Small molecules, LLL12 and FLLL32, inhibit STAT3 and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice
Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in osteosarcoma, and hence, may serve as a therapeutic target. In order to target STAT3, we tested two new STAT3 inhibitors, LLL12 and FLLL32. LLL12 and FLLL32 inhibit STAT3 phosphorylation and STAT3 downstream targets. LLL12 and FLLL32 also inhibit IL-6 induced STAT3 phosphorylation. The inhibition of STAT3 by LLL12 and FLLL32 resulted in the induction of apoptosis, reduction of plating efficiency, and migration in osteosarcoma cells. Furthermore, LLL12 and FLLL32 inhibited SJSA osteosarcoma cells and OS-33 tumor growth in murine xenografts. These results provide evidence that constitutive STAT3 signaling is required for osteosarcoma survival and migration in vitro and tumor growth in vivo. Blocking persistent STAT3 signaling by LLL12 and FLLL32 may be a novel therapeutic approach for osteosarcoma.
KeywordsSTAT3 Osteosarcoma Small molecule inhibitors
Osteosarcoma is the most common malignant bone tumor in children and young adults with approximately 750–900 new cases diagnosed annually in the United States (American Cancer Society). It generally presents as a painful mass that has a mixed lytic and sclerotic appearance on radiography and is characterized histologically by the presence of malignant cells that produce osteoid or immature bone. The treatment of osteosarcoma has not changed significantly over the last 20 years and consists of a combination of chemotherapy and aggressive surgical resection of the primary tumor and all sites affected by the metastasis of the disease. Unfortunately, the improvement in outcomes seen for many other cancers has not been demonstrated in osteosarcoma, with 50%–60% five-year disease free survival rates for patients with localized disease and less than 20% for those with metastasis at diagnosis [1, 2]. Also, because the chemotherapy regimens consist of high doses of anthracycline and cisplatin, they carry a significant risk of long-term toxicities, such as cardiotoxicity and ototoxicity. It is clear that there is a need for new and more effective therapy for osteosarcoma to help improve outcomes for these patients.
The Signal Transducers and Activators of Transcription (STAT) proteins family is a group of related proteins that play a role in relaying signals from cytokines and growth factors [3, 4, 5]. Ligand dependent activation of STAT3 regulatory cascade is often associated with the modulation of cell growth and differentiation. Hence, the abnormal activation of STAT proteins is becoming more frequently associated with unrestricted cell growth and malignant transformation . Constitutively activated STAT3 has been described in human and canine osteosarcoma cell lines [7, 8]. STAT3 is classified as a proto-oncogene because an activated form of STAT3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice [4, 9]. Constitutive STAT3 signaling may participate in oncogenesis by stimulating cell proliferation, promoting angiogenesis, mediating immune evasion, and conferring resistance to apoptosis induced by conventional therapies .
STAT3 activation occurs when the Tyrosine 705 (Tyr705) residue is phosphorylated, leading to dimerization and translocation from the cytoplasm to the nucleus [11, 12, 13]. In the nucleus, STAT3 binds with the promoters of downstream target genes and induces the transcription and up-regulation of proliferation and anti-apoptotic associated proteins [4, 9, 11, 14]. Therefore, constitutive STAT3 signaling is involved in stimulating cell cycle progression and preventing apoptosis, which both contribute to malignant progression [4, 11]. In addition, persistently activated STAT3 plays a role in impairing both the innate and adaptive immune responses by enhancing immunologic tolerance and enabling cancer cells to evade immune surveillance . Furthermore, the survival of these tumor cells appears to depend on the presence of STAT3 signaling [3, 16].
The implications of constitutive STAT3 signaling in tumors have presented it as a possible target for cancer treatment. Experiments aimed at blocking STAT3 signaling using dominant-negative STAT3, RNA interference, and STAT3 antisense oligonucleotides have provided further evidence of the potential of STAT3 as a target for treating cancer [3, 5, 17]. Inhibiting STAT3 using the stated approaches has been successful, resulting in the inhibition of growth and the induction of death in tumors. It was also determined that, in normal cells, blocking STAT3 is neither harmful nor toxic to the cells [3, 16]. Given the oncogenic functions of STAT3 and the promise of inhibiting it, directly targeting STAT3 signaling represents a potential therapeutic approach to treating cancer.
Two newly developed compounds, LLL12 and FLLL32, were evaluated for the ability to inhibit STAT3 phosphorylation (Tyr705) and STAT3 activities, down-regulate STAT3 downstream targets, inhibit proliferation, colony formation, cell migration, and induce apoptosis in osteosarcoma cells.Tumor xenografts were also used to demonstrate the anti-tumor growth effects of LLL12 and FLLL32 in mice.
Materials and methods
STAT3 inhibitors and chemicals
LLL12, a STAT3 inhibitor , and FLLL32, a JAK2/STAT3 inhibitor , were synthesized in Dr. Pui-Kai Li and Dr James Fuchs’s laboratory (College of Pharmacy, Ohio State University). Curcumin was obtained from Sigma-Aldrich (St. Louis MO). LLL3 and WP1066 were obtained from Dr Pui-Kai Li’s laboratory. St S3I-201 and AG490 were obtained from Cal biochem EMD4Biosciences (San Diego, CA). The chemicals MTT, Tris, glycine, Nacl, SDS, Cremaphor, Solubilization solution (N, N-dimethylformamide), and DMSO were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). The annexin V-FITC apoptosis detection kit was procured from BD sciences (San Jose, CA).
Cell culture and treatments
Human osteosarcoma cell lines (U2OS, SAOS2, and SJSA) and human skeletal muscle cells (HSMM) were purchased from the American type culture collection (ATCC—Manassas, VA). OS-33 xenograft from a patient specimen was kindly provided by Dr Houghton’s laboratory at the Research Institute of Nationwide Children’s Hospital. All cell lines were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum (FBS), 4.5 g/L L-glutamine, sodium pyruvate, and 1% penicillin/streptomycin. All cell lines were maintained in a humidified 37°C incubator with 5% CO2. LLL12 and FLLL32 were dissolved in sterile dimethyl sulfoxide (DMSO) to make a 20 mM stock solution. Aliquots of the stock solution were stored at −20°C until they were ready to use.
Cell viability assay
Osteosarcoma cell lines (U2OS, SAOS2, and SJSA) were seeded in 96-well plates (3,000 cells per well) and treated with 0.5–5 μmol/L of LLL12 or FLLL32 in triplicates. The cells were incubated at 37°C for 72 h. 25 μl of MTT was added to each sample and incubated for 3.5 h.
After this, 100 μl of N, N-dimethylformamide solubilization solution was added to each well.
The absorbance at 450 nm was read the following day. Half-maximal inhibitory concentrations (IC50) were determined using Sigma Plot 9.0 software (Systat Software Inc—Chicago, IL).
Flow cytometry analysis for the detection of apoptosis
FITC V Annexin staining precedes the loss of membrane integrity, which accompanies the later stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with FITC Annexin V is typically used in conjunction with a vital dye such as PI dye in order to allow early identification of early apoptotic cells. Human osteosarcoma cell lines (SAOS2 and SJSA) were separately seeded in plates at a density of 1 × 10 5 cells per well and incubated for 24 h. Cells were then treated with LLL12 or FLLL32 (10 μM), or left untreated for 24 h. The cells were then harvested and stained with FITC dye and Propidium dye (PI), following the manufacturer’s protocol (BD Biosciences). Cells were analyzed using Dodecapus flow cytometry (BD Biosciences) to identify early apoptotic cells (PI negative Annexin V positive).
Human osteosarcoma cell lines were treated with LLL12 (5 μM or 10 μM) or DMSO at 70%–90% confluence in the presence of 10% FBS for 24 h, and then lysed in cold RIPA lysis buffer containing protease inhibitors and subjected to SDS-PAGE. Membranes were probed with a 1:1000 dilution of antibodies (Cell Signaling Tech—Danvers, MA) against phospho-specific STAT3 (Tyrosine 705), phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), cleaved caspase-3, cyclin D, Bcl-2, survivin, and GAPDH. Immunoreactive bands were visualized using ECF solution and premium autoradiography film (Denville Scientific Inc- Metuchen NJ). Human Skeletal muscle cell line (HSMM) was treated with LLL12 (5 μM or 10 μM) or DMSO at 70%–90% confluence in presence of 10% FBS for 24 h, and then lysed in cold RIPA lysis buffer containing protease inhibitors and subjected to SDS-PAGE.
Membranes were probed with a 1:1000 dilution of antibodies (Cell Signaling Tech—Danvers, MA) against phospho-specific STAT3 (Tyrosine 705), STAT3, cleaved caspase-3, and GAPDH. Immunoreactive bands were visualized using ECF solution and premium autoradiography film (Denville Scientific Inc—Metuchen, NJ).
Reverse transcription PCR analysis
RNA was collected from SAOS2 and SJSA cells with RNeasy Kits (Qiagen—Valencia, CA) following 24 h of treatment with LLL12 or FLLL32. cDNA was generated from the 500 ng sample RNA using Omniscript RT (Qiagen—Valencia, CA).
IC50 values (μM) of LLL12 and other STAT3/JAK2 inhibitors in human osteosarcoma cells
IL-6 induction of STAT3 phosphorylation
SJSA osteosarcoma cells were seeded in 10 cm plates and allowed to adhere overnight. The following day, the specimen was serum starved for 24 h. The specimen was then treated with LLL12 (5 μM–10 μM) or FLLL32 (10 μM–20 μM), curcumin (10 μM–20 μM) or DMSO. After 4 h, the treated specimen was stimulated with IL-6 (50 ng/mL) or interferon gamma (IFN-γ). Cells were then harvested after 30 min and analyzed by Western blot.
Wound healing/cell migration assay
U2OS, SAOS2, and SJSA osteosarcoma cells were seeded in six-well plates. Approximately 24 h later, when the specimen became 100% confluent, the monolayer was scratched using a 1 mL pipette tip, then washed once to remove any non-adherent cells. New mediums containing LLL12, FLLL32 (2.5–10 μM), or DMSO were then added. Treatments were removed 4 h later and a fresh medium was then added. After an additional 20 h without treatment, the specimen was observed under a microscope. When the wound in the control group was closed, the inhibition of migration in treated cultures was assessed. MTT cell viability studies were also conducted over the same period of time.
Focus formation assay
Osteosarcoma cells (U2OS, SAOS2, and SJSA) with 10% FBS in DMEM were treated for 5 h with LLL12 (2.5–10 μM), FLLL32 (5 μM–10 μM), or DMSO.
The specimen was then trypsinized; viable cells were counted and then plated at a density of 500 cells per dish. The specimen was maintained at 37°C and allowed to grow for 2 weeks. Any colonies were stained using a crystal violet dye (5 ml per plate). Photomicrographs of any colonies were taken using a Leica MZ 16FA inverted microscope (Leica Microsystems—Deerfield, IL) with a 7.4 Slider Camera (Diagnostic Instruments Inc—Sterling Heights MI). Colonies were scored by counting and then numbers were normalized as a percentage of colonies formed in DMSO controls.
All animal studies were conducted in accordance with the principles and standard procedures approved by IACUC of the Research Institute at Nationwide Children’s Hospital. SJSA cells (1 × 107) in Matrigel (BD Science Franklin Lakes, NJ) were injected subcutaneously into the flank region of four to five week-old female athymic nude mice. After tumors developed, the mice were randomly sorted into three treatment groups consisting of six mice per group: DMSO vehicle group, 5 mg/kg LLL12, and 50 mg/kg FLLL32. These doses were based on the maximal tolerated dose in mice (data not shown). The inhibitors were formulated with Cremaphor, DMSO, and 5% dextrose water to enhance delivery and limit toxicity encountered with DMSO alone as the mixing base.
OS-33 xenografts were surgically transplanted into 24 female athymic mice. Following tumor engraftment, the mice were randomly sorted into three treatment groups consisting of eight mice per group and treated as above. Tumor growth was determined by measuring the major (L) and minor (W) diameter with a caliper. The tumor volume was calculated according to the formula: Tumor volume = 0.5236 × L × W2. After 14 days of treatment, tumors were harvested from dead mice, frozen in liquid nitrogen, and stored at −80°C.
Western blotting was then performed on tumor tissue homogenates to examine the expression of STAT3 phosphorylation and the induction of apoptosis in vehicle and inhibitor treated mice.
Statistical analysis was performed using an independent, one-sided t-test (Microsoft Excel 2007). Probability (p) values less than 0.05 were considered statistically significant.
LLL12 and FLLL32 inhibit constitutive STAT3 phosphorylation and induce apoptosis in osteosarcoma cell lines
LLL12 and FLLL32 induce apoptosis in osteosarcoma cell lines and inhibit cell viability
Osteosarcoma cell lines were treated for 24 h and then stained with Annexin V dye/PI dye to detect apoptosis.
Flow cytometry analyses revealed that both agents induced a two to six times increase in apoptosis over the DMSO control-treated cell population of osteosarcoma cell lines SJSA and SAOS2 (*p < 0.05) (Fig. 1e).
LLL12 or FLLL32 also shows greater potency (IC50) than the five other STAT3/JAK2 small molecule inhibitors (Table 1). This demonstrates that LLL12 or FLLL32 exhibits potent activity to inhibit cell viability of osteosarcoma cells (U2OS, SAOS2, and SJSA) when compared with LLL3, WP1066, Stattic, S3I-201, and AG490.
LLL12 and FLLL32 are potent inhibitors of STAT3-mediated gene transcription
LLL12 and FLLL32 suppress IL-6 induced STAT3 phosphorylation
LLL12 and FLLL32 suppress focus formation assay in osteosarcoma cell lines
LLL12 and FLLL32 reduced the colony forming ability of osteosarcoma cells
Numbers of colony
LLL12 and FLLL32 inhibit wound healing that occurs via cell migration
LLL12 and FLLL32 suppress tumor growth in vivo
The outcome for patients with advanced or metastatic osteosarcoma continues to be dismal, necessitating novel therapies, and STAT3 inhibitors are promising agents. STAT3 has been classified as an oncogene because activated STAT3 can mediate oncogenic transformation in cultured cells and tumor formation in nude mice . STAT3 activation results in the expression of downstream genes, which promote cell proliferation and provide resistance to apoptosis, such as cyclin D1 and Bcl-2, respectively [4, 9, 11, 14]. In its active form, STAT3 is found predominantly in the cytoplasm. Phosphorylation at Tyr-705 results in dimerization and translocation to the nucleus, where STAT3 binds to specific promoter sequences on target genes. Chen et al., 2007, showed that STAT3 phosphorylation levels were elevated in osteosarcoma, rhabdomyosarcoma, and other soft-tissue sarcoma tissues and cell lines. The mechanisms underlying the elevated STAT3 phosphorylation in sarcoma tissues are not clear. Possibilities include constant upstream activation by cytokines and growth factors, down regulation of counter balancing signal transduction pathways, such as SOCS1, or both.
In this study, the inhibitory efficacy of LLL12 and FLLL32 in human osteosarcoma cells was assessed. LLL12 is an optimal analog of LLL3  and is more potent in inhibiting STAT3 phosphorylation and cell viability (Table 1). FLLL32 is derived from the dietary agent curcumin, and is the first STAT3 inhibitor from curcumin that is designed to selectively target STAT3 SH2 . The inhibitory effects on STAT3 by LLL12 and FLLL32 were observed in human osteosarcoma cells expressing elevated levels of STAT3 phosphorylation. By inhibiting the same target, STAT3, both small molecular inhibitors demonstrate similar activity to inhibit STAT3 phosphorylation at tyrosine residue 705, which caused the decreasing cell viability, induction of apoptosis, and inhibition of colony forming ability and cell migration. LLL12 and FLLL32 are quite potent and their IC50 in three osteosarcoma cell lines are between 0.33 and 0.76 μM for LLL12, and 0.53 and 0.77 μM for FLLL32. We also found that LLL12 and FLLL32 did not induce detectable cleavage of caspase-3 in normal cells (HSMM) in which STAT3 is not constitutively activated.
We observed that the phosphorylation of ERK was increased by LLL12 and FLLL32 treatments. However, now we do not know the molecular mechanism(s) by which STAT3 inhibition may induce ERK phosphorylation in U2OS and Saos-2 osteosarcoma cell lines. Furthermore, LLL12 and FLLL32 demonstrated significant inhibition of tumor growth from both SJSA and OS-33 mice tumor xenografts. OS-33 is a xenograft derived from a primary osteosarcoma from the patient, and has never been grown in tissue culture. Therefore, using human OS-33 osteosarcoma makes this study more clinically relevant. The fact that both small molecular inhibitors LLL12 and FLLL32 hinder OS-33 tumor growth and reduce tumor mass in vivo suggests a potential therapeutic application.
In summary, LLL12 and FLLL32 should be suitable agents for targeting osteosarcoma and possibly certain types of cancer cells with constitutively activated STAT3, due to their ability to inhibit STAT3 phosphorylation and STAT3 activities as well as their potent growth suppressive activity both in vitro and in vivo. In this study, we demonstrated that STAT3 is an attractive therapeutic target in osteosarcoma. Targeting STAT3 with small molecule inhibitors such as LLL12 and FLLL32 has shown potential as a cancer therapeutic approach and deserves further exploration into the use of these inhibitors as potential agents in the treatment of osteosarcoma.
This work was supported by a pilot grant from the Experimental Therapeutics Program at the Ohio State University Comprehensive Cancer Center and a grant from the Hematology and Oncology Department at the Nationwide Children’s Hospital.
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