Fig 1.
Dose-dependent cytotoxicity of SQ40 on human normal and prostate cancer cell lines.
RWPE-1 normal prostate cells (black), WRL 68 normal liver cells (blue), LNCaP (red) and PC-3 prostate cancer cells (green) were treated with increasing concentrations (2.5–100 μg/mL) of SQ40 for 72 hours and cell viability was measured using MTT reduction assay. Data were expressed as mean ± SEM of four independent experiments. IC50 values of SQ40 on RWPE-1, WRL 68, LNCaP and PC-3 cells at 72 hours treatment were 59.26 μg/mL, 27.69 μg/mL, 5.97 μg/mL and 87.94 μg/mL, respectively.
Fig 2.
The effect of SQ40 on cell viability of DHT-stimulated LNCaP cells.
LNCaP cells were treated with increasing concentrations of DHT with or without 3, 6 and 12 μg/mL of SQ40 for 72 hours in RPMI 1640 supplemented with 5% CSS and cell viability was measured using MTT reduction assay. Percentage of viable cells was calculated relative to vehicle control cells without DHT treatment. Data were expressed as mean ± SEM of three independent experiments.
Fig 3.
The effects of SQ40 on LNCaP cells anchorage-independent growth.
LNCaP cells were pre-treated with SQ40 or vehicle control for 72 hours and then plated on the soft agar media for another 3 weeks. Representative colonies of (A) vehicle control and (B) SQ40-treated LNCaP cells are shown. (C) Graphical representations of soft agar colony formation efficiency. Data were expressed as mean ± SEM of three independent experiments for vehicle control cells and six independent experiments for SQ40-treated cells. * indicates p<0.05 versus vehicle control.
Fig 4.
Growth profile of LNCaP and RWPE-1 cells upon treatment with SQ40.
The growth kinetics of (A) LNCaP and (B) RWPE-1 cells were examined real-time using RTCA. The impedance values were recorded in real-time and were expressed as the Cell Index (CI). Cells treated with growth media alone were referred as vehicle control while 5 μM paclitaxel-treated cells were referred as positive control. (C) SQ40-treated LNCaP cells were stained with 0.4% trypan blue solutions in a ratio of 1:1 after 72 and 96 hours of treatment respectively. Cells treated with growth media alone were referred as vehicle control while 1 μM paclitaxel-treated cells were referred as positive control. Data were expressed as means ± SEM of three independent experiments. ** indicates p<0.01 versus 72-hour treated vehicle control. ## indicates p<0.01; ###, p<0.001 versus 96-hour treated vehicle control.
Fig 5.
Cell cycle distribution of LNCaP cells upon SQ40 treatment.
LNCaP cells were treated with growth media (vehicle control), 3, 6 and 12 μg/mL of SQ40. Cell distribution in (A) G0/G1, (B) S and (C) G2/M phase at 24, 48 and 72 hours treatment were analysed by FACSCanto II flow cytometry and evaluated using ModFit cell cycle analysis software. Data were expressed as means ± SEM of three independent experiments. * indicates p<0.05; **, p<0.01; ***, p<0.001 versus vehicle control. (D) Protein expression of cleaved-PARP in LNCaP cells upon SQ40 treatment for 72 and 96 hours. β-actin served as a loading control.
Fig 6.
Protein expression of G1/S regulatory proteins in LNCaP cells treated with SQ40.
LNCaP cells were first cultured in growth media supplemented with 5% CSS for 48 hours and then treated with 3, 6 and 12 μg/mL of SQ40 with the presence of 100 nM DHT for 72 hours. Immunoblotting was performed on protein extracts to detect CDK4, CDK2, Cyclin D1, Cyclin D3, p21Waf1/Cip1 and p27Kip1. β-actin served as a loading control.
Fig 7.
Quantitative measurement of the level of nuclear AR and PSA secretion in SQ40-treated LNCaP cells.
LNCaP cells were first cultured in growth media supplemented with 5% CSS for 48 hours and then treated with 3, 6 and 12 μg/mL of SQ40 in the presence or absence of 100 nM DHT for 72 hours. Nuclear fraction of AR obtained from the cell lysate and concentrations of PSA secreted into culture medium were measured using commercial available ELISA kit. (A) AR level was normalized to the total nuclear protein level while (B) the concentration of PSA were normalised to the total cell number. Data were expressed as means ± SEM of three independent experiments and indicated as percentage of 100 nM DHT-stimulated cells set at 100%. ** indicates p<0.01; ***, p<0.001 versus unstimulated control. ###, p<0.001 versus 100 nM DHT-stimulated cells.
Fig 8.
Anti-tumor activity of SQ40 against subcutaneous LNCaP cell tumors.
LNCaP cells at 2 x 106 were injected subcutaneously into right flank of NCr nude mice. SQ40 treatment was initiated when the tumor was palpable. Vehicle control (saline) and SQ40 were given intraperitoneally thrice a week for 6 weeks with a total of 18 doses. Graph of (A) mean body weight and (B) tumor volume for each treatment versus the number of days after initial injection of LNCaP cells. (C) Representative images of tumors isolated from vehicle control, 5 mg/kg and 10 mg/kg of SQ40-treated animals. Each point represents the mean ± SEM of data (n = 6). ** indicates p<0.01 versus vehicle control.
Fig 9.
A schematic representation describing the anti-proliferation activities of SQ40 in regulation of cell cycle proteins in LNCaP cells.