Human activated hepatic stellate cell line LX-2 was a gift from Dr. Friedman of Mount Sinai School of Medicine. LX-2 cells were a low-passaged human cell line from normal human stellate cells that were spontaneously immortalized[8,9]. The cells exhibited typical features of stellate cells and expressed α-SMA under all culture conditions and were regarded as at least partially activated even after immediate replating. LX-2 cells underwent further activation during growth and expansion on plastic surfaces[8].

Human hepatocellular carcinoma cell line HepG2 was purchased from the Chinese Academy of Medical Sciences (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine and streptomycin were purchased from Gibco, Invitrogen, Carlsbad, CA. Fetal bovine serum (FBS) was from Hyclone, USA. Penicillin and phenazine methosulfate (PMS) were from Sigma, USA. MTS and oligo (dT)₁₅ primers were acquired from Promega, USA. TRIzol reagent and Moloney murine leukemia virus reverse transcriptase (M-MLV RT) were the products of Invitrogen, CA. SYBR Green I was purchased from OPE Technology Development Company, Shanghai, China. Hotstar Taq DNA polymerase was purchased from TW-Biotech, China.

LX-2 cells were cultured in DMEM supplemented with 50 mL/L heat-inactivated FBS, 200 mmol/L L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. HepG2 cells were cultured in DMEM containing 100 mL/L FBS, 200 mmol/L L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin.

MTS assay was used to determine the effect of Cpd 861 on the proliferation of LX-2 and HepG2 cells. In metabolically active cells, MTS was reduced by dehydrogenase enzymes into an aqueous, soluble formazan product. The absorbance was measured directly at 490 nm from 96-well assay plates without additional processing. The quantity of formazan was considered to be directly proportional to the number of viable cells in the culture[10,11].

LX-2 and HepG2 cells were seeded into 96-well tissue culture plates containing 100 µL DMEM containing 50 mL/L FBS (5000 LX-2 cells/well and 8000 HepG2 cells/well). After 24 h of incubation (37 °C, 50 mL/L CO₂), the medium was carefully removed and 100 µL fresh medium containing various concentrations of Cpd 861 was added into the wells. The cells were treated continuously with Cpd 861 for 1, 2, 3, 5 and 7 d and the medium containing Cpd 861 was changed every other day. At the end of experiments, 20 µL/well of combined MTS/PMS solution was added. After 3 h of incubation at 37 °C in a humidified incubator, the absorbance was analyzed on a VERSAmax microplate reader at 490 nm. Absorbance values were the mean ± SE of 3 replicates for each treatment. The cells in only controls and compound controls were included.

LX-2 cells were seeded into 60 mm dishes in DMEM containing 50 mL/L FBS. After 3 d of seeding, the medium was carefully removed and fresh medium containing Cpd 861 (0.01 mg/mL) was added. After 24, 48 and 72 h of Cpd 861 treatment, total RNA was extracted from LX-2 cells using TRIzol reagent as the lysis buffer. Complementary DNA (cDNA) was synthesized using oligo (dT)₁₅ primers and M-MLV RT.

After the reverse-transcription reaction, cDNA templates were amplified by quantitative real-time PCR. The reaction was performed using the iCycler iQ real-time PCR detection system (Bio-Rad, USA) with SYBR Green I. Human α-SMA and G3PDH (glyceraldehyde-3-phosphate dehydrogenase) primers were designed using Primer 3 software (Table 1).

Each experiment was performed in 25 µL of reaction volume [1.25 µL of 20 × SYBR Green, 1 µL of first strand cDNA (50 ng RNA), 2 µL of each 5 nmolar primer, 0.75 U Hotstar Taq DNA polymerase, 2.5 µL of 10 × amplification buffer (Mg⁺⁺ free), 2.5 µL of 25 mmol/L MgCl ₂, 0.5 µL of 10 mmol/L solution of four dNTP and 15.1 µL of dH ₂O]. In the last tube, 1 µL of ddH ₂O was added as a non-template control. The conditions of amplification cycles were as follows: 40 cycles consisting of denaturation at 95 °C for 40 s, annealing at 59 °C (for α-SMA) or at 60 °C (for G3PDH) for 40 s, and extension at 72 °C for 30 s.

The iCycler apparatus was used to measure the fluorescence of each sample in every cycle at the end of the extension[12]. A series of consecutive 10 fold dilution of α-SMA and G3PDH plasmid DNA ranging from 10⁹ copies/µL to 10³ copies/µL were used as the templates for the standard curves. The iCycler software was used to construct the calibration curve by plotting the Ct (threshold cycle) vs the logarithm of the number of copies for each calibrator. The number of copies in unknown samples was calculated by comparing their Cts with the standard calibration curve. The quality and quantities of samples were normalized based on that of G3PDH.

After PCR, a melting curve was obtained by increasing the temperature from 55 °C to 95 °C with a temperature transition rate of 0.1 °C/s. The melting curves of all final PCR products were analyzed. The differences in melting temperature of PCR products allowed us to distinguish genuine products from nonspecific products, and primer dimers. To ensure that the correct product was amplified in the reaction, all samples were also separated on 20 g/L agarose gel electrophoresis. All PCR conditions and primers were optimized to produce a single product of the correct basepair size.

Data were expressed as mean ± SE. Statistical analysis was performed using GraphPad Prism (version 3.0) software. The t test was used for comparison between the groups. P < 0.05 was considered statistically significant.

We examined the effects of Cpd 861 on the proliferation of LX-2 and HepG2 cells (Figure 1, Figure 2). Cells were incubated with 0.1 mg/mL, 0.03 mg/mL, 0.01 mg/mL and 0.003 mg/mL of Cpd 861 for different days (Figure 1). Cell proliferation was performed by MTS assay. As shown in Figure 2, the effects of Cpd 861 on LX-2 cell proliferation seemed to be correlated with the dose. A significant proliferation inhibition was observed when Cpd 861 concentration was over 0.03 mg/mL (Figure 2). As shown in Figure 1A, 0.1 mg/mL Cpd 861 significantly inhibited LX-2 cell proliferation (15% decrease relative to control, P < 0.05) after 3 d of incubation. The inhibition effects seemed to increase with the treatment time (25% decrease after 5 d of incubation and 35% decrease after 7 d of incubation, P < 0.01). The inhibition of cell proliferation Cpd 861 at 0.03 mg/mL concentration was also observed on d 3 (15% decrease relative to control) and on d 5 (21% decrease relative to control) (data not shown), and still could be observed on d 7 (18% decrease relative to control) (Figure 2). However, Cpd 861, at 0.01 mg/mL and 0.003 mg/mL concentrations, did not inhibit LX-2 proliferation evidently. The effects of Cpd 861 on LX-2 cell proliferation seemed low at early time points (on day 1 and day 2). We also examined the effects of Cpd 861 on the proliferation of HepG2 cells. As shown in Figure 1B and Figure 2, Cpd 861 did not affect HepG2 cell proliferation.

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Figure 1 Regulation of proliferation in LX-2 and HepG2 cells by Cpd 861. A: Effect of 0.1 mg/mL Cpd 861 on LX-2 cell proliferation. B: Effect of 0.1 mg/mL Cpd 861 on HepG2 cell proliferation. Data are expressed as the mean ± SE of 3 replicates. ^aP < 0.05 vs LX-2 cell controls after 3 d of incubation. ^dP < 0.01 vs LX-2 cell controls after 5 d of incubation. ^fP < 0.01 vs LX-2 cell controls after 7 d of incubation.

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Figure 2 Dose dependent effects of Cpd 861 on cell prolifera-tion in LX-2 and HepG2 7 d after incubation. Data are expressed as the mean ± SE of 3 replicates. ^bP < 0.01 vs LX-2 cell controls.

We examined the effect of Cpd 861 on the expression of α-SMA, a phenotypic marker of activated HSCs. The PCR amp/cycle graph and standard curve graph of α-SMA are shown in Figure 3. The standard curve showed a correlation coefficient > 0.99, indicating a precise log-linear relationship. As shown in melt curve graph (Figure 4), single and sharply defined melting curves with narrow peaks were obtained for PCR products of α-SMA gene. Bands visible after electrophoresis on 2% agarose gel and ethidium bromide staining correlated well with the quantitative PCR results. As shown in Figure 5, Cpd 861 could reduce the expression levels of α-SMA in LX-2 cells after treatment. When LX-2 cells were exposed to Cpd 861 for 48 or 72 h, the expression levels of α-SMA decreased significantly (59% decrease relative to control at 48 h, P < 0.05, and 60% decrease relative to control at 72 h, P < 0.01) (Figure 5).

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Figure 3 Fluorescence data (A) and standard curve (B) for α-SMA generated on iCycler iQ real-time PCR detection system. Plasmids containing the fragments for α-SMA were diluted from 10⁹ to 10³ copies.

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Figure 4 Melting curve for all PCR products after amplification.

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Figure 5 Effects of Cpd 861 on expression of α-SMA mRNA in LX-2 cells. LX-2 cells were incubated with 0.01 mg/mL Cpd 861 for different time. The relative number of molecules of α-SMA mRNA after normalization to G3PDH was calculated. The data represent mean ± SE of 4 replicates for each treatment. ^aP < 0.05 vs LX-2 cell controls after 48 h of incubation. ^dP < 0.01 vs LX-2 cell controls after 72 h of incubation.