the "in vitro" modulatory activity of a series of pyrazolotriazolopyrimidine derivatives (PTP-d) in sensitizing malignant melanoma cells to the chemotherapic drugs: taxol and vindesine. To that end, we have described the impact of chemotherapeutic agents on the cell cycle and on the induction of apoptosis when used alone or in combination with PTP-d. We have demonstrated that four PTP-d reduced chemotherapic drugs EC(50) doses of the G(2)/M accumulation with an average of 1.7-fold for taxol and 9.5-fold for vindesine when challenged on A375 human melanoma cell line. This sensitization activity was also confirmed by analyzing the apoptosis degree induced by the chemotherapic drugs. Interestingly, PTP-d had no effects on the response to cytotoxic agents by skin-derived human keratinocyte cells, NCTC 2544. Therefore, we have investigated the signaling pathway sustaining the sensitizing effect of PTP-d, providing functional evidence that active compounds are able to inhibit multidrug resistance-associated ATP-binding cassette drug transporter. These results suggested that PTP-d hold great promise for the treatment of multidrug resistance in cancers, leading to potential new therapies for melanoma.     

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.     

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC₅₀ of approximately 80 μM GA at 24 h whereas an IC₅₀ of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 μM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). The percents of MMP (ΔΨ_m) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.     

Modulation of Bax/Bcl-2 and caspases by probiotics during acetaminophen induced apoptosis in primary hepatocytes

Oxidative stress is an important factor in drug induced hepatotoxicity and antioxidants from natural sources have potential to ameliorate it. The present study was aimed to investigate cyto-protective potential of probiotic Enterococcus lactis IITRHR1 (El_SN) and Lactobacillus acidophilus MTCC447 (La_SN) lysate against acetaminophen (APAP) induced hepatotoxicity. Cultured rat hepatocytes pretreated with El_SN/La_SN showed higher cell viability under APAP stress. Pre-treatment with El_SN, restored glutathione level and reduced ROS generation significantly which are major biomarkers of oxidative stress. It also reduced NO level, MDA formation and enhanced SOD activity. Pre-treatment with probiotic lysates significantly inhibited the translocation of pro-apoptotic protein (Bax), enhanced anti-apoptotic (Bcl-2) protein levels and prevented release of cyt c to cytosol; suggesting involvement of mitochondrial proteins in protection against APAP induced oxidative cellular damage. Loss in mitochondrial membrane potential due to APAP treatment was prevented in the presence of probiotic lysates. Protective action of El_SN/La_SN pretreatment was further supported by prevention of procaspase-3 activation, DNA fragmentation and chromatin condensation, in turn inhibiting APAP induced apoptotic cell death. The results indicate that probiotic preparations modulate crucial end points of oxidative stress induced apoptosis and may be used for management of drug induced liver injury.     

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.     

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.     

LL-202, a newly synthesized flavonoid,inhibits tumor growth via inducing G2/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo

We recently established that LL-202, a newly synthesized flavonoid, exhibited obvious anticancer effects against human breast cells in vivo and in vitro. The underlying mechanism of its anticancer activity remains to be elucidated. In this study, we demonstrated that LL-202 inhibited the growth and proliferation of human breast cancer MCF-7 cells in a concentration and time-dependent manner. We reported that LL-202 induced both mitochondrial- and death-receptor-mediated apoptosis, which were characterized by the dissipation of mitochondrial membrane potential (ΔΨ_m), cytochrome c (Cyt c) release from mitochondria to cytosol, the activation of several caspases and induction of poly (ADP-ribose) polymerase (PARP) and Bid cleavage. N-acetylcysteine (NAC), a general ROS scavenger, partly blocked the LL-202-induced ROS levels and apoptosis. In addition, LL-202 induced arrest in cell cycle progression at G2/M phase in MCF-7 cells. After the treatment with LL-202, the expression of cell cycle-related proteins, such as cyclin B1, cyclin A, and p-CDK1 (Thr161) were down-regulated, whereas the expression of p21^WAF1/Cip1 and p-CDK1 (Thr14/Tyr15) were up-regulated. Finally, in vivo studies, LL-202 significantly suppressed the growth of MCF-7 breast cancer xenograft tumors in a dose-dependent manner with low systemic toxicity. In conclusion, the results showed that LL-202 had significant anticancer effects against human breast cells via the induction of apoptosis and G2/M phase arrest and it may be a novel anticancer agent for treatment of breast cancer.     

Structure-specific control of differentiation and apoptosis of human promyelocytic leukemia (HL-60) cells by A-ring diastereomers of 2-methyl- 1α,25-dihydroxyvitamin D3 and its 20-epimer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has been shown to modulate not only proliferation and differentiation but also apoptosis of malignant cells, indicating that it would be useful for the treatment of hyperproliferative diseases such as cancer and psoriasis. Little information is available concerning structural motifs of the 1α,25(OH)₂D₃ molecule responsible for modulation of differentiation and apoptosis. We synthesized all possible A-ring diastereomers of the 2-methyl-1α,25(OH)₂D₃ and its 20-epimer and evaluated their biological activities in human promyelocytic leukemia (HL-60) cells. Surprisingly, the potent analogues could be clearly divided into two groups: (i) those bearing the 1α- and 3β-hydroxyl groups on the A-ring were potent inducers of differentiation and growth inhibitors of HL-60 cells and (ii) those bearing the 1β-hydroxyl group together with either 3α- or 3β-hydroxyl groups on the A-ring were potent stimulators of apoptosis in these cells. We have clearly identified for the first time the structural motifs on the basis of the stereochemistry of both hydroxyl groups at positions 1 and 3 of the A-ring of the 1α,25(OH)₂D₃ molecule responsible for the induction of differentiation and apoptosis of HL-60 cells. These findings provide useful information not only for structure–function studies of 1α,25(OH)₂D₃ analogues but also for the development of therapeutic agents for the treatment of leukemia and other cancers.     

Differential response of MG132 cytotoxicity against small cell lung cancer cells to changes in cellular GSH contents

The effect of the depletion or oxidation of cellular GSH on cytotoxicity of MG132 was assessed. Viability loss and decrease in GSH contents in small cell lung cancer (SCLC) cells treated with MG132 was attenuated by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk). Thiol compounds (N-acetylcysteine and N-(2-mercaptopropionyl)glycine) and free radical scavengers reduced MG132-induced cell death. Antioxidants, including N-acetylcysteine, inhibited the MG132-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c and caspase-3 activation. Depletion of GSH due to buthionine sulfoxime did not affect the cell viability loss, ROS formation and GSH depletion due to MG132 in SCLC cells. A thiol oxidant monochloramine, p-chloromercuribenzoate and N-ethylmaleiamide also did not affect cytotoxicity of MG132. The results suggest that the toxicity of MG132 on SCLC cells is mediated by activation of caspase-8, -9 and -3. Removal of free radicals and recovery of GSH contents may attenuate MG132-induced apoptotic cell death. Nevertheless, depletion or oxidation of cellular GSH may not affect toxicity of MG132.