Establishment of type II 5α-reductase over-expressing cell line as an inhibitor screening model

Jang, Sunhyae; Lee, Young; Hwang, Seong-Lok; Park, Su Jin; Kang, Sung Soo; Roh, Seok-Seon; Seo, Young‐Joon; Park, Jang-Kyu; Lee, Jeung‐Hoon; Kim, Chang Deok

doi:10.1016/j.jsbmb.2007.03.039

Cited by 13 publications

(12 citation statements)

References 32 publications

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“…A further advantage of TLC is that radioactive steroids can be identified by placing the plate in contact with x-ray film, producing an autoradiogram. Dalla Valle et al, 2004;Jang et al, 2007), although metabolites of [ 3 H]progesterone, for example, have been separated by TLC on silica gel, and located by cutting the plates into 1-cm bands and subsequent liquid scintillation counting (Carey et al, 1994). The availability of microparticulate silica has enabled the introduction of the so-called 'high-performance' TLC, which is claimed to have increased resolving power.…”

Section: Thin-layer Chromatographymentioning

confidence: 99%

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

Makin

Honour

Shackleton³

et al. 2010

Steroid Analysis

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Section: Thin-layer Chromatographymentioning

confidence: 99%

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

Makin

Honour

Shackleton³

et al. 2010

Steroid Analysis

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“…Jang et al . [ 22 ] reported that the HEK293 cells were stably transfected with 5AR2 and they suggested RT-PCR data to validate that the stable transfection has been achieved. They determined that 10 μg of stable cell extract completely converted 1 μCi of T into DHT in an in vitro enzymatic assay.…”

Section: Resultsmentioning

confidence: 99%

“…According to the method reported [ 22 ], human embryonic kidney cells (HEK293) over-expressing type II 5α-reductase were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Stable HEK293 cells (1 × 10 6 ) were planted in 24-well cell plate and incubated in 5% CO 2 incubator for 24 hours at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of Pregnane Derivatives, Their Cytotoxicity on LNCap and PC-3 Cells, and Screening on 5α-Reductase Inhibitory Activity

Kim

2009

Molecules

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A series of epoxy- and/or 20-oxime pregnanes were synthesized from commercially available pregnenolone. Compounds 1, 3, 7, 8 and 11-13 were evaluated for cytotoxicity activity towards LNCaP (androgen-dependent) and PC-3 (androgen-independent) prostate cancer cells. Compound 13 showed the highest activity on both LNCaP (IC50 15.17 μM) and PC-3 (IC50 11.83 μM) cell lines. Compound 11 showed weak activity on LNCaP cells (IC 50 71.85 μM) and 8 showed the weak activity on PC-3 cells (IC50 68.95 μM), respectively. The 5α-reductase II (5AR2) inhibitory effects of compounds 1-3, 5 and 7-13 were investigated in a convenient screening model, in which compounds 5, 8, 11 and 12 were observed to be potential inhibitors of 5α-reductase, in particular, the 4-azasteroid 11, that also inhibited cell proliferation of androgen-dependent cells and 8, that in addition inhibited PC-3 cells more potently than LNCaP cells.

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“…However, the IC 50 value of mangiferin (370 mg/ml) measured in our study compared with the reported inhibitor (11.85 mM). 6,10,18,27) From the molecular structure of mangiferin, a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside), we speculate that the inhibitory eŠect of mangiferin on hSRD5A2 is due to direct inhibition of hSRD5A2 itself rather than its non-speciˆc anti-oxidant potential. Inhibition activity of mangiferin may derive from the precipitation activity of the protein.…”

Section: Discussionmentioning

confidence: 96%

“…This enzyme also catalyzes NADPHdependent reduction of the D4 double bond of several other steroid substrates. 5,6) So far, two distinct forms of hSRD5A isoenzymes, called type I and type II, have been characterized based on their diŠerent pH optimum. 7,8) Although the structure of the type I and type II SRD5A genes is similar, the protein sequence homology is relatively low (about 47％).…”

Section: Introductionmentioning

confidence: 99%

Establishment of a Novel Model for Studying the Effects of Extracts of Chinese Herb Medicine on Human Type II 5α-Reductase <i>in Vitro</i>

et al. 2010

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Human steroid 5a-reductase type II (hSRD5A2) and dihydrotestosterone (DHT) play important roles in benign prostatic hyperplasia (BPH). The aim of our study was to establish a novel model to investigate the inhibitory eŠects of extracts and compounds of Chinese herb medicine on hSRD5A2. The gene, hSRD5A2, was artiˆcially synthesized and cloned into pcDNA3.1(＋) vector, which was transfected into CHO cells by liposome. Transfected cells were screened through G418 and MTX. The expressed protein of hSRD5A2 by cells was puriˆed and detected by western blotting. A minim reactive system comprising hSRD5A2 and testosterone (T) as substrate together with NADPH as hydrogen donor was established for screening inhibitors of hSRD5A2. The reaction system was optimized in the concentrations of T, NADPH, and hSRD5A2 and reaction temperature, time, and activity of hSRD5A2 were determined by the production of DHT. Furthermore, we screened some extracts and compounds of Chinese herb medicine using this model. The concentrations of T, NADPH, and hSRD5A2 were 0.02 mM, 0.8 mM, and 0.05 U/ml, respectively, in the model; maximum activity of hSRD5A2 was achieved at 37°C and 60 min reaction, and mangiferin had signiˆcant inhibitory eŠect on the activity of hSRD5A2. The model in this study is convenient and reliable for screening and evaluation of inhibitors of hSRD5A2; mangiferin may be a potential medicine for the treatment of BPH.

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Establishment of type II 5α-reductase over-expressing cell line as an inhibitor screening model

Cited by 13 publications

References 32 publications

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

Synthesis of Pregnane Derivatives, Their Cytotoxicity on LNCap and PC-3 Cells, and Screening on 5α-Reductase Inhibitory Activity

Establishment of a Novel Model for Studying the Effects of Extracts of Chinese Herb Medicine on Human Type II 5α-Reductase <i>in Vitro</i>

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