• Open Access

Novel small molecule, XZH-5, inhibits constitutive and interleukin-6-induced STAT3 phosphorylation in human rhabdomyosarcoma cells

Authors

  • Aiguo Liu,

    1. Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio
    2. Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
    3. Department of Pediatrics, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
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  • Yan Liu,

    1. Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio
    2. Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
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    • Present address: Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA.

  • Zhenghu Xu,

    1. Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio
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  • Wenying Yu,

    1. Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio
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  • Hong Wang,

    1. Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio
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  • Chenglong Li,

    1. Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio
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  • Jiayuh Lin

    Corresponding author
    1. Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio
    2. Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA
    3. Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, Ohio, USA
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To whom correspondence should be addressed. E-mail: lin.674@osu.edu

Abstract

Signal transducers and activators of transcription 3 (STAT3) signaling is constitutively activated in many types of human cancers and cancer cell lines and represents a promising target for cancer therapy. We previously reported that the STAT3 signaling pathway is constitutively activated in human rhabodomyosarcoma cell lines (RH28, RH30 and RD2). We also demonstrated that inhibition of the STAT3 pathway led to apoptosis in human rhabdomyosarcoma cells. In the present study, we investigated the inhibitory effects of a novel small molecule, XZH-5, on the STAT3 signaling pathway in human rhabdomyosarcoma cells. XZH-5 was designed based on STAT3 structure, and our idea was to design peptide mimics to bind to the phosphorylated Tyr705 site and the side pocket. We found that XZH-5 downregulated STAT3 phosphorylation. The inhibition of STAT3 by XZH-5 was confirmed by the inhibition of STAT3 DNA binding ability and the downregulation of STAT3 downstream genes, such as Bcl-2, Bcl-xL, Cyclin D1 and Survivin; we also demonstrated that blockade of STAT3 phosphorylation in human rhabdomyosarcoma cells with XZH-5 caused apoptosis and suppressed colony-forming ability and cell migration. In addition to reducing constitutive STAT3 phosphorylation, XZH-5 also exhibited the potency to block interleukin-6 (IL-6)-induced STAT3 phosphorylation and nuclear translocation but did not inhibit the stimulation of STAT1 phosphorylation by interferon (IFN)-γ. Our findings indicate that XZH-5 has the potential for targeting human rhabdomyosarcoma cells expressing constitutive STAT3. (Cancer Sci 2011; 102: 1381–1387)

Signal transducers and activators of transcription 3 (STAT3) signaling pathways are activated in response to cytokines and growth factors.(1,2) STAT3 has been classified as an oncogene because activated STAT3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice.(3) In contrast, STAT3-deficient fibroblasts were shown to be resistant to transformation by a variety of oncogenes.(4,5) Constitutive STAT3 signaling participates in oncogenesis by stimulating cell proliferation, mediating immune evasion, promoting angiogenesis and conferring resistance to apoptosis induced by conventional therapies.(6–10) The possible molecular mechanisms of STAT3 promoted oncogenesis might be through upregulation of its downstream target genes, Bcl-2, Survivin, Cyclin D1, Bcl-xL, and others.(3,6,11) Blocking signaling to STAT3 by dominant negative STAT3 (dnSTAT3) mutant, STAT3 small interfering RNA or antisense STAT3 oligonucleotides inhibits cell growth, demonstrating that STAT3 is crucial to the survival and growth of tumor cells and can serve as a therapeutic target for cancer.(11–14) Furthermore, studies using normal mouse fibroblasts demonstrated that disrupting STAT3 signaling has a much less profound effect in normal human and murine cells,(15–19) suggesting that blocking STAT3 signaling might not be grossly toxic.

Osteosarcoma, rhabdomyosarcoma and other soft tissue sarcomas are childhood and adult cancers and their molecular mechanisms are not fully understood. We previously reported that STAT3 is detected in osteosarcoma, rhabdomyosarcoma and other soft tissue sarcoma tissues as well as cell lines; targeting STAT3 using dnSTAT3 suppresses cell growth and induces apoptosis through caspase cleavage pathways in osteosarcoma and rhabdomyosarcoma cell lines.(20) In the present study, we evaluated the inhibitory effects of a STAT3 model-based novel compound, XZH-5, on STAT3 in human rhabdomyosarcoma cells. XZH-5 suppressed STAT3 phosphorylation in a dose-dependent fashion and led to apoptosis. We also demonstrated that IL-6-induced STAT3 phosphorylation, not IFN-γ-induced STAT1 phosphorylation, was blocked by the treatment of XZH-5, indicating that XZH-5 selectively inhibited STAT3. Therefore, XZH-5 might be a lead compound for further developing STAT3 inhibitors.

Materials and Methods

Cell culture.  The human rhabdomyosarcoma cell lines RD2, RH28, RH30 and SMS-CTR were gifts from Dr Peter Houghton at Nationwide Children’s Hospital. Normal fibroblast cell line WI-38 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were maintained in Dulbecco’s modification of Eagle’s medium (DMEM; MediaTech Inc., Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were maintained at 37°C with 5% CO2.

RT-PCR.  RNA was extracted using a RNeasy kit (Qiagen, Valencia, CA, USA). Reverse transcription was done using a Omniscript reverse transcription kit (Qiagen). Polymerase chain reaction (PCR) amplification was performed under the following conditions: 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C with a final extension of 10 min at 72°C.

Western blot analysis.  Cells were lysed in cold radio-immunoprecipitation assay buffer containing proteasome inhibitor cocktail and phosphatase inhibitor cocktail. The lysates were resolved by SDS-PAGE and transferred to a PVDF membrane. Membranes were probed with specific primary antibody and HRP conjugated secondary antibody. Antibodies against phosphorylated STAT3 (Tyr705, p-STAT3Y705), STAT3, phosphorylated STAT1 (Tyr701, p-STAT1Y701), STAT1, phosphorylated JAK1 (Y1022/1023, p-JAK1), JAK1, phosphorylated JAK2 (Y1007/1008, p-JAK2), JAK2, phosphorylated AKT (Ser473, p-AKT), phosphorylated ERK 1/2 (Thr202/Tyr204, p-ERK1/2), Cleaved poly (ADP-ribose) polymerase (PARP), Cleaved Caspase-3, GAPDH, IL-6, IFN-γ and secondary antibody were from Cell Signaling Technology (Beverly, MA, USA).

DNA binding assay.  DNA binding was performed using a STAT3 DNA binding ELISA kit (Active Motif, Carlsbad, CA, USA). Cells were seeded in a 10-cm plate and treated as indicated in the text. Then nuclear protein was extracted and mixed with a STAT3-specific DNA probe. The complex of protein and DNA was then transferred into an ELISA assay plate. After the incubation of primary and secondary antibodies, the developing solution was added to the well. The stop solution (1% SDS) was added after the color was well developed. Absorbance was read at 450 nm.

Immunofluorescence.  Cells were seeded on a glass slide and pretreated with XZH-5 for 2 h followed by IL-6 for 30 min. After the treatment, cells were fixed with cold methanol for 15 min at −20°C. The slide was then blocked in PBS buffer with 5% normal goat serum and 0.3% Triton X-100 for at least 1 h at room temperature, incubated with specific primary antibody overnight at 4°C, and Alexa Fluor 594 secondary antibody (red) (Molecule Probe, Invitrogen, Eugene, Oregon, USA) for 1 h at room temperature. Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were captured by Leica Microsystems (Bannockburn, IL, USA).

Cell viability assay.  Cell viability was measured using a CyQUANT NF kit (Molecule Probe, Invitrogen). Cells were seeded into a 96-well plate. After specific treatment, as indicated in the text, the medium was removed and 100 μL of dye solution was added to each well. The plate was incubated at 37°C for 30 min, and the fluorescence was measured at an excitation/emission wavelength range of 485/530 nm.

Apoptosis assay.  Apoptosis was performed using caspase 3/7 assay (Promega, Madison, WI, USA). Cells were seeded into a 96-well plate. After the treatment, 100 μL of Apo-One Caspase 3/7 reagent was added to the well. The plate was incubated at 37°C for 30 min, and fluorescence was measured at an excitation/emission wavelength range of 485/530 nm.

Colony formation assay.  To determine the long-term effects, cells were seeded in DMEM with 10% FBS for 24 h, then pretreated with XZH-5, Stattic or dimethyl sulfoxide (DMSO) at 60–80% confluent for 2 h. After being digested, 500 cells per 100 mm dish were seeded in fresh medium to grow for 14 days to form colonies, which were then stained with crystal violet (10 g/L; Fisher Scientific, Fair Lawn, NJ, USA). The colony numbers were counted.

Wound healing assay.  When cells were 100% confluent, the monolayer was scratched using a pipette tip. The cells were then treated with XZH-5, Stattic or DMSO for 2 h. After 48-h culture without treatment, images were captured by Leica Microsystems.

Small molecular compounds.  XZH-5 was synthesized in Dr Hong Wang’s laboratory. Stattic was purchased from Calbiochem (San Diego, CA, USA).

Statistical analysis.  Statistical significance was calculated by Student’s t-test. A P-value of <0.05 was considered significant. * and ** indicates < 0.05 and < 0.01, respectively.

Results

Inhibition of STAT3 phosphorylation by Stattic.  To explore whether blocking STAT3 phosphorylation in human rhabdomyosarcoma cells expressing persistently phosphorylated STAT3 would lead to an increase in apoptosis and a decrease in cell viability, we treated RD2, RH28 and RH30 cells with Stattic, a previously reported STAT3 inhibitor.(21) After the treatment, we examined p-STAT3Y705, STAT3, Cleaved PARP and Cleaved Caspase-3. Treatment with Stattic decreased p-STAT3Y705 and induced Cleaved PARP and Cleaved caspase-3 (Fig. S1A). We also performed a cell viability assay to determine the inhibitory effects of Stattic on cell viability. The results showed that blocking p-STAT3Y705 with Stattic effectively decreased cell viability (Fig. S1B).

Development of the novel STAT3 inhibitor.  Phosphorylated Tyr705 (pTyr705) in STAT3 is required for STAT3 dimerization. Persistent tyrosine phosphorylation is therefore required for constitutive activation of STAT3 in cancer cells.(3) Consequently, blockade of Tyr705 phosphorylation in STAT3 can inhibit cancer cell growth and cause apoptosis. Our idea was to design peptide mimics to bind to the pTyr705 site and the side pocket (Fig. 1A,B). We used urea linker to capture H-bonds because the space between the two sites is rich in H-bond acceptors and donors. We designed 10 compounds and found that XZH-5 fits the idea best: (i) carboxylate mimics the phosphate of pTyr705; (ii) fluorobenzene has decent hydrophobic interaction with the side pocket; and (iii) a combination of urea and peptidyl linkers offers the right distance. The synthetic steps are described in Figure 1(C).

Figure 1.

 Structure and synthesis of XZH-5. (A) XZH-5 chemical structure. (B) XZH-5 and signal transducers and activators of transcription 3 (STAT3) interaction model. XZH-5 docked to STAT3 SH2 binding sites (pTyr705 site and the side pocket). (C) XZH-5 synthesis.

XZH-5 inhibits STAT3 phosphorylation and induces apoptosis.  To examine whether XZH-5 might inhibit STAT3 phosphorylation in rhabdomyosarcoma cells expressing persistently activated STAT3, RD2, RH28 and RH30 cells were treated with two different concentrations of XZH-5. Phosphorylated STAT3 was reduced in a XZH-5 dose-dependent fashion (Fig. 2A). In addition to phosphorylated STAT3, phosphorylated ERK, phosphorylated AKT, phosphorylated JAK1 and phosphorylated JAK2 were investigated in XZH-5-treated cells. The results showed that all these molecules were not affected (Fig. S2A). To investigate whether the downregulation of p-STAT3 might disrupt the DNA binding ability of STAT3, we performed a STAT3 DNA binding assay in XZH-5-treated RH30 cells. Our result showed that the STAT3 DNA binding ability decreased in XZH-5-treated cells compared with DMSO-treated controls (Fig. 2B). To further analyze whether the treatment of XZH-5 might affect the expression of STAT3 downstream genes, we looked at the mRNA levels of Bcl-2, Bcl-xL, CyclinD1 and Survivin in XZH-5-treated cells. In all three cell lines, XZH-5 visibly reduced the expression of STAT3 targeted genes over the DMSO control (Fig. 2C).

Figure 2.

 XZH-5 reduces signal transducers and activators of transcription 3 (STAT3) phosphorylation. (A) RD2, RH28 and RH30 cells were treated with different concentrations of XZH-5 for 8 h. p-STAT3 and STAT3 were analyzed using western blot. (B) RH30 cells were treated with 25 μM of XZH-5 for 8 h, and then the STAT3 DNA binding ability was evaluated by ELISA. (C) RD2, RH28 and RH30 cells were treated with XZH-5 for 8 h. The mRNA expression of Bcl-2, Bcl-xL, CyclinD1, Survivin and GAPDH was analyzed by RT-PCR.

We also observed that decreases in STAT3 phosphorylation were concomitant with increased cleavage of PARP and Caspase-3 (Fig. 3A). In addition to the increased levels of Cleaved Caspase-3, the activity of Caspase-3/7 was also enhanced in XZH-5-treated cells (Fig. 3B), leading to decreased cell viability (Fig. 3C). However, the levels of Cleaved PARP and Caspase-3, the activity of caspase 3/7 and cell viability were not affected in SMS-CTR cells exhibiting little phosphorylated STAT3 (Fig. 3A–C).

Figure 3.

 XZH-5 treatment decreases cell viability. (A) RD2, RH28, RH30 and SMS-CTR cells were treated with XZH-5 for 8 h. Cleaved PARP and Cleaved Caspase-3 were analyzed by western blot. (B) Caspase-3/7 activity was measured in XZH-5-treated RD2, RH28, RH30 and SMS-CTR cells. The data represent three independent results. (C) RD2, RH28, RH30 and SMS-CTR cells were treated with XZH-5 for 8 h. Cell viability was measured in XZH-5 treated cells. The data represent three independent results.

XZH-5 inhibits STAT3 phosphorylation and nuclear translocation induced by IL-6.  Interleukin-6 has been shown to induce STAT3 phosphorylation and might play a role in cancer development.(22–25) First of all, we found that SMS-CTR cells had lower levels of IL-6 than RD2, RH28 and RH30 cells, whereas all four cell lines expressed IL-6R and gp130 (Fig. 4A). We then examined whether XZH-5 might inhibit IL-6-induced STAT3 activation. SMS-CTR cells were pretreated with XZH-5 for 2 h followed by treatment with IL-6 (50 ng/mL, 30 min). IL-6 stimulated STAT3 phosphorylation, whereas XZH-5 pretreatment blocked IL-6-induced STAT3 activation (Fig. 4B). Compared with the inhibitory effects on IL-6-induced STAT3 phosphorylation, XZH-5 did not affect IFN-γ-induced STAT1 phosphorylation (Fig. 4C). These results suggest that XZH-5 selectively inhibits IL-6-induced STAT3 phosphorylation. Furthermore, XZH-5 also blocked IL-6-induced STAT3 nuclear translocation (Fig. 4D).

Figure 4.

 XZH-5 inhibits IL-6-induced signal transducers and activators of transcription 3 (STAT3) phosphorylation and nuclear translocation. (A) The mRNA expression of IL-6, IL-6R and gp130 in RD2, RH28 and RH30 cells was examined by RT-PCR. (B,C) SMS-CTR cells were pretreated with XZH-5 for 2 h, followed by 50 ng/mL of IL-6 (B) or IFN-γ (C) for 30 min. p-STAT3 (B) and p-STAT1 (C) were analyzed using western blot. (D) SMS-CTR cells were treated as described in (B). The distribution of STAT3 was analyzed by immunofluorescence.

XZH-5 reduces colony formation and migration.  To investigate whether XZH-5 treatment might inhibit colony formation, we treated RD2, RH28 and RH30 cells with XZH-5 for 2 h. After the treatment, the same numbers of viable cells were seeded and cultured in fresh medium without XZH-5 for 2 weeks. As shown in Figure 5, XZH-5 treatment remarkably reduced colony formation.

Figure 5.

 XZH-5 reduces colony-forming ability. RD2, RH28 and RH30 cells were treated with XZH-5 for 2 h. After the treatment, living cells were counted and the same numbers of cells were seeded and cultured for 2 weeks. Colonies were fixed by ice-cold methanol and stained with 1% crystal violet. The data represent three independent results.

STAT3 has been shown to be involved in wound healing and migration of cancer cells, which might lead to invasion and metastasis.(26,27) We evaluated whether XZH-5 might affect cell migration. When cells were 100% confluent, the monolayer was scratched using a pipette tip. The cells were then treated with XZH-5 or DMSO for 2 h. After 48 h, we observed that XZH-5 treatment reduced migration ability (Fig. 6A), whereas cell viability was not reduced significantly (Fig. 6B).

Figure 6.

 XZH-5 inhibits cell migration. (A) When cells were 100% confluent, the monolayer was scratched using a pipette tip. The cells were then treated with XZH-5 or DMSO for 2 h. After the treatment, fresh medium was added and the cells were cultured for 48 h. (B) Cells were treated as indicated in (A), and cell viability was measured.

XZH-5 shows higher potency than Stattic in human rhabdomyosarcoma cell lines and low toxicity against normal fibroblasts.  Because XZH-5 is a new structure-based small molecule, we compared the inhibitory effects and toxicity of XZH-5 with another STAT3 inhibitor, Sttatic. We demonstrated that at the same dose (25 μM), XZH-5 induced more caspase 3 activation and cell death (Fig. S2B,C). XZH-5 is more potent than Stattic regarding cell migration and colony-forming ability (Fig. S2D,E). We also performed cell viability and caspase 3/7 activity assays in XZH-5- or Stattic-treated normal fibroblasts (WI-38), showing that both XZH-5 and Stattic have minimal toxicity against normal fibroblasts (Fig. S3).

Discussion

Human rhabdomyosarcoma is one of the most common soft-tissue sarcomas in children with a high relapse rate of 30%,(28,29) and the mortality rate of relapsing rhabdomyosarcoma is still more than 50%.(29) Therefore, development of high potency drugs is urgently needed to improve the survival rate for patients with rhabdomyosarcoma.

STAT3 is persistently activated in many types of human cancer and cancer cell lines, which represents a valid target for drug design. However, few studies investigated STAT3 in human sarcomas. Whether STAT3 signaling plays a role in the survival and growth of osteosarcomas, rhabdomyosarcomas and other soft-tissue sarcomas is not fully understood.

We previously analyzed sarcoma tissues and cell lines and found STAT3 phosphorylation is upregulated in approximately 20% of osteosarcoma, 27% of rhabdomyosarcoma and 15% of other soft-tissue sarcoma tissues, as well as in sarcoma cell lines. We also found that blockade of STAT3 with a dominant-negative STAT3 or a STAT3 small molecule inhibitor (STA-21(30)) in sarcoma cells decreased cell growth and caused apoptosis.(20) Recently, we reported that LLL12 and FLLL32 can inhibit STAT3 activity in human rhabdomyosarcoma cells and caused apoptosis.(31) We found similar results when treating the cells with Stattic (Fig. S1). Therefore, STAT3 might be a promising target for human rhabdomyosarcoma.

In the present study, we demonstrated that XZH-5, a newly developed small molecule, could inhibit STAT3 phosphorylation in rhabdomyosarcoma cell lines expressing constitutive STAT3. Inhibition of STAT3 by XZH-5 was confirmed by the downregulated STAT3 DNA binding ability and STAT3 downstream genes, such as Bcl-2, Bcl-xL, Cyclin D1 and Survivin. Meanwhile, treatment with XZH-5 induced apoptosis and reduced cell viability, as well as colony-forming ability and cell migration.

In addition to inhibiting constitutively activated STAT3, the pretreatment with XZH-5 could also block IL-6-induced STAT3 phosphorylation. IL-6 is a major mediator of inflammation and STAT3 activator and might prevent apoptosis to keep cells alive under very toxic conditions. Unfortunately, the same signaling might also protect cancer cells from chemotherapeutic agents.(32) IL-6 has been detected in the serum of patients with a variety of cancers, as well as in many cancer cell lines.(33–36) We have demonstrated IL-6-induced anti-apoptotic effects in human hepatocellular carcinoma cells, which can be reversed by blocking the IL-6/STAT3 pathway.(37) In the present study, we showed that three out of four rhabdomyosarcoma cell lines expressed IL-6, and all four cell lines expressed IL-6 receptor and gp130. Exogenous IL-6 induced STAT3 phosphorylation and nuclear translocation in rhabdomyosarcoma cells expressing lower IL-6 and phosphorylated STAT3, whereas the XZH-5 pretreatment could block this process. To explore whether XZH-5 selectively targeted STAT3 phosphorylation, we also examined IFN-γ-induced STAT1 phosphorylation. Our results showed XZH-5 did not affect the activity of this tumor suppressor. Compared with LLL12 and FLLL32 that we published previously,(31,34) solubility of XZH-5 in ethanol is much better, although we still used DMSO as a solvent.

In addition to phosphorylated STAT3, phosphorylated ERK, phosphorylated AKT, phosphorylated JAK1 and phosphorylated JAK2 were also investigated in XZH-5-treated cells. Our results showed that all of these molecules were not affected, indicating that XZH-5 is more selective to STAT3 phosphorylation. We also compared the inhibitory effects and toxicity of XZH-5 with another STAT3 inhibitor, Sttatic, because XZH-5 is a new structure-based small molecule. Figure S2(B,C) shows that at the same dose (25 μM), XZH-5 induced more caspase 3 activation and cell death. Our results also showed that XZH-5 is more potent than Stattic regarding cell migration and colony-forming ability. Moreover, XZH-5 did not show more toxicity than Stattic against normal fibroblasts (WI-38) (Fig. S3). Although several STAT3 inhibitors have been discovered, few of them were designed based on STAT3 structure. For example, AG490 blocks STAT3 phosphorylation by suppressing JAK2 phosphorylation.(38) Sorafenib is a multi-kinase inhibitor.(39) FLLL31 and FLLL32 are JAK2/STAT3 inhibitors.(40)

In conclusion, XZH-5 has potency in inhibiting STAT3 activation in human rhabdomyosarcoma cells expressing phosphorylated STAT3. Furthermore, in an in vivo mouse tumor model, pharmacodynamic and pharmacokinetic studies should be conducted to explore the future clinical potential of XZH-5.

Disclosure Statement

The authors have no conflict of interest.

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