Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, Lipofectamine™ LTX with PLUS™ reagent, TRIzol reagent were purchased from Invitrogen (Carlsbad, CA, USA). Bcl-2, cyclin D1, SOCS3 and phospho-STAT3 (Tyr⁷⁰⁵) antibodies, horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell Signaling (Beverly, MA, USA). SuperScript II reverse transcriptase and Dual-Luciferase Reporter Assay System were obtained from Promega (Madison, WI, USA). The Hoechst staining kit was obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). Cignal STAT3 Reporter (luc) kit was obtained from SABiosciences (Qiagen, Hilden, Germany). All the other chemicals, unless otherwise stated, were obtained from Sigma Chemicals (St. Louis, MO, USA).

PZH was obtained from and authenticated by the sole manufacturer Zhangzhou Pien Tze Huang Pharmaceutical Co. Ltd., China (Chinese FDA approval no. Z35020242). Stock solution of PZH was prepared immediately prior to use by dissolving the PZH power in phosphate-buffered saline (PBS) to a concentration of 20 mg/ml. The working concentrations of PZH were made by diluting the stock solution in the culture medium.

Human colon carcinoma HT-29 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin in a 37°C humidified incubator with 5% CO₂.

HT-29 cells were first grown in complete DMEM (10% FBS) until ~50% confluency and then continuously cultured in FBS-free medium overnight. The medium was replaced with DMEM complete with 10% FBS and cells were pre-treated with various concentrations of PZH for 1 h followed by stimulation with 10 ng/ml of IL-6 for the indicated periods of time in different experiments as described below.

Viability of HT-29 cells was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HT-29 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well in 0.1 ml medium. Cells were treated with PZH and/or IL-6 as described above for 24 h. MTT (100 μl) (0.5 mg/ml in PBS) was added to each well, and the samples were incubated for an additional 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 μl DMSO. The absorbance was measured at 570 nm using an ELISA reader (Model ELX800; BioTek, Winooski, VT, USA).

HT-29 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium. After treatment with PZH and/or IL-6 as described above for 24 h, cells were harvested and diluted in 2 ml fresh medium without PZH and IL-6, and then reseeded into 6-well plates at a density of 1×10³ cells/well. Following incubation for 8 days in a 37°C humidified incubator with 5% CO₂, formed colonies were fixed with 10% formaldehyde, stained with 0.01% crystal violet and counted. Cell survival was calculated by normalizing the survival of the control cells as 100%.

HT-29 cells were seeded into 12-well plates at a density of 1×10⁵ cells/well in 1 ml medium. After treatment with PZH and/or IL-6 as described above for 24 h, cell apoptosis was evaluated by Hoechst staining kit according to the manufacturer’s instructions. Briefly, at the end of treatment, cells were fixed with 4% polyoxymethylene and then incubated in Hoechst solution for 5 min in the dark. The staining images were recorded using a phase-contrast fluorescent microscope (Olympus, Japan). The images were captured at a magnification of ×400.

HT-29 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium. After cells were treated with PZH and/or IL-6 as described above for 24 h, total-RNA was isolated with TRIzol reagent. Oligo(dT)-primed RNA (1 μg) was reverse-transcribed with SuperScript II reverse transcriptase according to the manufacturer’s instructions. The obtained cDNA was used to determine the mRNA amount of Bcl-2 and cyclin D1 by PCR. GAPDH was used as an internal control.

HT-29 cells were seeded into 6-well plates at a density of 2×10⁵ cells/well in 2 ml medium. Cells were treated with PZH and/or IL-6 as described above. IL-6 stimulation was performed for 15 min for pSTAT3 detection, or 24 h for examination of SOCS3, Bcl-2 and cyclin D1 protein expression. Treated cells were lysed with mammalian cell lysis buffer containing protease and phosphatase inhibitor cocktails. The lysates were resolved in 12% SDS-PAGE gels and electroblotted. The PVDF membranes were blocked with 5% skimmed milk and probed with primary antibodies against phosphor-specific STAT3 (Tyr⁷⁰⁵), SOCS3, Bcl-2, cyclin D1 and β-actin (1:1,000) overnight at 4°C and then with the appropriate HRP-conjugated secondary antibody followed by enhanced chemiluminescence detection.

HT-29 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well in 0.1 ml complete DMEM until ~50% confluency and then continuously cultured in FBS- and antibiotics-free medium overnight. Cells were transfected with a mixture of inducible STAT3-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct using Lipofectamine LTX with PLUS™ reagent. Six hours after transfection the medium was changed back into DMEM complete with FBS, penicillin and streptomycin. After 24 h of transfection, cells were treated with various concentrations of PZH for 1 h followed by IL-6 for another 24 h. Cell extracts were prepared and analyzed using Promega Dual Luciferase Reporter Assay System according to the manufacturer’s instructions. The measured firefly luciferase activity was normalized to the activity of Renilla luciferase in the same well.

Data were analyzed using the SPSS package for Windows (Version 11.5). Statistical analysis of the data was performed with the Student’s t-test and one-way ANOVA. Differences with P<0.05 were considered statistically significant.

Several cultured human cancer cell lines including HT-29 do not express constitutively phosphorylated STAT3 in vitro; we therefore stimulated STAT3 activation with IL-6 in HT-29 cells. We first determined STAT3 activation by performing western blotting to examine its phosphorylation level using an antibody that recognizes phosphorylated STAT3 (pSTAT3) at Tyr⁷⁰⁵. As shown in Fig. 1, stimulation with 10 ng/ml of IL-6 for 15 min significantly increased the level of pSTAT3 in HT-29 cells, which, however, was profoundly inhibited by PZH in a dose-dependent manner. The levels of non-phosphorylated STAT3 remained unchanged after the treatment with IL-6 and/or PZH. To further confirm the inhibitory effect of PZH on the activation of STAT3, we performed Dual Luciferase Reporter Assay to examine STAT3 transcriptional activity. Results in Fig. 2 showed that PZH significantly and dose-dependently inhibited IL-6-stimulated increase of STAT3 transcriptional activity. Taken together, our data suggest that PZH is potent in inhibiting IL-6-mediated STAT3 activation in human colon carcinoma cells.

The effect of PZH on HT-29 cell viability in the presence of IL-6 was determined by MTT assay. As shown in Fig. 3A, although IL-6 stimulation increased the viability of HT-29 cells to 115% compared to control cells (P<0.05), treatment with 0.25–1 mg/ml of PZH for 24 h decreased the viability of IL-6-stimulated cells from 60 to 22% (P<0.05 vs. PZH-untreated cells). To further verify these results, we examined the effect of PZH on HT-29 cell survival using a colony formation assay. As shown in Fig. 3B, treatment with 0.25, 0.5 and 1 mg/ml of PZH for 24 h dose-dependently reduced the survival rate of IL-6-stimulated cells by 27, 73 and 81% (P<0.05). Collectively, these data demonstrate that PZH inhibits HT-29 cell proliferation in the presence of IL-6 stimulation.

Cell apoptosis was evaluated by observing nuclear morphological changes by staining the cell nuclei with DNA-binding dye Hoechst. As shown in Fig. 4, PZH-treated cells showed condensed chromatin and fragmented nuclear morphology that are typical apoptotic morphological features, whereas the untreated cell nuclei showed homogenous staining and were less intense than PZH-treated cells, suggesting that PZH promotes HT-29 cell apoptosis in the presence of IL-6 stimulation.

To further investigate the underlying mechanism of PZH’s activities, we performed RT-PCR and western blot analyses to examine the effect of PZH on the expression of the pro-proliferative cyclin D1 and the anti-apoptotic Bcl-2, two important target genes of the STAT3 signaling pathway. The results in Fig. 5 show that the mRNA and protein expression of cyclin D1 and Bcl-2 was clearly increased by IL-6 stimulation. However, PZH treatment profoundly inhibited IL-6-induced upregulation of cyclin D1 and Bcl-2 expression, at both the transcriptional and translational levels.

We next investigated the effect of PZH on another STAT3 transcriptional target, the SOCS3 protein, a critical negative feedback inhibitor of the IL-6/STAT3 pathway. As shown in Fig. 5B, IL-6 stimulation induced SOCS3 expression, which is consistent with previous studies (39,40). Notably, PZH treatment further increased the protein expression of SOCS3, suggesting that PZH suppresses the IL-6/STAT3 pathway in HT-29 cells partially via promoting SOCS3 expression.