Wogonoside exerts growth-suppressive effects against T acute lymphoblastic leukemia through the STAT3 pathway

Abstract

Wogonoside is the main flavonoid of the traditional Chinese medicinal herb Scutellaria baicalensis Georgi and has been found to induce growth suppression in myelogenous leukemia cells. However, its activity in T acute lymphoblastic leukemia (T-ALL) is still unclear. In this study, T-ALL cell lines MOLT-3 and Jurkat were exposed to different concentrations of wogonoside for 48 h, and cell viability, cell cycle distribution, and apoptosis were measured. The involvement of signal transducers and activators of transcription 3 (STAT3) signaling in the activity of wogonoside was checked. The in vivo effect of wogonoside on T-ALL growth was investigated in a xenograft mouse model. Wogonoside significantly inhibited the viability of MOLT-3 and Jurkat cells, with the IC₅₀ (the half maximal concentration) of 68.5 ± 3.8 and 52.6 ± 4.3 μM, respectively. However, healthy T lymphocytes were unaffected. Wogonoside-treated Jurkat cells exhibited a G1-phase cell cycle arrest and significant apoptosis, which was coupled with inactivation of STAT3 signaling. Overexpression of constitutively active STAT3 reversed wogonoside-mediated growth suppression and apoptosis and restored the expression of cyclin D1, Mcl-1, and Bcl-xL. In vivo studies demonstrated that wogonoside retarded tumor growth and suppressed STAT3 phosphorylation in Jurkat xenografts. In conclusion, wogonoside suppresses the growth of T-ALL through the STAT3 pathway and may have therapeutic benefits in this disease.

Introduction

T acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy arising from T cells. T-ALL accounts for 15–20% of childhood ALL cases. ¹ Chemotherapy is the primary treatment option for most leukemic patients. Despite a high response rate to modern intensive chemotherapy, relapse occurs in about 25% of children with T-ALL. ² Constitutive activation of signal transducers and activators of transcription 3 (STAT3) signaling is associated with the pathogenesis of T-ALL. ^3,4 Inhibition of STAT3 activity has been shown to exert growth-suppressive effects against T-ALL cells. ^5,6 Therefore, STAT3 signaling represents a potential therapeutic target for T-ALL.

Natural compounds are gaining considerable attention as a new source of anticancer drugs. ⁷ Wogonoside is the main flavonoid compound extracted from Scutellaria baicalensis Georgi, a traditional Chinese medicinal herb. ⁸ Wogonoside exhibits multiple biological properties including anticancer, ⁹ anti-inflammatory, ¹⁰ and antifibrotic ¹¹ activities. Several lines of evidence indicate that wogonoside has the capacity to induce growth suppression in acute and chronic myelogenous leukemia cells. ^12,13 However, its activity in T lymphoblastic leukemia is still unknown.

In this work, we aimed to explore the biological effects of wogonoside on T-ALL cell growth both in vitro and in vivo. To gain insight into the mechanism for the action of wogonoside, we checked if the STAT3 pathway was involved.

Materials and methods

Cell culture and wogonoside treatment

Jurkat and MOLT-3 cells were obtained from the Shanghai Institute of Cell Biology of Chinese Academy of Sciences (Shanghai, China). Normal T lymphocytes were magnetically isolated from peripheral blood of healthy volunteers after informed consent. The study protocol was approved by the Ethical Committee of Sichuan Provincial People’s Hospital, Chengdu, China. Cells were allowed to grow in RPMI-1640 medium containing 10% fetal calf serum (Sigma-Aldrich, St. Louis, Missouri, USA). Wogonoside (≥98% in purity) was purchased from ChemFaces (Wuhan, China) and dissolved in dimethyl sulfoxide (DMSO). For wogonoside treatment, ^12,13 Jurkat and MOLT-3 cells were exposed to wogonoside at a final concentration ranging from 20 μM to 120 μM for 48 h and examined for cell viability, cell cycle distribution, apoptosis, and gene expression.

Cell viability analysis

Cells were seeded onto 96-well plates (5 × 10³ cells per well) and cultured in fresh media (0.2 mL) containing different concentrations of wogonoside for 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide solution (0.5 mg/mL; Sigma-Aldrich) was added and incubated for 4 h at 37°C. Absorbance at 570 nm was recorded. Absorbance readings were plotted against the concentrations of wogonoside and the IC₅₀ value was determined from the graph.

Cell cycle and apoptosis analysis

Cells were seeded onto 24-well plates (4 × 10⁴ cells per well) and incubated with 120 μM of wogonoside in 1 mL of fresh media for 48 h. The cells were trypsinized and fixed with ice-cold 70% ethanol. For cell cycle analysis, cells were incubated with propidium iodide solution containing RNase A (Sigma-Aldrich) for 30 min in the dark. Apoptosis was detected using the Annexin V fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGEN Biotechnology, Nanjing, China). Stained cells were analyzed with a flow cytometer.

Western blot analysis

Cells were washed and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Pierce, Rockford, Illinois, USA) on ice for 30 min. Cells were centrifuged at 12,000 × g for 10 min to remove cell debris. Xenograft tumor samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors and spun at 12,000 × g for 10 min to eliminate cell debris. Protein concentrations were determined using a bicinchoninic acid assay (BCA) protein assay reagent kit (Pierce). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed to separate proteins. The primary antibodies were as follows: anti-cyclin D1, anti-Bcl-xL, anti-Mcl-1, anti-phospho-STAT3 (Tyr705), anti-STAT3, and anti-β-actin antibodies (Abcam, Cambridge, Massachusetts, USA). Membranes were individually incubated with the primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Pierce). Proteins were visualized using the enhanced chemiluminescence system (Pierce) and quantified by densitometry using Quantity One software (Bio-Rad Laboratories, Hercules, California, USA).

STAT3 transcriptional activity

STAT3 transcriptional activity was determined using STAT3 reporter assay, as described previously. ¹⁴ In brief, cells were seeded onto 24-well plates (3 × 10⁴ cells per well) and transfected with an STAT3 reporter plasmid pLucTKS3 (1 μg) and an internal control plasmid pRL-TK (20 ng) using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). Twenty-four hours later, transfected cells were treated with 120 μM of wogonoside in fresh media (1 mL) for additional 48 h. The cells were lysed and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA).

Overexpression of STAT3

A plasmid expressing constitutively active STAT3 (CA-STAT3) was obtained from Addgene Inc. (Cambridge, Massachusetts, USA). Cells were seeded onto 24-well plates (3 × 10⁴ cells per well) and transfected with the CA-STAT3 plasmid (1 μg) or empty vector for 24 h and then exposed to 120 μM of wogonoside in fresh media (1 mL) for 48 h. The cells were then tested for cell viability, apoptosis, and gene expression.

Animal experiments

Male athymic nude BALB/c mice (4–5 weeks old) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). The mice were subcutaneously injected Jurkat cells (5 × 10⁶ cells/mouse) to form palpable tumors (100 mm³). The tumor-bearing animals were randomly assigned into two groups (n = 5 for each group): control group (0.1% DMSO) and wogonoside group. Wogonoside (80 mg/kg) in 100 μL of DMSO ¹² was administered by intraperitoneal injection every 3 days for 18 days. Tumor volume was measured for each mouse. At the 18th day after the initial treatment, mice were killed and tumors were excised. One part of tumor samples was processed for immunostaining for Ki-67 using anti-Ki-67 antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA). The other part was subjected to Western blot analysis of STAT3 phosphorylation. The animal experiments were approved by the Ethics Committee for the Use and Care of Laboratory Animals from Sichuan Provincial People’s Hospital (Chengdu, China).

Statistical analysis

Data are expressed as mean ± standard error of the mean. Statistical significance was determined by one-way analysis of variance followed by Tukey’s test. A p value of <0.05 was considered statistically significant.

Results

Wogonoside inhibits the viability of T-ALL cells

Two human T-ALL cell lines MOLT-3 and Jurkat were treated with different concentrations of wogonoside for 48 h and cell viability was determined. Compared to vehicle-treated MOLT-3 cells, wogonoside treatment led to a concentration-dependent reduction of the viability of MOLT-3 cells (Figure 1(a)). The IC₅₀ for wogonoside in MOLT-3 cells was 68.5 ± 3.8 μM. Similar findings were detected in Jurkat cells, with the IC₅₀ of 52.6 ± 4.3 μM (Figure 1(b)). However, the viability of healthy T lymphocytes was not affected by wogonoside up to the maximal concentration used (Figure 1(c)).

Figure 1.

Wogonoside inhibits the viability of T-ALL cells. (a) MOLT-3, (b) Jurkat, and (c) normal T lymphocytes were treated with indicated concentrations of wogonoside for 48 h and cell viability was measured. *p < 0.05 versus vehicle-treated cells. T-ALL: T acute lymphoblastic leukemia.

Wogonoside induces cell cycle arrest and apoptosis in Jurkat cells

Cell cycle analysis demonstrated that treatment with 120 μM wogonoside for 48 h resulted in an increase of G1-phase cells (72.6 ± 2.7% versus 55.4 ± 1.8%) and a decrease of S-phase (13.3 ± 0.9% versus 23.7 ± 1.0%) and G2/M-phase (14.1 ± 0.9% versus 20.8 ± 1.1%) cells in Jurkat cells, compared to vehicle controls (Figure 2(a)). Wogonoside treatment also caused a significant G1 arrest in MOLT-3 cells (data not shown). Apoptosis analysis revealed that wogonoside-treated Jurkat cells showed a 3.8-fold more apoptotic cells than vehicle-treated cells (p < 0.05; Figure 2(b)). To confirm the effects of wogonoside on cell cycle progression and apoptosis, we examined several regulatory proteins involved in cell cycle and apoptosis. The results showed that wogonoside exposure significantly decreased the expression of cyclin D1, Mcl-1, and Bcl-xL, compared to control cells (Figure 2(c)).

Figure 2.

Wogonoside induces cell cycle arrest and apoptosis in Jurkat cells. (a) Analysis of cell cycle distribution in Jurkat cells treated with 120 μM wogonoside or vehicle for 48 h. Left, representative histograms from three independent experiments. (b) Detection of apoptosis in Jurkat cells with the same treatments as shown in (a). Left, representative dot plots of apoptosis assays involving annexin V and PI staining. (c) Western blot analysis of indicated proteins. Left, representative blots from three independent experiments. *p < 0.05 versus vehicle-treated cells. PI: propidium iodide.

Wogonoside suppresses T-ALL cell viability via inactivation of STAT3 signaling

Next, we asked whether the anticancer activity of wogonoside is associated with suppression of STAT3 signaling. As shown in Figure 3(a), there was a significant decline in the levels of phosphorylated STAT3 in MOLT-3 and Jurkat cells, compared to vehicle controls. Moreover, the STAT3-dependent transcriptional activity was inhibited by wogonoside (Figure 3(b)). Rescue experiments with CA-STAT3 indicated that CA-STAT3 transfection significantly attenuated the growth suppression and apoptosis induction by wogonoside in Jurkat cells (Figure 3(c)), which was coupled with increased expression of cyclin D1, Mcl-1, and Bcl-xL (Figure 3(d)). These results suggest that inhibition of STAT3 signaling contributes to the anticancer activity of wogonoside in T-ALL cells.

Figure 3.

Wogonoside suppresses T-ALL cell viability via inactivation of STAT3 signaling. (a) Western blot analysis of STAT3 phosphorylation in T-ALL cells after treatment with wogonoside for 48 h. Top, representative blots from three independent experiments. (b) MOLT-3 and Jurkat cells were transfected with an STAT3 reporter plasmid pLucTKS3 and an internal control plasmid pRL-TK for 24 h and treated with 120 μM wogonoside for another 48 h. Luciferase activities were measured. *p < 0.05 versus vehicle-treated cells. (c) and (d) Jurkat cells were transfected with the CA-STAT3 plasmid or empty vector for 24 h and then exposed to 120 μM of wogonoside for additional 48 h. Cell viability (top) and apoptosis (bottom) in (c) were determined. (d) Western blot analysis of indicated proteins. *p < 0.05 versus control; ^# p < 0.05 versus wogonoside-treated, vector-transfected cells. T-ALL: T acute lymphoblastic leukemia; STAT3: signal transducers and activators of transcription 3.

Wogonoside inhibits tumor growth in T-ALL xenografts

Finally, we investigated the in vivo effect of wogonoside on the growth of T-ALL xenograft tumors in nude mice. As shown in Figure 4(a), wogonoside treatment significantly retarded the growth of Jurkat xenografts. At the 18th day after the initial treatment, the tumor volume was decreased by about 60% in the wogonoside treatment group, relative to the control group (305 ± 28 mm³ vs. 743 ± 45 mm³; p < 0.05). Immunohistochemistry for Ki-67 further demonstrated that the Ki-67 proliferation index was significantly lower in the tumors treated with wogonoside than that treated with vehicle (p < 0.05; Figure 4(b)). Western blot analysis confirmed the lower levels of phosphorylated STAT3 in wogonoside-treated tumors, compared with vehicle-treated tumors (Figure 4(c)). These observations confirm the therapeutic potential of wogonoside in T-ALL growth.

Figure 4.

Wogonoside inhibits tumor growth in T-ALL xenografts. (a) Tumor growth curves were determined for Jurkat xenograft tumors treated with wogonoside (80 mg/kg) or vehicle every 3 days for 18 days. (b) Immunohistochemical staining for Ki-67. (c) Western blot analysis of STAT3 phosphorylation in Jurkat xenograft tumors treated with wogonoside (80 mg/kg) or vehicle for 18 days. *p < 0.05 versus vehicle. T-ALL: T acute lymphoblastic leukemia; STAT3: signal transducers and activators of transcription 3.

Discussion

It has been documented that wogonoside has antiproliferative activity in human myelogenous leukemia cells. ^12,13 In several types of solid tumor cells such as breast cancer cells ¹⁵ and osteosarcoma cells, ¹⁶ wogonoside also shows growth-suppressive activities. In this study, we extended the observation to T-ALL cells and showed that wogonoside treatment caused a concentration-dependent reduction of T-ALL cell viability. However, the viability of normal T lymphocytes was not suppressed by wogonoside, suggesting that this compound was selectively cytotoxic to malignant cells. The factors determining the susceptibility to wogonoside are still unclear. Wogonoside has been shown to cause a cell cycle arrest in different types of cancer cells. ^12,16,17 In U937 and HL-60 acute myeloid leukemia cells, wogonoside exposure led to a G1 cell cycle arrest. ¹² However, in osteosarcoma ¹⁶ and hepatocellular carcinoma cells, ¹⁸ wogonoside was found to arrest cells at the G2/M phase. Our data showed that wogonoside treatment increased the G1 cell fraction and decreased the S and G2/M cell fraction, indicating a G1 cell cycle arrest. Therefore, the cytotoxic effect of wogonoside seems not to be cell cycle specific. In addition to induction of cell cycle arrest, wogonoside exposure significantly caused apoptosis in T-ALL cells. In vivo xenograft studies confirmed the in vitro findings that wogonoside has growth-suppressive activity in T-ALL cells. It has been documented that Scutellaria baicalensis exhibits antiproliferative and proapoptotic activity in ALL, lymphoma, and myeloma cells. ¹⁹ Baicalin, as a bioactive component of Scutellaria baicalensis, has been shown to trigger apoptotic response in leukocytes of children with acute lymphocytic leukemia. ¹⁸ Our data provide evidence that the antileukemia activity of Scutellaria baicalensis is also partially ascribed to wogonoside.

Mechanistically, wogonoside impaired the activation of STAT3 in T-ALL cells and interfered with its transcriptional activity. Overexpression of CA-STAT3 significantly abrogated the antiproliferative and proapoptotic activities of wogonoside, suggesting that the anticancer effects of wogonoside on T-ALL are causally linked to inactivation of STAT3 signaling. Multiple downstream genes of STAT3 are involved in cell proliferation and survival, such as cyclin D1, Bcl-xL, and Mcl-1. ²⁰ Our data demonstrated that wogonoside treatment decreased the expression of cyclin D1, Bcl-xL, and Mcl-1 in T-ALL cells, which was reversed by delivery of CA-STAT3. Cyclin D1 is a key regulator of cell cycle G1/S phase progression and its downregulation contributes to induction of cell cycle arrest. ²¹ Both Bcl-xL and Mcl-1 are well known as antiapoptotic proteins. ²² Downregulation of these genes by wogonoside provides a molecular explanation for its anticancer properties in T-ALL.

However, it should be noted that delivery of CA-STAT3 failed to completely reverse the wogonoside inhibition of T-ALL cell proliferation and survival. Therefore, it is possible that some other signaling pathways may also be involved in the activity of wogonoside. Indeed, wogonoside has shown the ability to suppress the PI3K/Akt/mTOR ¹¹ and MAPK-mTOR ¹⁵ pathways in different biological settings.

In conclusion, wogonoside exerts cytotoxic effect against T-ALL cells by inducing G1 cell cycle arrest and apoptosis. Inhibition of STAT3 signaling partially accounts for the growth-suppressive properties of wogonoside in T-ALL. Our findings warrant further investigation of the therapeutic potential of wogonoside for T-ALL.