Investigational New Drugs

, Volume 30, Issue 3, pp 916–926 | Cite as

Small molecules, LLL12 and FLLL32, inhibit STAT3 and exhibit potent growth suppressive activity in osteosarcoma cells and tumor growth in mice

  • Grace-Ifeyinwa Onimoe
  • Aiguo Liu
  • Li Lin
  • Chang-Ching Wei
  • Eric B. Schwartz
  • Deepak Bhasin
  • Chenglong Li
  • James R. Fuchs
  • Pui-kai Li
  • Peter Houghton
  • Amanda Termuhlen
  • Thomas Gross
  • Jiayuh Lin
PRECLINICAL STUDIES

Summary

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.

Keywords

STAT3 Osteosarcoma Small molecule inhibitors 

Introduction

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 [6]. 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 [10].

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 [15]. 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 [18], and FLLL32, a JAK2/STAT3 inhibitor [19], 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).

Western blotting

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).

Primer sequences and source information can be found in supplemental Table 1.
Table 1

IC50 values (μM) of LLL12 and other STAT3/JAK2 inhibitors in human osteosarcoma cells

 

LLL3

LLL12

FLLL32

WP1066

Stattic

S3I-201

AG490

U2OS

16.53

0.33

0.53

2.81

1.39

49.08

52.12

SAOS2

1.64

0.43

0.54

5.25

2.58

49.1

2.38

SJSA

16.60

0.76

0.77

4.38

2.40

>60

>60

LLL12 and FLLL32 inhibit tumor cell viability. The half-maximal inhibitory concentrations (IC50) were obtained for STAT3 inhibitors in osteosarcoma cells. All values reflect concentrations calculated following 72 h of treatment in an MTT cell viability assay

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.

Mouse xenografts

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

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.

Results

LLL12 and FLLL32 inhibit constitutive STAT3 phosphorylation and induce apoptosis in osteosarcoma cell lines

STAT3 activity is ultimately dependent upon phosphorylation of residue Y705. The ability of LLL12 and FLLL32 to inhibit STAT3 activation in osteosarcoma cells was investigated. Western blot analysis with a phospho-Y705-specific STAT3 antibody was used in osteosarcoma cell lines U2OS, SAOS2, and SJSA. LLL12 and FLLL32 effectively reduced levels of Y705-phosphorylated STAT3 (hereafter referred to as P-STAT3) in each osteosarcoma cell line. The effect of LLL12 was concentration dependent, and FLLL32 (10 μM) significantly inhibited P-STAT3 (Fig. 1a, b, and c). The expressions of STAT3 downstream target genes such as Cyclin D1 (4, 34, 35) were likewise downregulated (Fig. 1a). Decreases in P-STAT3 were concomitant with increased cleavage of pro-caspase-3. We also found that LLL12 and FLLL32 did not induce detectable cleavage of caspase-3 in normal human skeletal muscle cells (HSMM), in which STAT3 is not constitutively activated (Fig. 1d). We observed that the phosphorylation of ERK was increased by LLL12 and FLLL32 treatments in U2OS and SAOS2 cells. However, at the present time, we do not know the exact molecular mechanism(s) by which STAT3 inhibition by LLL12 and FLLL32 induce ERK phosphorylation in U2OS and SAOS2 osteosarcoma cell lines.
Fig. 1

LLL12 and FLLL32 inhibit constitutive STAT3 phosphorylation and induce tumor cell apoptosis. a, b, c Three osteosarcoma cell lines (U2OS, SAOS2, and SJSA) were treated with LLL12 (5 μM–10 μM) or FLLL32 (10 μM) for 24 h and then whole-cell extracts were prepared and phosphor-STAT3 was detected by western blot. There was a decrease in the levels of expression of STAT3 phosphorylation. Apoptosis is indicated by the induction of cyclin D1 or cleaved caspase-3. d LLL12 and FLLL32 did not induce detectable cleavage of caspase-3 in normal human skeletal muscle cells (HSMM) in which STAT3 is not constitutively activated. e Flow cytometry analyses of SJSA and SAOS2 cells treated with LLL12 (10 μM) or FLLL32 (10 μM) for 24 h; results are depicted in bar graphs that show a two- to six-fold increase in apoptotic cells compared to the control (*p < 0.05)

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

As mentioned above, STAT3 binding to target genes induces the transcription of several proliferation and anti-apoptotic associated proteins. Several STAT3 downstream target genes such as Cyclin D1, Bcl-2, Bcl-xL, and Survivin [20, 21, 22] were downregulated by LLL12, compared to the DMSO control-treated cell population of osteosarcoma cell lines (Fig. 2a and b). FLLL32 downregulated Bcl-XL, Bcl-2, Cyclin D1 in SAOS2 cells (Fig. 2a) and Bcl-2, Cyclin D1 and Survivin in SJSA cells (Fig. 2b). The reduction of these downstream target genes provides a molecular mechanism for LLL12 and FLLL32 inhibition in osteosarcoma cells.
Fig. 2

LLL12 and FLLL32 suppress STA3-regulated gene products involved in proliferation, survival, and angiogenesis. a–b Osteosarcoma cell lines (SAOS2 and SJSA) were treated with LLL12 (10 μM) or FLLL32 (10 μM) for 24 h. Reverse transcriptase PCR reveals decreased expression of STAT3 target genes over a DMSO control and negative preparation (did not contain RNA) following treatment with LLL12 and FLLL32. Cyclin D1, Bcl-2, Bcl-xL, and Survivin expressions were downregulated by LLL12 (10 μM) compared to a DMSO control in osteosarcoma cell lines. FLLL32 (10 μM) downregulated Bcl-XL, Bcl-2, and Cyclin D1 in SAOS2 cells (a) and Bcl-2, Cyclin D1, and Survivin in SJSA cells (b). The reduction of these downstream target genes provides a molecular mechanism of LLL12 and FLLL32 inhibition in osteosarcoma cells

LLL12 and FLLL32 suppress IL-6 induced STAT3 phosphorylation

Activation of STAT3 can be induced by IL-6 [12, 13]. Osteosarcoma cells (SJSA) were used to determine if LLL12 was capable of inhibiting IL-6 induced STAT3 phosphorylation. We found that the treatment with LLL12 inhibited the induction of STAT3 phosphorylation by IL-6 (Fig. 3a). FLLL32 was also able to inhibit the induction of STAT3 phosphorylation by IL-6, and its action was found to be more potent than curcumin (Fig. 3a). The specificity of LLL12 and FLLL32 actions on the STAT3 pathway was shown via their inability to inhibit IFN-γ mediated STAT1 phosphorylation (Fig. 3b).
Fig. 3

LLL12 and FLLL32 inhibit STAT3 phosphorylation induced by IL-6. STAT1 IFN-γ induced phosphorylation was not inhibited by LLL12 or FLLL32. a SJSA cells were serum starved overnight and then were treated with LLL12 (5 μM–10 μM), FLLL32 (10 μM–20 μM), Curcumin (10 μM–20 μM), or DMSO. After 4 h, the untreated and treated cells were stimulated by IL-6 (50 ng/mL). The cells were harvested after 30 min and analyzed by Western blot. LLL12 and FLLL32 action is selective for STAT3. b SJSA cells were treated with LLL12 (5 μM–10 μM) or FLLL32 (10–20 μM) for 4 h. Subsequently, the cells were stimulated with IFN-γ. Whole-cell extracts were then prepared and analyzed for phosphor-STAt1 by western blotting. IFN-γ induced phosphorylation was not inhibited by LLL12 or FLLL32

LLL12 and FLLL32 suppress focus formation assay in osteosarcoma cell lines

To determine the focus formation activity of cells treated with LLL12 or FLLL32, a focus formation assay was performed. U2OS, SAOS2, and SJSA cells were treated for 5 h with LLL12 or FLLL32 and the focus formation was then compared to the DMSO-treated control. Results displayed in Fig. 4a, b, and Table 2, show that a concentration-dependent inhibition of focus formation occurred.
Fig. 4

LLL12 and FLLL32 inhibit focus formation. a–b U2OS, SAOS2, and SJSA cells were treated with LLL12 (5 uM–10 uM) or FLLL32 (5–10 uM) for 5 h. The cells were then harvested. A cell density of 500 mm3 was then re-plated in several dishes for each concentration and allowed to grow for 2 weeks. Crystal violet staining was then performed, showing a decrease in the ability of U2OS, SAOS2, and SJSA cells to form colonies in comparison to a DMSO control

Table 2

LLL12 and FLLL32 reduced the colony forming ability of osteosarcoma cells

Cell line

Drug

Dose

Numbers of colony

U2OS

DMSO

 

536

LLL12

2.5 μM

0

5 μM

0

10 μM

0

FLLL32

5 μM

8

10 μM

0

SAOS2

DMSO

 

224

LLL12

2.5 μM

0

5 μM

0

10 μM

0

FLLL32

5 μM

0

10 μM

0

SJSA

DMSO

 

332

LLL12

2.5 μM

0

5 μM

0

10 μM

0

FLLL32

5 μM

7

10 μM

0

The colony numbers were counted 2 weeks post treatment, with LLL12 or FLLL32 in osteosarcoma cells

LLL12 and FLLL32 inhibit wound healing that occurs via cell migration

Cell migration is important in physiologic processes, such as wound healing and tumor metastasis. Wound healing and cancer are both characterized by cell proliferation, the remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and the modulation of blood coagulation [23]. It has been shown that STAT3 regulates a common set of genes involved in wound healing and cancer [23]. To assess the effects of LLL12 and FLLL32 on cell migration, a wound healing assay was performed. Following the creation of a wound, cells were treated with various concentrations of LLL12 or FLLL32. The drugs were removed after 4 h. Cells were allowed to migrate into the denuded area for 24 h. Treatment with LLL12 or FLLL32 at a concentration of 2.5 μM or higher caused a significant decrease in cell migration in U2OS, SAOS2, and SJSA cells (Fig. 5a and b). The results of the 4 h MTT cell viability assay show that inhibition of cell migration by LLL12 may be due to a combination of the inhibition of cell migration and cell growth at the doses of 5 μM and 10 μM (Fig. 5a). The inhibition of cell viability by FLLL32 is minimal (Fig. 5b); hence the inhibition of cell migration in these cell lines is unlikely to be due to the inhibition of cell viability.
Fig. 5

LLL12 and FLLL32 inhibit wound healing that occurs via cell migration. a–b A wound was created in U2OS, SAOS2, and SJSA cells at 100% confluency and was then treated with LLL12 (5 uM–10 uM) and FLLL32 (5 uM–10 uM) for 4 h. After the wound in the DMSO treated cells had closed, wound healing/cell migration was then analyzed in the treated cells assay, showing that LLL12 and FLLL32 have a significant impact on SAOS2, SJSA, and U2OS osteosarcoma cell migration. The cells’ ability to migrate is increasingly inhibited by an increase in doses of LLL12 and FLLL32. MTT cell viability studies conducted over a 4 h period show that LLL12 may exert cell inhibition via a combination of inhibition of cell migration and cell viability (a), while the inhibition of cell migration by FLLL32 is unlikely to be due to the inhibition of cell viability (b)

LLL12 and FLLL32 suppress tumor growth in vivo

In order to examine the potential of using LLL12 and FLLL32 for osteosarcoma therapy, we next tested the inhibition of tumor growth in vivo. After inoculation of SJSA or OS-33 tumor cells and tumor development, treatment groups were given daily intraperitoneal doses of 50 mg/kg FLLL32 or 5 mg/kg LLL12. DMSO was given to vehicle control groups. The administration of LLL12 or FLLL32 resulted in a significant reduction in tumor volume and tumor mass in the OS-33 (*p < 0.05) and SJSA xenografted mice, relative to the vehicle (Fig. 6a, b, and c) and (Fig. 7a, b, and c). Western blots performed with SJSA tumor tissue samples harvested from these mice at the end of the experiment (2 h after the last dose of drug inhibitors) also showed decreases in total levels of P-STAT3 by LLL12 and FLLL32 (Fig. 7c).
Fig. 6

The effect of LLL12 and FLLL32 on tumor growth in mouse xenografts with OS-33 osteosarcoma cells. a, b, and c After the development of tumors in the OS-33 mouse xenograft, daily intraperitoneal dosages of 5 mg/kg LLL12, 50 mg/kg FLLL32, or DMSO were injected intraperitoneally on a daily basis for a period of 2 weeks (*p < 0.5)

Fig. 7

The effect of LLL12 and FLLL32 on mouse xenografts with SJSA osteosarcoma cells. a–b Following tumor development, LLL12 (5 mg/kg), FLLL32 (50 mg/kg), or DMSO were injected intraperitoneally into the SJSA murine mice model on a daily basis for 2 weeks (*p < 0.5). c Tumor tissue samples from the mice analyzed by Western blotting showed a decrease of P-STAT3

Discussion

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 [9]. 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 [18] 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 [19]. 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.

Notes

Acknowledgements

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.

Supplementary material

10637_2011_9645_MOESM1_ESM.tif (92 kb)
ESM Table 1(TIF 91.7 kb)

References

  1. 1.
    Damron TA, Ward WG, Stewart A (2007) Osteosarcoma, chondrosarcoma and Ewing’s sarcoma. Clin Orthop Relat Res 459:40–47. doi:10.1097/Blo.0b013e318059b8c9 PubMedCrossRefGoogle Scholar
  2. 2.
    Abate ME, Longhi A, Galletti S, Ferrari S, Bacci G (2010) Non-metastatic osteosarcoma of the extremities in children aged five years or younger. Pediatr Blood Cancer 55(4):652–654. doi:10.1002/Pbc.22567 PubMedCrossRefGoogle Scholar
  3. 3.
    Buettner R, Mora LB, Jove R (2002) Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 8(4):945–954PubMedGoogle Scholar
  4. 4.
    Bromberg J, Darnell JE Jr (2000) The role of STATs in transcriptional control and their impact on cellular function. Oncogene 19(21):2468–2473PubMedCrossRefGoogle Scholar
  5. 5.
    Turkson J, Jove R (2000) STAT proteins: novel molecular targets for cancer drug discovery. Oncogene 19(56):6613–6626PubMedCrossRefGoogle Scholar
  6. 6.
    Burke W, Jin X, Liu R, Huang M, Reynolds RK, Lin J (2001) Inhibition of constitutively active Stat3 pathway in ovarian and breast cancer cells. Oncogene 20:7925–7934PubMedCrossRefGoogle Scholar
  7. 7.
    Chen C, Loy A, Cen L, Chan C, Hsieh F, Cheng G, Wu B, Qualman S, Kunisada K, Yamauchi-Takihara K, Lin J (2007) Signal transducer and activator of transcription 3 is involved in cell growth and survival of human rhabdomyosarcoma and osteosarcoma cells. BMC Cancer 7:111PubMedCrossRefGoogle Scholar
  8. 8.
    Fossey SL, Liao AT, McCleese JK, Bear MD, Lin J, Li PK, Kisseberth WC, London CA (2009) Characterization of STAT3 activation and expression in canine and human osteosarcoma. BMC Cancer 9:81. doi:10.1186/1471-2407-9-81 PubMedCrossRefGoogle Scholar
  9. 9.
    Bromberg J, Wrzeszczynska M, Devgan G, Zhao Y, Pestell R, Albanese C, Darnell JJ (1999) Stat3 as an oncogene. Cell 98(3):295–303PubMedCrossRefGoogle Scholar
  10. 10.
    Chen CL, Hsieh FC, Lieblein JC, Brown J, Chan C, Wallace JA, Cheng G, Hall BM, Lin J (2007) Stat3 activation in human endometrial and cervical cancers. Br J Cancer 96(4):591–599. doi:10.1038/sj.bjc.6603597 PubMedCrossRefGoogle Scholar
  11. 11.
    Bowman T, Garcia R, Turkson J, Jove R (2000) STATs in oncogenesis. Oncogene 19(21):2474–2488PubMedCrossRefGoogle Scholar
  12. 12.
    Kaptein A, Paillard V, Saunders M (1996) Dominant negative stat3 mutant inhibits interleukin-6-induced Jak-STAT signal transduction. J Biol Chem 271(11):5961–5964PubMedCrossRefGoogle Scholar
  13. 13.
    Faruqi T, Gomez D, Bustelo X, Bar-Sagi D, Reich N (2001) Rac1 mediates STAT3 activation by autocrine IL-6. Proc Natl Acad Sci U S A 9014–9019Google Scholar
  14. 14.
    Real PJ, Sierra A, De Juan A, Segovia JC, Lopez-Vega JM, Fernandez-Luna JL (2002) Resistance to chemotherapy via Stat3-dependent overexpression of Bcl-2 in metastatic breast cancer cells. Oncogene 21(50):7611–7618PubMedCrossRefGoogle Scholar
  15. 15.
    Wang T, Niu G, Kortylewski M, Burdelya L, Shain K, Zhang S, Bhattacharya R, Gabrilovich D, Heller R, Coppola D, Dalton W, Jove R, Pardoll D, Yu H (2004) Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells. Nat Med 10(1):48–54PubMedCrossRefGoogle Scholar
  16. 16.
    Inghirami G, Chiarle R, Simmons WJ, Piva R, Schlessinger K, Levy DE (2005) New and old functions of STAT3: a pivotal target for individualized treatment of cancer. Cell Cycle (Georgetown, Tex) 4(9):1131–1133CrossRefGoogle Scholar
  17. 17.
    Ling X, Arlinghaus RB (2005) Knockdown of STAT3 expression by RNA interference inhibits the induction of breast tumors in immunocompetent mice. Cancer Res 65(7):2532–2536PubMedCrossRefGoogle Scholar
  18. 18.
    Lin L, Hutzen B, Li P, Ball S, Zuo M, DeAngelis S, Foust E, Sobo M, Friedman L, Bhasin D, Cen L, Li C, Lin J (2010) A novel small molecule, LLL12, inhibits STAT3 phosphorylation and activities and exhibits potent growth-suppresive activity in human cancer cells. Neoplasia 12(1):39–50. doi:10.1593/neo.91196 PubMedGoogle Scholar
  19. 19.
    Lin L, Hutzen B, Zuo M, Ball S, Deangelis S, Foust E, Pandit B, Ihnat MA, Shenoy SS, Kulp S, Li PK, Li C, Fuchs J, Lin J (2010) Novel STAT3 phosphorylation inhibitors exhibit potent growth-suppressive activity in pancreatic and breast cancer cells. Cancer Res 70(6):2445–2454. doi:10.1158/0008-5472.CAN-09-2468 PubMedCrossRefGoogle Scholar
  20. 20.
    Yu H, Jove R (2004) The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 4(2):97–105PubMedCrossRefGoogle Scholar
  21. 21.
    Frank DA (2007) STAT3 as a central mediator of neoplastic cellular transformation. Cancer Lett 251:199–210PubMedCrossRefGoogle Scholar
  22. 22.
    Kanda N, Seno H, Konda Y, Marusawa H, Kanai M, Nakajima T, Kawashima T, Nanakin A, Sawabu T, Uenoyama Y, Sekikawa A, Kawada M, Suzuki K, Kayahara T, Fukui H, Sawada M, Chiba T (2004) STAT3 is constitutively activated and supports cell survival in association with surviving expression in gastric cancer cells. Oncogene 23(28):4921–4929PubMedCrossRefGoogle Scholar
  23. 23.
    Dauer DJ, Ferraro B, Song L, Yu B, Mora L, Buettner R, Enkemann S, Jove R, Haura EB. Stat3 regulates genes common to both wound healing and cancerGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Grace-Ifeyinwa Onimoe
    • 1
    • 6
  • Aiguo Liu
    • 1
    • 2
  • Li Lin
    • 1
    • 3
  • Chang-Ching Wei
    • 4
  • Eric B. Schwartz
    • 5
  • Deepak Bhasin
    • 5
  • Chenglong Li
    • 5
  • James R. Fuchs
    • 5
  • Pui-kai Li
    • 5
  • Peter Houghton
    • 1
    • 6
  • Amanda Termuhlen
    • 6
  • Thomas Gross
    • 6
  • Jiayuh Lin
    • 1
    • 6
    • 7
  1. 1.Center for Childhood CancerThe Research Institute at Nationwide Children’s HospitalColumbusUSA
  2. 2.Department of Pediatrics, Tongji HospitalHuazhong University of Science and TechnologyWuhanChina
  3. 3.Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
  4. 4.Department of PediatricsChina Medical University HospitalTaichungTaiwan
  5. 5.Division of Medicinal Chemistry and Pharmacognosy, College of PharmacyOhio State UniversityColumbusUSA
  6. 6.Department of Pediatrics, College of MedicineOhio State University and Nationwide Children’s HospitalColumbusUSA
  7. 7.Department of Pediatrics, Center for Childhood CancerThe Research Institute at Nationwide Children’s HospitalColumbusUSA

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