Human prostate cancer cell lines PC-3, LNCaP, and 22RV1 were purchased from DS Pharma Biomedical (Osaka, Japan) and cultured in RPMI 1640 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS (Moregate BioTech, Bulimba, Australia). PC-3 is an androgen receptor-negative human prostate cancer cell line (7). LNCaP-LA cells, which were generated from LNCaP cells, were cultured in medium containing 10% charcoal-stripped fetal bovine serum (FBS) for more than 3 months.

Cells were cultured on a 6-well plate and incubated overnight in medium containing 10% FBS. Cells were then incubated with or without simvastatin (5 µM). After 48 h, androstenedione (100 µM) was added to the medium. After 24 h, culture medium was collected, and testosterone and DHT concentrations were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (ASKA Pharmaceutical Medical Co., Ltd., Kawasaki, Japan). RIPA buffer was added to wells and protein concentration was measured by the DC Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Testosterone and DHT levels were calculated by dividing the results of the protein assay by the total protein concentration.

Transcript levels were quantified using the Applied Biosystems 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Total RNA was extracted, cDNA was synthesized (8), and polymerase chain reaction (PCR) amplification was performed, using 2 µl cDNA and the StAR, CYP11A1, CYP17A1, aldo-keto reductase family 1 member C3 (AKR1C3), HSD3B1, HSD3B2, SRD5A1, SRD5A2, and AKR1C2 primers (No. Hs00986559_g1, Hs00167984_m1, Hs01124136_m1, Hs00366267_m1, Hs00426435_m1, Hs00605123_m1, Hs00602694_mH, Hs00165843_m1, and Hs00912742_m1, respectively; Applied Biosystems). Next, PCR was performed for one cycle of 10 min at 95°C followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. b-Actin (No. 4326315E, Applied Biosystems) transcript levels were used as the internal control. mRNA fold changes were quantified using ΔΔCq.

Cells were plated onto a 96-well plate in 100 µl culture medium containing 10% FBS. After 24 h, cells were incubated with medium containing simvastatin (5 µM) and/or meclofenamic acid (50 µM). After incubation for 48 h, the number of living cells was measured using the MTS assay.

Cells were plated onto a 12-well plate and grown to confluence. A 1,000-µl tip was used to make a denuded area. Cells were washed twice with PBS and incubated with medium containing various concentrations of simvastatin for 48 h. Mitomycin C (0.5 µM) was added to the medium to inhibit cell proliferation. Photographs were taken at 0 and 48 h, and the distance of cell migration was determined by subtracting the values obtained at 0 h from those obtained at 48 h. Migration distance is expressed as fold change over the control.

Cells were transfected with ON-TARGETplus Non-targeting Pool (no. D-001810-10-05; Dharmacon, Waltham, MA, USA) or ON-TARGETplus AKR1C3 siRNA (No. L-008116-00-0005, Dharmacon) using DharmaFect 2 (Dharmacon). Cells were incubated for 48 h after transfection.

Cell lysates were prepared in RIPA buffer containing 1 mM sodium orthovanadate (Sigma-Aldrich; Merck KGaA) and Halt Protease Inhibitor Cocktail (Pierce; Thermo Fisher Scientific, Inc.). Samples were boiled for 5 min; an equal amount of protein (30 µg/lane) was subjected to 4–12% SDS-PAGE and transferred onto nitrocellulose membranes. Each membrane was incubated with the following primary polyclonal antibodies: rabbit anti-Akt (1:1,000), rabbit anti-phospho-Akt (Ser473) (1:1,000) (Cell Signaling Technology, Inc., Beverly, MA, USA). Blots were developed using a 1:2,000 dilution of the HRP-conjugated secondary antibody (Cell Signaling Technology, Inc.). Proteins were visualized using Immobilon (Merck Millipore, Darmstadt, Germany).

Data are expressed as the mean ± standard deviation. Differences between values were evaluated by one-way ANOVA using Tukey's post hoc analysis and Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

We examined PC-3, LNCaP-LA and 22Rv1 cells to determine whether simvastatin alters genes that encode steroidogenic enzymes in androgen-independent prostate cancer cells. After treatment with simvastatin, the expression of AKR1C3 was increased in PC-3 and LNCaP-LA cells (Figs. 1A and 2A) but not in 22Rv1 cells (data not shown). Moreover, the fold change was more than 60 times in PC-3 cells. Conversely, the expression of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1) was decreased in PC-3 and LNCaP-LA cells (Figs. 1A and 2A) but not in 22Rv1 cells (data not shown). Moreover, simvastatin increased steroid 5 alpha-reductase 1 (SRD5A1) expression in PC-3 (Fig. 1A) but not in LNCaP-LA or 22Rv1 cells (data not shown). The expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (HSD3B2), steroid 5 alpha-reductase 2 (SRD5A2), and aldo-keto reductase family 1 member C2 (AKR1C2) did not change following treatment with simvastatin (data not shown).

To determine whether increased levels of AKR1C3 affect the de novo synthesis of intracellular androgen, we measured the testosterone and DHT levels in culture medium following treatment with simvastatin by LC-MS/MS. Simvastatin significantly increased both testosterone (Fig. 2A) and DHT (Fig. 2B) levels after the addition of androstenedione. These data show that the up-regulation of AKR1C3 is functional.

PC-3 is an AR-negative human prostate cancer cell line. Therefore, there is a possibility that an increase in testosterone and DHT levels does not affect cell viability. In contrast, the overexpression of AKR1C3 promotes angiogenesis and aggressiveness in PC-3 cells (9). To further determine whether increased AKR1C3 expression affects simvastatin-induced cell viability, AKR1C3 expression was reduced by transfection with siRNA against AKR1C3. siRNA treatment inhibited the expression of AKR1C3 mRNA in PC-3 cells (Fig. 3A). The reduction in AKR1C3 expression in PC-3 cells following siRNA transfection was not associated with basal cell proliferation and migration; however, siRNA transfection with simvastatin significantly decreased both cell proliferation (Fig. 3B) and cell migration (Fig. 3C and D) compared to simvastatin alone.

Some drugs are reported to inhibit AKR1C3. Meclofenamic acid is an NSAID as well as one of the best inhibitors of AKRs, especially AKR1C3 (10). Therefore, we evaluated the combinatory effects of simvastatin and meclofenamic acid in PC-3 cells. Treatment with either simvastatin or meclofenamic acid alone inhibited cell proliferation (Fig. 4A) and migration (Fig. 4B and C). The combination of the two drugs further enhanced cell proliferation (Fig. 4A) and migration (Fig. 4B and C).

AKR1C3 overexpression induces Akt activation in PC-3 cells (9). We previously showed that simvastatin without IGF1 decreases IGF1R expression strongly in PC-3 cells (4). IGF1-Akt activation is a well-known pathway in prostate cancer. We hypothesized that inhibiting simvastatin-stimulated AKR1C3 expression with an AKR1C3 inhibitor would have a synergistic effect on simvastatin-blocked IGF1-induced Akt activation. Therefore, the effects of the combination of simvastatin and meclofenamic acid on IGF1-induced Akt activation were evaluated in PC-3 cells. Treatment with either simvastatin or meclofenamic acid alone attenuated IGF1-induced Akt activation, whereas the combination of simvastatin and meclofenamic acid further inhibited Akt activation (Fig. 5).