The online version of this article (doi:10.1186/1475-2867-12-1) contains supplementary material, which is available to authorized users.
FK received a scholarship from Bayer HealthCare during the conduction of the experiments for his doctoral thesis. BH and AS are employees of Bayer HealthCare. No conflict of interest was encountered.
FK conducted the experiments on A549 and NCI-H460 cells. AS performed Affymetrix microarray experiments and analyzed SRD5A1 and SRD5A2 in the Array Northern database. TK and GD participated in the experimental design and contributed to the statistical analysis of the data. BH designed, coordinated and supervised the experimental studies. All authors read and approved the final manuscript.
Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC.
siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used.
The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation.
In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines.
Additional File 1: Location of the siRNA 1-3 binding sites in the human SRD5A1 cDNA sequence. The position of the siRNA 1-3 binding sites in the human SRD5A1 sequence is shown. Letters with green background indicate non-coding regions of the human SRD5A1 sequence (Gene ID: 6715; RefSeq Seq: NM_001047.2), letters with white background represent the coding sequence. colored letters indicate the binding site of the specific siRNAs (blue: siRNA 1, green: siRNA 2, red: siRNA 3). (PPT 106 KB)
Additional File 2: Expression construct for CMV-driven expression of human V5-tagged SRD5A1 protein. A map of the vector used for expression of human SRD5A1 is shown. The SRD5A1 cDNA was cloned into the mammalian expression vector pcDNA/V5-His (Invitrogen) upstream of the V5 tag in order to generate a SRD5A1 fusion protein with the V5 tag at the C-terminus. (PPT 646 KB)
Additional File 3: Structure of 17-methylene-4-azasteroids. The general chemical structure formula of 17-methylene-4-azasteroids is shown . For a more detailed structure, also see the review by Aggarwal et al. . (PPT 55 KB)
Additional File 4: SRD5A1 expression in normal tissues and in cell lines. The expression pattern of SRD5A1 was analyzed in a panel of human tissues (A) and cell lines (B) in the Array Northern database. Probeset 204675_at which interrogates SRD5A1 on the Affymetrix HGU133Plus2.0 array is shown. On the x-axis the human tissues and cell lines are shown sorted by type. The number of replicates analyzed is indicated. The y-axis depicts arbitrary expression units. In normal tissues, the highest transcript levels were detected in skin, esophagus, liver, small intestine, and in neuronal tissue. A relatively weak expression was observed in normal lung tissue. The cell lines with highest SRD5A1 expression are derived from breast cancer. (PPT 309 KB)
Additional File 5: Relative gene expression of SRD5A1 normalized to cyclophilin 24 h after siRNA-mediated silencing in A549 (A) and NCI-H460 (B) cells. SRD5A1 silencing experiments were performed in A549 and NCI-H460 cells in triplicate with 10 pmol siRNA and 5 μl Lipofectamine™ 2000. (A) A549: *significant difference to untreated (mm 1: P = 0.001)¸ **significantly smaller than 5% (siRNA 1: P = 0.001, siRNA 2: P < 0.001, siRNA 3: P < 0.001). (B) NCI-H460: *significant difference to untreated (siRNA 1: P = 0.010, siRNA 2: P = 0.004, siRNA 3: P < 0.001). (PPT 118 KB)
Additional File 6: Proliferation assay after siRNA treatment of A549 (A) and NCI-H460 (B) cells. Proliferation experiments were conducted in triplicate with 10 pmol siRNA and 5 μl Lipofectamine™ 2000. Significantly reduced proliferation was observed in all Lipofectamine™ 2000-treated groups (P < 0.001-P = 0.04) in both cell lines. (PPT 142 KB)
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- 5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis
Friedrich G Kapp
- BioMed Central
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