Functional Characterization of Drosophila melanogaster CYP6A8 Fatty Acid Hydroxylase

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni2+ -nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type І spectral changes, with Kd values 28 ± 4 and 144 ± 20 μM, respectively. Ultra-performance liquid chromatography–mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a kcat value of 0.038 ± 0.002 min –1 and a Km value of 10 ± 2 μM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a kcat value of 0.135 ± 0.007 min –1 and a Km value of 21 ± 4 μM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

Synergistic Induction of Apoptosis by the Combination of an Axl Inhibitor and Auranofin in Human Breast Cancer Cells

Axl receptor tyrosine kinase has been implicated in cancer progression, invasion, and metastasis in various cancer types. Axl overexpression has been observed in many cancers, and selective inhibitors of Axl, including R428, may be promising therapeutic agents for several human cancers, such as breast, lung, and pancreatic cancers. Here, we examined the cell growth inhibition mediated by R428 and auranofin individually as well as in combination in the human breast cancer cell lines MCF-7 and MDAMB-231 to identify new advanced combination treatments for human breast cancer. Our data showed that combination therapy with R428 and auranofin markedly inhibited cancer cell proliferation. Isobologram analyses of these cells indicated a clear synergism between R428 and auranofin with a combination index value of 0.73. The combination treatment promoted apoptosis as indicated by caspase 3 activation and poly (ADP-ribose) polymerase cleavage. Cancer cell migration was also significantly inhibited by this combination treatment. Moreover, we found that combination therapy significantly increased the expression level of Bax, a mitochondrial proapoptotic factor, but decreased that of the X-linked inhibitor of apoptosis protein. Furthermore, the suppression of cell viability and induction of Bax expression by the combination treatment were recovered by treatment with N-acetylcysteine. In conclusion, our data demonstrated that combined treatment with R428 and auranofin synergistically induced apoptosis in human breast cancer cells and may thus serve as a novel and valuable approach for cancer therapy.

G0/G1 Switch 2 Induces Cell Survival and Metastasis through Integrin-Mediated Signal Transduction in Human Invasive Breast Cancer Cells

Human breast cancer cell line, MDA-MB-231, is highly invasive and aggressive, compared to less invasive cell line, MCF-7. To explore the genes that might influence the malignancy of MDA-MB-231, DNA microarray analysis was performed. The results showed that G0/G1 switch 2 (G0S2) was one of the most highly expressed genes among the genes upregulated in MDA-MB-231. Although G0S2 acts as a direct inhibitor of adipose triglyceride lipase, action of G0S2 in cancer progression is not yet understood. To investigate whether G0S2 affects invasiveness of MDA-MB-231 cells, G0S2 expression was inhibited using siRNA, which led to decreased cell proliferation, migration, and invasion of MDA-MB-231 cells. Consequently, G0S2 inhibition inactivated integrin-regulated FAK-Src signaling, which promoted Hippo signaling and inactivated ERK1/2 signaling. In addition, G0S2 downregulation decreased β-catenin expression, while E-cadherin expression was increased. It was demonstrated for the first time that G0S2 mediates the Hippo pathway and induces epithelial to mesenchymal transition (EMT). Taken together, our results suggest that G0S2 is a major factor contributing to cell survival and metastasis of MDA-MB-231 cells.

Induction of Integrin Signaling by Steroid Sulfatase in Human Cervical Cancer Cells

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a pivotal role in the regulation of estrogens and androgens that promote the growth of hormone-dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not clear. To elucidate the role of STS in cancer cell proliferation, we investigated whether STS is able to regulate the integrin signaling pathway. We found that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), one of the main metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. Further, STS expression and DHEA treatment enhanced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Moreover, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS activates the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up-regulation of integrin β1 and activation of FAK.

Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells.

Functional Significance of Cytochrome P450 1A2 Allelic Variants, P450 1A2*8, *15, and *16 (R456H, P42R, and R377Q)

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of approximately 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (k(cat)) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (k(cat)/K(m)) increased up to 2.5 fold with a slight increase of its K(m) value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

Keratinocytic Vascular Endothelial Growth Factor as a Novel Biomarker for Pathological Skin Condition

Skin is an emerging target tissue in pharmaceutical and cosmetic science. Safety assessment for dermal toxicity is a critical step for development of topically applicable pharmaceutical agents and ingredients in cosmetics. Urgent needs exist to set up toxicity testing methods for dermal safety, and identification of novel biomarkers for pathological cutaneous alteration is highly required. Here we will discuss if vascular endothelial growth factor (VEGF) has a potential as a biomarker for dermal impairment. Experimental and clinical evidences for induction of keratinocytic VEGF under pathological conditions will be reviewed.

Role of Annexin A5 on Mitochondria-Dependent Apoptosis Induced by Tetramethoxystilbene in Human Breast Cancer Cells

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a trans-stilbene analogue, induces apoptosis in human cancer cells. However, the detailed mechanisms of mitochondria-dependent apoptosis induced by TMS are not fully understood. In the present study, the possible roles of annexin A5 in TMS-mediated apoptosis were investigated in MCF7 human breast cancer cells. Quantitative real-time PCR analysis and Western blot analysis showed that the expression of annexin A5 was strongly increased in TMS-treated cells. TMS caused a strong translocation of annexin A5 from cytosol into mitochondria. Confocal laser scanning microscopic analysis clearly showed that TMS induced translocation of annexin A5 into mitochondria. TMS increased the expression and oligomerization of voltage-dependent anion channel (VDAC) 1, which may promote mitochondria-dependent apoptosis through disruption of mitochondrial membrane potential. When cells were treated with TMS, the levels of Bax, and Bak as well as annexin A5 were strongly enhanced. Moreover, we found that the cytosolic release of apoptogenic factors such as cytochrome c, or apoptosis-inducing factor (AIF) in mitochondria was markedly increased. Annexin A5 depletion by siRNA led to decreased proapoptotic factors such as Bax , Bak, and annexin A5. Taken together, our results indicate that annexin A5 may play an important role in TMS-mediated mitochondrial apoptosis through the regulation of proapoptotic proteins and VDAC1 expression.

Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

Annexin A5 as a New Potential Biomarker for Cisplatin-Induced Toxicity in Human Kidney Epithelial Cells

Cisplatin is a member of platinum-containing anti-cancer drugs that causes cross-linking of DNA and ultimately cancer cell apoptosis. The therapeutic function of cisplatin on various types of cancers has been widely reported but the side effects have been discovered together and nephrotoxicity has been regarded as major side effect of cisplatin. To select candidates for new sensitive nephrotoxicity biomarker, we performed proteomic analysis using 2-DE/MALDI-TOF-MS followed by cisplatin treatment in human kidney cell line, HK-2 cells, and compared the results to the gene profi le from microarray composed of genes changed in expression by cisplatin from formerly reported article. Annexin A5 has been selected to be the most potential candidate and it has been identifi ed using Western blot, RT-PCR and cell viability assay whether annexin A5 is available to be a sensitive nephrotoxic biomarker. Treatment with cisplatin on HK-2 cells caused the increase of annexin A5 expression in protein and mRNA levels. Overexpression of annexin A5 blocked HK-2 cell proliferation, indicating correlation between annexin A5 and renal cell toxicity. Taken together, these results suggest the possibility of annexin A5 as a new biomarker for cisplatin-mediated nephrotoxicity.

Induction of steroid sulfatase expression by tumor necrosis factor-alpha through phosphatidylinositol 3-kinase/Akt signaling pathway in PC-3 human prostate cancer cells

Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates and has a pivotal role in regulating the formation of biologically active estrogens. STS may be considered a new promising drug target for treating estrogen-mediated carcinogenesis. However, the molecular mechanism of STS expression is not well-known. To investigate whether tumor necrosis factor (TNF)-alpha is able to regulate gene transcription of STS, we studied the effect of TNF-alpha on STS expression in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that TNF-alpha significantly induced the expression of STS mRNA and protein in a concentration- and time-dependent manner. Treatment with TNF-alpha resulted in a strong increase in the phosphorylation of Akt on Ser-473 and when cells were treated with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, or Akt inhibitor (Akt inhibitor IV), induction of STS mRNA expression by TNF-alpha was significantly prevented. Moreover, activation of Akt1 by expressing the constitutively active form of Akt1 increased STS expression whereas dominant-negative Akt suppressed TNF-alpha-mediated STS induction. We also found that TNF-alpha is able to increase STS mRNA expression in other human cancer cells such as LNCaP, MDA-MB-231, and MCF-7 as well as PC-3 cells. Taken together, our results strongly suggest that PI 3-kinase/Akt activation mediates induction of human STS gene expression by TNF-alpha in human cancer cells.

Distribution of Malassezia Species on the Scalp in Korean Seborrheic Dermatitis Patients

BACKGROUND: Malassezia species play an important role in the pathogenesis of seborrheic dermatitis. In particular, M. restricta and M. globosa are considered to be the predominant organisms in seborrheic dermatitis of Western countries. However, species distribution of Malassezia in seborrheic dermatitis has not been clearly determined yet in Asia. OBJECTIVE: To identify the distribution of Malassezia species on the scalp of seborrheic dermatitis patients in Korea using 26S rDNA PCR-RFLP analysis. METHODS: A total of 40 seborrheic dermatitis patients and 100 normal healthy volunteers were included in this study. For the identification of Malassezia species, the scalp scales of the subjects were analyzed by 26S rDNA PCR-RFLP analysis. RESULTS: The most commonly identified Malassezia species were M. restricta in the seborrheic dermatitis patients, and M. globosa in the normal controls. In the seborrheic dermatitis group, M. restricta was identified in 47.5%, M. globosa in 27.5%, M. furfur in 7.5%, and M. sympodialis in 2.5% of patients. In the healthy control group, M. globosa was identified in 32.0%, M. restricta in 25.0%, M. furfur in 8.0%, M. obtusa in 6.0%, M. slooffiae in 6.0%, and M. sympodialis in 4.0% of subjects. CONCLUSION: M. restricta is considered to be the most important Malassezia species in Korean seborrheic dermatitis patients.

A Study of Potential Application of Rubus coreanus Miquel Extract for Seborrheic Dermatitis Treatment / 대한의진균학회지

BACKGROUND: Seborrheic dermatitis is chronic relapsing inflammatory skin disorder. Bokbunja (Rubus coreanus Miquel) is a wild berry to Rosaceae genus and also known to have an anti-inflammation effect. OBJECTIVE: We were to determine the effect of Rubus coreanus Miquel extract for seborrheic dermatitis in vivo and in vitro. METHODS: Seven patients with mild seborrheic dermatitis were enrolled in this study. PCR and culture were performed to identify subtypes of six Malassezia species (M. restricta, M. globosa, M. furfur, M. slooffiae, M. sympodialis, M. obtusa). Topical application of Rubus coreanus Miquel Extract was applied twice daily for 2 weeks. Clinical improvement and safety assessment were performed initially and 2 weeks later. Minimum inhibitory concentration (MIC) was evaluated on Malassezia globosa comparing with ketoconazole and itraconazole. Sebum production was also checked prior the experiment and 2 weeks later. RESULTS: Five of seven patients showed improvement. No significant adverse effects were found during the clinical trial. Mild dryness was reported in 2 patients but they resolved spontaneously without any treatment. Rubus coreanus Miquel Extract didn't show antimicrobial effect to Malassezia globosa. However, Rubus coreanus Miquel Extract showed anti-inflammatory effect. CONCLUSION: In this study, we were verified that Rubus coreanus Miquel Extract can be applied for seborrheic dermatitis treatment. And this action mechanism is not related with antimicrobial effect.

The Development and Evaluation of Multiplex PCR Technique for Identification of Malassezia Yeast / 대한의진균학회지

BACKGROUND: Malassezia yeasts as major pathogenic fungi causes the common skin diseases including dandruff, psoriasis, seborrheic dermatitis and atopic dermatitis etc. various molecular techniques were developed to identify and classify the Malassezia species until now. But, these methods were discovered the problems. So, the development of the better molecular methods required to identify and classify of Malasseiza species. OBJECTIVE: We sought to develop of molecular techniques to identify and classify of six Malassezia species (M. restricta, M. globosa, M. furfur, M. slooffiae, M. sympodialis, M. obtusa). METHODS: We designed primers about ITS1 (Internal transcribed space 1) region that were well-known region useful to identify of Malassezia species. Because, ITS1 region that is located between 18S and 5.8S rDNA of ribosomal DNA was comparatively mutated quickly. The mono PCR using ITS1 primers was performed to confirm the specificity of ITS1 primers with six Malassezia standard strains. Then, Malassezia Multiplex detection kit was developed on the basis of technique using ITS1 regions. Malassezia Multiplex detection kit was used to perform multiplex PCR with six Malassezia standard strains and clinical isolates. RESULTS: The results of mono PCR using ITS1 primers about six Malassezia standard strains was detected each Malassezia standard strains. Also, the multiplex PCR using developed Malassezia Multiplex detection kit was confirmed to classify about six Malassezia standard strains and clinical isolates. CONCLUSION: In this study, we verified that six Malassezia yeasts was classified using Malassezia Multiplex detection kit from Malasszia standard strains and clinical isolates. And we anticipate that Malassezia Multiplex detection kit is able to do accurate diagnosis about six Malassezia yeasts (M. restricta, M. globosa, M. furfur, M. slooffiae, M. sympodialis, M. obtusa).

Clinical Efficacy for 1% Zinc Pyrithione Shampoo for the Treatment of Dandruff / 대한피부과학회지

BACKGROUND: Dandruff is a common complaint, and is suffered by up to 50% of the population at some time. Malassezia yeasts, which comprise part of the normal skin flora, might be a critical factor in this disease, as they have been found in higher proportions in patients with seborrheic dermatitis or dandruff, its milder form. OBJECTIVE: The aim of this study was to evaluate the clinical efficacy of 4 weeks of treatment with 1% zinc pyrithione (ZP) shampoo. METHODS: A randomized, double-blind, 4-week treatment period was preceded by a 1-week run-in period. A total of 30 patients were enrolled in this study. Assessments included the patient's subjective score (PSS) and the investigator's assessment score (IAS), images of the affected scalp area, the severity of sebum production, and the erythema and moisturizing effect of the shampoo. RESULTS: 1% ZP shampoo significantly reduced the extent and severity of scaling, as measured by folliscope imaging on visit 2 (p=0.0391) and visit 3 (p=0.0381), as well as pruritus related to the disease as measured by the grading systems, PSS (p=0.0352) and IAS (p=0.0142). Additionally, the results of this study show that a treatment regimen with 1% ZP shampoo significantly reduced scalp sebum production as measured by a sebumeter. Erythema measured by the chromameter was not as meaningful. The corneometric values were slightly increased in the group treated with 1% ZP shampoo but not in the group treated with ZP-free shampoo. Side effects of the ZP shampoo were quite mild and tolerable, and were observed only in a small group of patients. CONCLUSION: 1% ZP shampoo appears to be both effective and well-tolerated when used for the treatment of dandruff.

Optimal Culture Condition for Antifungal Susceptibility Tests of Malassezia globosa / 대한의진균학회지

BACKGROUND: Although numerous culture conditions for Malassezia species were suggested, there were not so many objective evaluation articles in the literature. OBJECTIVES: We examined the various culture conditions for Malassezia globosa. METHODS: Malassezia globosa culture conditions were assessed by Dixon's agar, modified Leeming-Notman medium in diverse oil content and temperature conditions. RESULTS: Maximum growth rate of Malassezia globosa was achieved at 3% olive oil. The optimal temperatures for the maximal growth of M. globosa were observed at 32~34degrees C. CONCLUSION: In this study, we established the optimal culture condition for M. globosa, and confirmed its excellent utility for the antifungal susceptibility tests for M. globosa and M. restricta. Our results can help the investigators plan to do the prospective researches involving Malassezia species, such as the susceptibility test for newly developed antifungal agents.

Effect of Global Ischemic Preconditioning After Cardioplegic Arrest: Langendorff Isolated Heart Study / 대한흉부외과학회지

Ischemic preconditioning is known to have protective effect on myocardial function at prolonged ischemic insult but the mechanism of the effect is not clearly known. The effect of the preconditioning on the global ischemia using cardioplegic solution is not well known. To evaluate the effect of global myocardial preconditioning on the functional recovery after cardioplegic arrest and two hours of hypothermic storage, we used the isolated rat heart and two hours cardioplegic arrest time at 0degree C. In the experimental group (n=10), after baseline functional data was obtained, ischemic preconditioning was induced with 1 min of global normothermic ischemia for three times before the arrest period. In the control group (n=10), hearts underwent no ischemic precondi- tioning. After 2 hrs of cardioplegic arrest and storage in the 0degree C cardioplegic solution reperfusion was done and hemodynamic data were collected at post-reperfusion 20 min. Heart with ischemic preconditioning showed improved functional recovery at post reperfusion 20 min in peak developed pressure and dP/dT. In percent change of the peak pressure, preconditioning group showed 93.20+/-15.7% recovery rate compared to baseline data, and control group showed 67.3+/-15.6% recovery rate. In percent change of the dP/dT, control group showed 54.7+/-18.2% recovery rate and preconditioning group showed 78.1+/-15.1% recovery rate. Percent changes in heart rate and coronary flow showed no significant difference between two groups and there was no significant differences in amount of cardioplegic delivery between groups. Our data suggest ischemic preconditioning may have protective effect on recovery state after cardioplegic arrest and 2 hr ischemic storage of isolated rat heart and its mechanism is not related to the amount of the cardioplegic delivery amount.

Thoracoscopic resection of mediastinal tumor: two cases report / 대한흉부외과학회지

No abstract available.

Carcinosarcoma of the lung: two cases report / 대한흉부외과학회지

No abstract available.