Soon Cheol Ahn

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.

Androgen Receptor: Structural Domains and Functional Dynamics after Ligand-Receptor Interaction

Abstract: Androgens are C‐19 steroids secreted primarily from the testes and adrenals that play a critical role in reproduction. Reproductive functions of androgens are mediated through coordination of diverse physiological processes ranging from brain functions to specific cell proliferation and apoptosis. At the molecular level, most of these regulatory influences are exerted by altered expression of appropriate genes by the androgen receptor (AR), a member of the nuclear receptor (NR) superfamily. The unliganded AR is a cytoplasmic protein and, upon ligand binding, it translocates into the nucleus. Thereafter, in conjunction with other transcription factors and coactivators, the AR influences transcription of target genes through a multistep process that includes its clustering in a subnuclear compartment. Here, we describe the genomic organization of the AR, the role of individual structural domains in specific AR function, and the influence of agonistic/antagonistic ligands in the intracellular movement of the receptor. We also show that the AR is capable of undergoing multiple rounds of nucleocytoplasmic recycling after ligand binding and dissociation. Xenobiotic ligands, considered as selective androgen receptor modulators (SARMs), can modulate AR activity by inhibiting either its nuclear translocation or its subnuclear clustering and subsequent transactivation function.

Interplay of reactive oxygen species, intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca u2009i2+ surge, increased mitochondrial membrane potential (MMP, ΔΨm), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca u2009i2+ surge, on the other hand the Ca2+ chelator BAPTA inhibited the Ca u2009i2+ overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca2+ flux. This suggested that both ROS and Ca u2009i2+ signaling play roles in the increased MMP via Ca u2009i2+ ‐dependent and/or ‐independent mechanisms, since ΔΨm elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca u2009i2+ ‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca2+ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013.

SULT2B1b Sulfotransferase: Induction by Vitamin D Receptor and Reduced Expression in Prostate Cancer

An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3β-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house-developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α-bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease progression.

Secretion of Bacillus α‐amylase from yeast directed by glucoamylase I signal sequence of Saccharomyces diastaticus

For the secretion of Bacillus stearothermophilus α ‐amylase from yeast, a recombinant plasmid pGAT17 was constructed by fusing B. stearothermophilus α‐amylase structural gene in frame to the promoter and signal sequence of Saccharomyces diastaticus glucoamylase I gene (STA1). The secretion of the heterologous α‐amylase from S. diastaticus transformed with pGAT17 was confirmed by the halo formation around colonies on selective starch agar medium. About 80% of the total α‐amylase activity was detected in the extracellular culture medium. The secreted α‐amylase was glycosylated and its molecular weight increased from 61 kDa to 75 kDa. The thermostability of the glycosylated α‐amylase was markedly enhanced, compared with that of the non‐glycosylated enzyme from E. coli.

Biological Activities of Pharbitis nil and Partial Purification of Anticancer Agent from Its Extract

This study aimed to evaluate several biological activities of Pharbitis nil and to isolate an anticancer agent from its methanol extract. Pharbitis nil seeds were extracted with methanol (PNM). Then, PNM was fractionated into solvent layers such as ethyl acetate fraction (PNE), butanol fraction (PNB), and water fraction (PNW). The biological activities of the fractions were analyzed for tyrosinase inhibition, lipase inhibition, DPPH-free radical scavenging, and cell growth inhibition. PNM showed strong growth inhibition of prostate cancer PC-3 cells. PNM was subjected to Diaion HP-20 and eluted stepwise with 50%, 80%, and 100% methanol. Then, for activity-guided fraction, each fraction was analyzed for growth inhibition of prostate cancer PC-3 cells by using an MTT assay. Because the 100% fraction showed significantly strong inhibitory activity, the fraction was further separated in the reverse phase C18, which was eluted with 80% and 90% methanol. The 90% fraction was further subjected to Sephadex LH-20 using a mobile solvent of 100% methanol. Finally, the compound PN was partially purified for HPLC analysis. PN showed cell growth inhibitory activity and induced the apoptosis and cell cycle arrest of prostate cancer PC-3 cells, as measured by flow cytometry. The results together suggest that Pharbitis nil possesses various biological activities, especially the inhibitory activity for the proliferation of prostate cancer PC-3 cells, suggesting the possibility of its use as an anticancer agent.

Evaluation of allergic sensitivity to Acanthamoeba allergen in patients with chronic cough.

BACKGROUNDnAcanthamoeba and their proteins can elicit severe allergic airway inflammation in experimental mice.nnnOBJECTIVEnAlthough Acanthamoeba can induce severe allergic airway inflammation in mice, there is no allergenicity data for humans.nnnMETHODSnWe performed a skin-prick test on 65 patients with chronic cough by using 54 previously known allergens and Acanthamoeba excretory-secretory proteins and enzyme-linked immunosorbent assay on 34 patients to evaluate Acanthamoeba-specific serum immunoglobulin (Ig) levels. To detect a novel Acanthamoeba allergen, Western blot analysis was performed on serum from patients who reacted positively to Acanthamoeba or some pollen allergens.nnnRESULTSnAfter skin-prick testing, 29 patients (44.6%) showed positive reactions to one or more common aeroallergens. Acanthamoeba allergenicity was evaluated in 4 of 65 subjects (6.1%). An Acanthamoeba-positive reaction was closely related to several pollen allergens, especially willow tree, poplar, elm, oak, velvet grass, and cockroach. Average levels of Acanthamoeba-specific IgG subtypes in patient serum did not differ compared with healthy subjects; however, Acanthamoeba-specific IgE titers of patients were significantly higher than in healthy subjects. IgE antibodies of patients who tested positive in the skin-prick test reacted strongly to the 15 kDa excretory-secretory protein. Moreover, these antigens also reacted with those who tested positive in the skin-prick test to pollens.nnnCONCLUSIONnTaken together, our results indicated that some patients with allergy showed a positive response to the skin-prick test and that they also have high IgE serum levels. However, further experimental investigation is warranted because our preliminary findings indicated that Acanthamoeba might be a new allergen in humans.

Gene Expression Analysis of Immune Cell Activation Markers in Extracts of Platycodon grandiflorum Containing Medicinal Herbs

Platycodon grandiflorum Containing Medicinal Herbs Shin Ae Kang, Sung Sik Chun*, Shin Kwon Kang, Young Chul Chung, Eun Woo Cheon, Sang Uk Cho 4 , Kyung Hwa Jung 5 , Soon Cheol Ahn 2,6 and Hak Sun Yu 1,2 * Department of Parasitology, Pusan National University School of Medicine, Yangsan 626-870, Korea Immunoregulatory Therapeutics Group in Brain Busan 21 Project Department of Medicinal Food, International University of Korea, Jinju 660-759, Korea JangSaeng Doraji Research Institute of Biotechnology, JangSaeng Doraji Co. LTD., Jinju 660-833, Korea Department of Food Science and Biotechnology, Kyungsung Univeristy, Busan 608-736, Korea Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan 626-870, KoreaExtracts of Platycodon grandiflorum have been reported to show anti-inflammatory, antioxidant, anti-metastatic, and hepato-protective effects. This study was designed to evaluate T-cell activation and M1/M2 differential macrophage activation by extracts of P. grandiflorum or P. grandiflorum containing various medicinal herbs. Using real-time RT-PCR, we analyzed expression levels of c-fos, and CD40L(T-cell activation markers) in splenocytes and iNOS, Ym1, and ARG1 in RAW 246.7 cells after treatment of CC (hot water extract of P. grandiflorum), MAEK (hot water extract of P. grandiflorum [82%]and six different plants), and HWAL (hot water extract of P. grandiflorum [7%] and eight different plants. The results showed that MAEK significantly elevated the expression of T-cell activation markers of splenocytes, with the c-fos gene activated more than 10-fold and the CD40L gene activated more than 6-fold. Although CD40L was significantly increased by CC and HWAL, the increase was only about 2-fold. In addition, CC and HWAL did not significantly activate the expression of the c-fos gene. On the other hand, CC elevated the M1 activation marker iNOS, and HWAL elevated the M2 activation marker Ym1 and ARG1 gene expression. In conclusion, MAEK could be used as an immune stimulant because of its ability to activate T cells (elicited c-fos and CD40L gene expression), whereas HWAL could serve as an anti-inflammatory agent because of its differential activation of M2 macrophages.

Glucocorticoid Regulation of Gene Expression in Hippocampal CA3 and Dentate Gyrus

Glucocorticoids (GCs) alter metabolism, synaptogenesis, apoptosis, neurogenesis, and dendritic morphology in the hippocampus. To better understand how glucocorticoids regulate these aspects of hippocampal biology, we studied gene expression patterns in the CA3 (Hippocampal pyramidal cell field CA3) and dentate gyrus (DG). Litter-matched Lewis inbred rats treated for 20 days with either 9.5 ㎎ per day sustained-release corticosterone or placebo pellets were compared with high-density oligonucleotide microarray analysis (Rat Neurobiology U34 Arrays, Affymetrix). In placebo-treated rats, 32 genes were expressed at greater levels in CA3 than DG, whereas 3 genes were expressed at great levels in DG than CA3. Regional differences were also apparent in corticosterone-induced changes in the hippocampal transcriptome. Six genes in CA3 and 41 genes in DG were differentially regulated by corticosterone. As per the glucocorticoid effects on gene transcription in the brain, forty three of these genes were upregulated, and 4 genes were downregulated. Genes differentially expressed in hippocampus included those for 13 neurotransmitter proteins, 5 ion channel related proteins, 4 transcription factors, 3 neurotrophic factors, 1 cytokine, 1 apoptosis related protein, and 5 genes involved in synaptogenesis. Interestingly, GCs can have suppressive effects on brain BDNF mRNA transcription, one of the neurotrophic factors. These results indicate the diversity of targets affected by chronic exposure to corticosterone and highlight important regional differences in hippocampal neurobiology.

Biochemical Characterization of a Protease with Fibrinolytic Activity from Maggots of Protaetia brevitarsis

Fibrin clots remained in blood vessels can be one of the serious factor caused cardiovascular disease, such as ischemia, infarction and necrosis The development of an antithrombotic and thrombolysis solvent is necessary to prevent and treat these diseases. In this study, the fibirinolytic protease was prepared from the maggots of Protaetia brevitarsis using ammonium sulfate fractionation and desalting column. The optimum pH and temperature for the enzyme activity were pH 9.0 and 50℃, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below 60℃. The activity of the enzyme was strongly inhibited by phenylmethanesulfonyl fluoride. And the activity of the enzyme was inhibited by Ca²? and Zn²?, but it was not by Mg²? and Fe²? ions. In these experimental results, we have speculated that the enzyme derived from maggots of Protaetia brevitarsis is a serine protease with a strong fibrinolytic activity.