A new steroidal 5,7-diene derivative, 3beta-hydroxyandrosta-5,7-diene-17beta-carboxylic acid, shows potent anti-proliferative activity

Abstract

The new steroidal 5,7-diene, 3beta-hydroxyandrosta-5,7-diene-17beta-carboxylic acid (17-COOH-7DA), was synthesized from 21-acetoxypregnenolone, with the oxidative cleavage of the side chain being dependent on the presence of oxygen. In human epidermal (HaCaT) keratinocytes, 17-COOH-7DA inhibited proliferation in a dose-dependent manner, starting at a dose as low as 10(-11) M. This inhibition was accompanied by decreased expression of epidermal growth factor receptor, bcl2 and cyclin E2 mRNAs and by increased expression of involucrin mRNA. Inhibition of proliferation was associated with slowing of the cell cycle in G1/G0 phases but not with cell death. 17-COOH-7DA was significantly more potent than pregnenolone, 17-COOH-pregnenolone, 17-COOCH(3)-7DA and calcitriol. 17-COOH-7DA also inhibited proliferation of normal human epidermal melanocytes and human and hamster melanoma lines, however, with lower potency than for keratinocytes. In normal human dermal fibroblasts 17-COOH-7DA stimulated proliferation in serum-free media but inhibited it in the presence of 5% serum. 17-COOH-7DA inhibited cell colony formation of human and hamster melanoma cells, and induced monocyte-like differentiation of human HL60 leukemia cells. Thus, the new steroidal 5,7-diene, 17-COOH-7DA, can serve as an inhibitor of proliferation of normal keratinocytes and normal and malignant melanocytes, as a condition-dependent regulator of fibroblast proliferation and a stimulator of leukemia cell differentiation.

Scheme 1

Synthesis of 17-COOH-7DA and its methyl ester. Reagents and conditions: (a) Ac₂O, microwave, p-toluenesulfonic acid monohydrate; (b) Dibromantin, 2, 2′-azobisisobutyronitrile, benzene/hexane (1:1), 100°C, reflux; (c) Bu₄NBr, Bu₄NF, THF, room temperature; (d) K₂CO₃, O₂, MeOH-THF, room temperature, overnight; (e). DBU, MeI, THF, room temperature.

Scheme 2

Synthesis of 3β-hydroxyandrost-5-ene-17β-carboxylic acid. Reagents and conditions: (d) K₂CO₃, O₂, MeOH-THF, room temperature, overnight.

Fig. 1. 17-COOH-7DA inhibits DNA synthesis in HaCaT keratinocytes and has greater effect than pregnenolone

HaCaT cells were incubated with drugs for 72 h in DMEM containing 5% charcoal treated FBS. [³H]-thymidine was added for last 4 h of incubation. DNA synthesis was measured by counting the radioactivity incorporated into TCA precipitable material. Data are presented as means ± SEM (n = 4). The dose dependent inhibition was analyzed by one-way ANOVA with IA, P < 0.05 and IIA, P < 0.01. The differences between 17-COOH-7DA and pregnenolone was analyzed with student's t-test where II, P < 0.01 and III, P < 0.001.

Fig. 2. 17-COOH-7DA stimulates involucrin and represses cyclin E1, bcl-2 and EGFR mRNA expression in HaCaT keratinocytes

HaCaT keratinocytes were treated with 17-COOH-7DA for 6 (involucrin) or 24 h (cyclin E1, bcl-2 and EGFR), total RNA was extracted and subjected to real time RT-PCR analysis with cyclophilin B used as a housekeeping gene. Data are presented as means ± SEM (n = 3). III, P < 0.001; IV, P < 0.0001 in student's t-test.

Fig. 3. Cell cycle analysis with flow cytometry

HaCaT keratinocytes were cultured in DMEM plus 5% ctFBS and 10⁻⁷ M 17-COOH-7DA for 72 h. The cells were fixed with 70% ethanol and stained with propidium iodide as described in materials and methods. Cell cycle was determined using flow cytometry with 10,000 cells scored. Veh., DMSO control; 17-D, 17-COOH-7DA. I, P < 0.05 in student t-test.

Fig. 4. Comparison of the effects of 17-COOH-7DA, 17-COOH-pregnenolone (17-COOH-A) and 17-COOCH₃-7DA on DNA synthesis in HaCaT keratinocytes

Data are presented as means ± SEM (n = 4). The dose-dependent inhibition was analyzed by one-way ANOVA with IA, P < 0.05 and IIA, P < 0.01. The differences between 17-COOH-7DA and 17-COOH-A or 17-COOCH₃-7DA were analyzed with student's t-test where II, P < 0.01 and III, P < 0.001.

Fig. 5. 17-COOH-7DA inhibits DNA synthesis in human keratinocytes (HaCaT), normal melanocytes, melanoma cells (SKMEL-188, WM35, WM1341) and hamster AbC1 melanoma cells

Data are presented as means ± SEM (n = 4). I, P < 0.05; II, P < 0.01; III, P < 0.001 in student's t-test. IA, P < 0.05; IIA, P < 0.01 in one-way ANOVA analysis.

Fig. 6. The effect of 17-COOH-7DA on DNA synthesis in normal human dermal fibroblasts is dependent on the presence or absence of serum

Normal human dermal fibroblasts were cultured in DMEM containing 5 μg/ml insulin and 17-COOH-7DA in the absence (serum free media) or presence of 5% or 1% of charcoal treated FBS. After 68 h, [³H]-thymidine was added and after 4 h the incorporation of [³H]-thymidine into DNA was measured using a beta-counter. A, 5% FBS; B, 1% FBS; C, serum free media plus 0.1% BSA. Data are presented as means ± SEM (n = 4). II, P < 0.01; III, P < 0.001 in student's t-test. IA, P < 0.05; IIA, P < 0.01 in one-way ANOVA analysis.

Fig. 7. 17-COOH-7DA inhibits colony formation of SKMEL-188 (A), WM35 (B) and AbC1 (C) melanoma cells

The plates were incubated with drugs for 7 (SKMEL-188 and AbC1) or 17 days (WM35) and colonies were stained with crystal violet. The number of formed colony units (CFU) were counted for all colonies (>0.2 mm), medium size colonies (>0.5 mm) or large colonies (>1 mm). Data are presented as means ± SEM for colony forming assay (n = 4). I, P < 0.05; II, P < 0.01; III, P < 0.001 in student's t-test. Lower right corner shows representative plates of SKMEL-188 and AbC1 treated with vehicle control (−) or 10⁻⁹ M 17-COOH-7DA (+).

Fig. 8. Effects of 17-COOH-7DA, 17-COOH-pregnenolone (17-COOH-A) and 17-COOCH₃-7DA on monocyte-like differentiation of HL-60 leukemia cells

After 5 days of treatment, the cells were resuspended in NBT/TPA solution and incubated at 37°C for 60 min. The differentiated cells were identified by their intracellular blue formazan deposits. The NBT-positive and –negative cells were scored under light contrast microscopy (20x) with a minimum of 200 cells scored. N, untreated control; D, DMSO treated control; 17-D, 17-COOH-7DA; 17-Ac., 17-COOCH₃-7DA; 17-A, 17-COOH-A. Data are presented as means ± SEM (n = 3). I, P < 0.05; III, P < 0.001 in student's t-test.

Fig. 9. 17-COOH-7DA inhibits DNA synthesis in HaCaT keratinocytes, SKMEL-188 and AbC1 and has a larger effect than 1,25(OH)₂D₃

The cells were incubated with drugs for 72 h in DMEM (HaCaT) and Ham's F-10 (SKMEL-188 and AbC1) containing 5% charcoal-treated FBS. [³H]-thymidine was added for last 4 h of incubation. DNA synthesis was measured by counting the radioactivity incorporated into TCA precipitable material. A, HaCaT; B, SKMEL-188; C, AbC1. Data are presented as means ± SEM (n = 4). The dose dependent inhibition was analyzed by one-way ANOVA with IA, P < 0.05 and IIA, P < 0.01. The differences between 17-COOH-7DA and 1,25(OH)₂D₃ was analyzed with student's t-test where I, P < 0.05 and III, P < 0.001