Glycogen synthase kinase 3 beta inhibitors induce apoptosis in ovarian cancer cells and inhibit in vivo tumor growth

Abstract

Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3β (GSK3β), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3β inhibitors (GSK3βi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, the GSK3βi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4′6-diamidino-2-phenylindole (DAPI) positive nuclear condensation, poly (ADP-ribose) polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3βi upregulated phosphorylation of the inhibitory serine residue of GSK3β in the OVCA432 and SKOV3 cell lines as well as inhibited phosphorylation of the downstream target glycogen synthase. An in vivo xenograft study using SKOV3 cells demonstrated that tumor progression was hindered by 9ING41 in vivo. The maximum tolerated dose for 9ING41 was greater than 500 mg/kg in rats. Pharmacokinetic analysis showed 9ING41 to have a bioavailability of 4.5% and was well distributed in tissues. Therefore, GSK3β inhibitors alone or in combination with existing drugs may hinder growth of serous ovarian cancers.

Introduction

Ovarian cancer kills approximately 14,000 women annually in the United States (1). Standard drug therapy consists of treatment with paclitaxel/carboplatin (2), but most women will die from recurrence in 5 years due to drug resistance. Therefore, combination therapy or alternative strategies for resistant disease are of critical importance. Inhibitors of the Wnt signaling pathway including its downstream kinase, GSK3β (glycogen synthase kinase 3 beta), offer possible options for drug therapy in resistant ovarian cancers (3).

Wnt signaling controls embryogenesis, cell differentiation, proliferation, and migration (4, 5). Wnt ligands are secreted molecules that bind to frizzled receptors. Wnt signals through both canonical and noncanonical pathways, but all pathways initially begin with one of the nineteen ligands binding to one of the ten receptors in the human genome (6). In the canonical pathway, a frizzled receptor phosphorylates GSK3β. GSK3β is a dual regulated kinase in the Wnt pathway, originally found to regulate glycogen metabolism, but now is known to affect a variety of cellular events by phosphorylation of different substrates (7). Kinase activity is dependent on phosphorylation of GSK3β at tyrosine 216 and on the contrary, inhibition of kinase activity is dependent on phosphorylation of GSK3β at serine 9 (3). Activation of GSK3β phosphorylates β-catenin and glycogen synthase. Phosphorylation of β-catenin targets it for proteolytic degradation. If β-catenin is not phosphorylated and degraded, it enters the nucleus, where it is available for transcription of the T cell factor/lymphoid enhancer-binding factor 1 pathway (TCF/LEF1). Transcriptional targets downstream of this pathway include c-myc, E-cadherin, and cyclin D1 (8). Inhibition of GSK3β kinase activity could block phosphorylation of β-catenin, a signaling event that investigators have suggested would promote cancer. However, in cancers of the peritoneal cavity including ovarian surface epithelium, colon, and pancreatic cells, GSK3β inhibitors (GSK3βi) induce apoptosis or reduce cell viability (3, 9-12).

The induction of apoptosis by GSK3βi has been reported for ovarian cancer cells, and the goal of this research was to characterize novel maleimide inhibitors for increased efficacy (13). Lithium chloride (LiCl) is a common GSK3βi, which could perturb the pathway in a non-selective manner and with relatively weak potency. Previously LiCl induced pGSK3β^ser9 expression as well as increased apoptosis in immortalized ovarian surface epithelium cells (IOSE) (14). Tumor growth was inhibited by LiCl, which suggests that potent and more selective GSK3β inhibition could be effective in treating ovarian cancer (3, 14, 15). A series of novel GSK3βi were profiled against two cancer cell lines and evaluated in vitro and in vivo for efficacy towards slowing cell and tumor growth (13).

Materials and Methods

Cell Culture and Materials

OVCA432 ovarian cancer cells (RC Bast, MD Anderson) were grown in Minimum Essential Medium (MEM) supplemented with 10% FBS, 1% L-glutamine, 1% non-essential amino acid, 1% sodium pyruvate and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). SKOV3 cells from American Type Culture Collection (ATCC) were grown in McCoy's 5A (Sigma Aldrich, St Louis, MO), 10% FBS, and 1% penicillin-streptomycin. Both cell lines were incubated at 37 °C, 5% CO₂. Both SKOV3 and OVCA432 cell lines are sensitive to cisplatin treatment (16). All GSK3β inhibitors were synthesized by Dr. Kozikowski's group at University of Illinois at Chicago as previously described (13, 17, 18). SB216763 and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich and LiCl from Fisher Science (Hanover Park, IL).

Proliferation Assays

Cells were seeded into 96 well plates at 5 × 10³ cells/100 μL in MEM media. The next day, fresh media with DMSO or test compounds in Table 1 at various concentrations were added to plates and the cells were allowed to grow for 4 days. Proliferation was measured with CellTiter 96® Aqueous One Solution (Promega, Madison, WI) according to the manufacturer. Spectrophotometric analysis was completed using a Biotek EL312e microplate reader (Fisher Biotek, Pittsburgh, PA). All conditions were tested in six replicates in triplicate experiments. The IC₅₀ value was determined as the concentration that caused 50% reduction in survival of cells.

Table 1. Inhibitory concentration required for 50% cell death of GSK3β Inhibitors in ovarian cancer cells.

Compound	Name Code	IC50 (μM) OVCA432	IC50 (μM) SKOV3
	SB216763	>50	>50
	9-ING-41	11.2	10.5
	5-ING-135	33.6	74.5
	9-ING-49	20.0	26.7
	2-ING-173	13.0	36.7
	9-ING-87	10.7	42.7
	10-ING-52	26.6	>50
	FG2-007B	14.2	>50
	FG1-022A	58.3	>50
	FG1-029	>50	46.6

Data represent concentration required to kill 50% of the cells as measured using four concentrations of inhibitors and fit using sigmoidal dose response curve with variable slope.

DAPI and TUNEL staining of ovarian cancer cells

Cells were plated at 5 × 10⁴ cells per well in chamber culture slides from BD Biosciences (Bedford, MA). The cells were allowed to attach overnight at 37 °C, and then treated with 0.1% DMSO, 50 μM LiCl, 5 μM 9ING41, or 25μM SB216763. One day later, cells were fixed with 4% paraformaldehyde and washed with phosphate buffered saline (PBS). After fixation, slides were cover slipped with Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). For TUNEL staining, the DeadEnd Colorimetric TUNEL system (Promega) was used according to the manufacturer's protocol. All cells were imaged using a 20× objective (Nikon DS-Ri1, Huntley, IL). Apoptotic cells were counted using ImageJ (National Institutes of Health). Cell death was determined by the appearance of condensed nuclear DNA and nuclear membrane fragmentation visible on the DAPI stains or positive brown TUNEL staining in 3 fields from triplicate experiments.

Western Blot

Cell lysates were collected after 24 hour treatment with 0.1% DMSO, 50μM LiCl, 5μM 9ING41, or 25μM SB216763. Cells were lysed in 150 μL lysis buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Pierce, Rockford, IL)), supplemented with Complete Mini Protease Inhibitor Cocktail tablets (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail II (Sigma). Equal protein concentrations were confirmed through a bicinchoninic acid (BCA) assay (Pierce). Samples were run under reducing conditions in a tris-glycine buffer using 10% acrylamide Bis-Tris gels. Gels were transferred to a polyvinylidene fluoride (PVDF) membrane using the iBlot dry transferring system (Invitrogen, Carlsbad, CA). Membranes were blocked for 1 hour in 5% nonfat milk in Tris buffered saline with 0.1% tween (TBS-T), except for cleaved caspase 3, which was blocked in 5% bovine serum albumin (BSA) in PBS with 0.5% tween and cyclin D1 which was blocked in 5% BSA in TBS-T. Phospho-GSK3β, phospho-glycogen synthase, cyclin D1, cleaved caspase 3, and cleaved poly-ADP ribose polymerase (PARP) antibodies were purchased from Cell Signaling Technologies (Beverly, MA). Anti-actin was purchased from Sigma. The secondary antibody was a goat anti-rabbit horseradish peroxidase (Cell Signaling Technologies). Proteins were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL) on an Alpha Innotech gel documentation system. Blots were performed in triplicate and densitometry analysis was performed using Image J.

Xenograft

Female nude (nu/nu) mice aged 6-7 weeks were ordered from Charles River Laboratories (Wilmington, MA). Mice were placed in three separate categories (n=4); DMSO/0.85% saline solvent (9:7), LiCl (340mg/kg), and 9ING41 (40mg/kg) (15). Mice were injected with 4 × 10⁶ SKOV3 cells in the rear hind limb subcutaneously. Once the tumors were palpable, drugs were injected i.p. in 100 μL volume every other day for 21 days (3, 15). Tumor volumes were measured using digital calipers and quantified using the volume calculation (π/6)*(larger diameter)*(smaller diameter)². Body weights were measured every other day. At the end of the study, tumors were excised, weighed, and fixed in paraformaldehyde.

Range-finding Toxicity Studies

Sprague-Dawley rats were used for toxicity studies performed at the Stanford Research Institute (SRI) International in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals. 9ING41 was administered orally at 100 mg/kg or 500 mg/kg to three male and three female Sprague-Dawley rats. On day 3, blood was collected for evaluation of the hematology and clinical chemistry parameters. Animals were sacrificed on day 8 and macroscopic examinations were performed.

Bioavailability and Pharmacokinetic studies

Male Sprague-Dawley rats were used for clearance studies performed at Stanford Research Institute (SRI). Blood samples (∼ 300 μL) were collected (n=3) in tubes containing ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. Samples were kept on ice and processed within 30 min of collection. Plasma was prepared by centrifuging blood samples at 2,600 rpm at 2 to 8 °C for 15 min and stored at -80 °C. The extraction method included the addition of 100 μL acetonitrile containing 100 ng/ml hexylnicotinate (internal standard) to 50 μL of plasma. Samples were vortexed for 15 min, centrifuged for 10 min at 18000g, and 100 μL of the supernatant removed to a 150 μL glass insert in a 2 mL high performance liquid chromatography (HPLC) vial for subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Pharmacokinetic analysis was performed using non-compartmental methods and WinNonlin® Professional (Version 5.2, Pharsight Corp, Mountain View, CA). The human-Eag-relaed gene potassium channel assay was performed by Pharmaron inc. (www.pharmaron.com).

Statistical Analysis

All values are expressed as the mean +/- standard error from the mean. Students t-test was used to assess differences between compound and negative control samples assuming 2-tails. P -values of less than 0.05 were considered statistically significant. GraphPad Prism 4.02 was used to calculate half maximal inhibitory concentration (IC₅₀) values.

Results

Inhibition of GSK3β Blocks Ovarian Cancer Cellular Proliferation

Nine GSK3β inhibitors were tested from chemical variants of a maleimide that were shown to have selectivity and higher inhibition of GSK3β than SB216763 using in vitro kinase assays (13). The inhibitors were screened against two serous ovarian cancer cell lines, OVCA432 and SKOV3, for their ability to slow proliferation after 96 hours. OVCA432 are a more epithelial serous cell type with cuboidal shape and mutant p53 expression, while SKOV3 are a p53 null serous cell line with fibroblastic, invasive characteristics. The IC₅₀ values for the drugs compared to the commercially available inhibitor, SB216763, are reported in Table 1. Of the novel inhibitors, four of them were consistently more active than SB216763 in both cell lines. Overall, 9ING41 was the most cytotoxic in both cell lines and was chosen as the candidate for further evaluation. Based on IC₅₀ values taken from logarithmic doses spanning 5 concentrations, the optimal concentrations for in vitro assays were determined.

Inhibition of GSK3β Induces Cellular Apoptosis

To investigate possible mechanisms for inhibition of proliferation, apoptosis analyses on OVCA432 and SKOV3 cells were performed (Figure 1A-D). LiCl and SB216763 were chosen as positive controls, and 9ING41 was used based on its potency in the cell growth assays. In OVCA432 cells 50 μM LiCl, 5 μM 9ING41, and 25 μM SB216763 induced apoptosis. In SKOV3 cells only 5 μM 9ING41 induced apoptosis compared to DMSO control. Much higher doses of LiCl have the ability to induce apoptosis as demonstrated previously (3).

Figure 1.

(A, C) Induction of cellular apoptosis by GSK3βi. OVCA432 and SKOV3 cells were treated with 0.1% DMSO, 50 μM LiCl, 5 μM 9ING41, and 25 μM SB216763 for 24 hrs and stained with DAPI. DAPI-stained cells exhibiting condensed, pyknotic, or fragmented nuclei were representative of apoptotic cells. (B, D) Representative DAPI stained OVCA432 (B) and SKOV3 (D) cells. White arrow indicates ‘healthy’ and red arrow indicates ‘apoptotic cells’. Scale bar 20μm. (E, G) OVCA432 and SKOV3 cells were treated with GSK3βi for 24 hours and then stained for TUNEL-positive apoptotic cells. TUNEL-positive cells are stained brown. (F, H) Representative TUNEL-stained OVCA (F) and SKOV3 (G) cells. Black arrow indicates ‘healthy cell’ and red arrow indicates TUNEL-positive cells. All data represent average percentage of apoptotic cells +/- SEM in three fields from three or more independent experiments. * indicates significantly different than DMSO p < 0.05. Scale bar 20μm. (I) Cleaved caspase-3 and cleaved PARP protein expression from OVCA432 and SKOV3 cell lines treated for 24 hours with GSK3βi. The densitometry value from triplicate experiments is shown below each band.

To confirm that the cells were undergoing apoptosis, TUNEL staining was performed. Similar to DAPI analysis, 9ING41 significantly increased apoptosis in OVCA432 cells as compared to the negative control, DMSO (Figure 1E-F). In addition, 9ING41 significantly increased apoptosis in SKOV3 as compared to DMSO (Figure 1G-H). To establish the mechanism of apoptosis downstream of GSK3βi, caspase-3 cleavage was measured (Figure 1I). Western blot analyses showed an increase in cleaved caspase-3 in OVCA432 and SKOV3 cells treated with the GSK3βi 9ING41. Lastly, PARP cleavage was investigated (Figure 1I). The cleaved product of PARP was detected in both cell lines when treated with 9ING41 indicating that the cells were undergoing apoptosis.

GSK3β Inhibitors Block Downstream Signal Transduction

In order to understand if inhibition of GSK3β was directly responsible for cellular death, downstream targets of GSK3β were analyzed using western blots (Figure 2). A serine 9 phospho-specific antibody for GSK3β was used to probe cell lysates collected from OVCA432 and SKOV3 after being treated with 0.1% DMSO, 50 μM LiCl, 5 μM 9ING41, and 25 μM SB216763 for 24 hours. 9ING41 increased phosphorylation of the inhibitory residue, GSK3β^ser9, in OVCA432 and SKOV3 cells when normalized to total GSK3β. GSK3β phosphorylates glycogen synthase as one of its substrates. Therefore, the phosphorylation status of glycogen synthase was evaluated after treatment with compounds. Phosphorylation of glycogen synthase was significantly inhibited by 9ING41 in both cell lines demonstrating inhibition of GSK3β. Lastly, expression of cyclin D1 was evaluated because this is a target gene of TCF/LEF transcription following inhibition of GSK3β and stabilization of β-catenin. Cyclin D1 expression increased in SKOV3 cells treated with 9ING41.

Figure 2.

Protein expression in OVCA432 and SKOV3 ovarian cancer cells after treatment with GSK3βi for 24 hours. Western blots were analyzed with antibodies specific for phospho-glycogen synthase kinase 3β (pGSK3β), phospho-glycogen synthase (pGS), and cyclin D1. GSK3β and actin antibodies were used as internal controls. Western blots were quantified with densitometry (values below each band). All data represent average from three or more experiments.

GSK3β Inhibitors Slow Ovarian Xenograft Tumor Growth

In order to validate these in vitro findings, an in vivo xenograft experiment was performed using SKOV3 cells. Animals were given an i.p. injection of either LiCl, 9ING41, or solvent (DMSO:saline) every other day for 21 days (15). SKOV3 cells were selected based on previous experiments that suggested LiCl could reduce tumor burden in nude mice and because it is the most commonly used ovarian cancer cell line for xenografts (3). Animal body weight was measured every other day and no significant differences were found suggesting limited toxicity from treatment with either drug (Figure 3A). When the tumor volume was calculated and compared to the initial volume, the 9ING41-injected animals had significantly smaller tumor volumes at 7 and 14 days suggesting that inhibition of GSK3β slows tumor growth (Figure 3B). By the end of the study, all of the tumor volume ratios were similar. The tumors were excised and analyzed for protein expression by immunohistochemistry. Paraffin embedded excised tumors were immunohistochemically stained with cleaved caspase-3 antibody. The presence of brown DAB stained nuclei mark tumor cells undergoing apoptosis (Figure 3C).

Figure 3.

(A) Average body weights of animals treated with solvent, 9ING41, and LiCl. (B) Tumor progression in a SKOV3 xenograft injected i.p. with GSK3βi. All data represent the average +/- SEM from four independent animals treated with DMSO/0.85% saline solvent (9:7), 9ING41 (40mg/kg), or LiCl (340mg/kg) every 2 days for 21 days. Tumor volumes were calculated on first day palpable and then as a ratio over time compared to initial volume. * Indicates significantly different than control p < 0.05. (C) Immunohistochemical analysis of activated cleaved caspase 3 in xenograft tumors dissected from mice after treatment. Scale bar 10 μm.

Bioavailability and Pharmacokinetic Study

To determine if the inhibitors could be developed as in vivo drug therapies for ovarian cancer, a series of metabolic clearance and pharmacokinetics studies were performed. Although in vivo efficacy was detected for a reduction in tumor growth, higher doses would likely result in more effective chemotherapy. Therefore, the maximum dose that could be administered in vivo was determined. To establish the maximum tolerated dose (MTD) and potential toxic effects, 9ING41 was administered orally at 100 mg/kg or 500 mg/kg to three male and three female Sprague-Dawley rats and adverse effects were monitored throughout the testing period (8 days). These studies showed that 9ING41 was well tolerated, and no differences between animals treated with the compound and those treated with the control were observed in any of the following parameters: mortality/morbidity, clinical observations, body weights, clinical pathology, and gross necropsy findings (data not shown). Because no adverse effects were seen, the MTD is considered to be greater than 500 mg/kg in rats.

To determine the bioavailability and plasma pharmacokinetics of 9ING41, male Sprague-Dawley rats were administered the compound at doses of 10 mg/kg (iv), and 400 mg/kg (po). Mean plasma drug levels for the compound evaluated are summarized in Table 2. Pharmacokinetic analysis of the plasma level data (Table 3) indicated that the bioavailability of 9ING41 was 4.5% and the volumes of distribution were ≥ 26 L/kg suggesting that this drug was well distributed to the tissues. The elimination half-life was 4.0-4.85 hr.

Table 2. Plasma levels for 9ING41.

Time (h)	Ca (ng/ml)
	9ING41(iv) 10 mg/kg	9ING41 (po) 400 mg/kg
0.083	1390	4.7
0.167	1410	18.2
0.5	1190	156
1	690	540
2	367	820
4	139	934
6	77	340
8	41	123
24	6.7	8.3

^aBlood was collected from three rats per group, per time point.

Table 3. Pharmacokinetics parameters^a of 9ING41 in rats.

Compound	Cob (ng/ml)	T1/2 (h)	AUC (h· ng/ml)	CL (ml/h/kg)	V (L/kg)	F (%)
9ING41 (iv) 10 mg/kg	1390	4.85	2740	3690	26.2
9ING41 (po) 400 mg/kg	1040	4.0	4950	132600	810	4.5

^aMean value of three animals

^bC_o, plasma concentration at T = 0; T_1/2, apparent elimination half-life; AUC, area under the concentration-time curve; CL, systematic clearance; V, volume of distribution; F, bioavailability

The inhibition potential of 9ING41 on human-Eag-related gene (hERG) potassium channel was assessed to determine cardiac side effects. In this assay, the hERG-channel was overexpressed in U2OS cells and the activity was evaluated by measuring the permeability of the potassium channel to thallium and specific thallium dye. The FDA criterion for defining a drug as hERG- positive is IC₅₀ < 1 μM. The potency of 9ING41 to inhibiting hERG channel is low (IC₅₀ 3.8 μM).

Discussion

The Wnt pathway has been implicated in many cancers including ovarian cancer; however, very few studies have used chemical inhibitors of this pathway for possible cancer therapies (6, 19). In advanced stages of ovarian cancer, an increased level of GSK3β is detected (20, 21). The overexpression of GSK3β increased proliferation of ovarian cancer cell lines indicating that this protein has a unique role in ovarian cancer. SKOV3 was growth inhibited by LiCl and SB216763, but 9ING41 was significantly better at slowing cellular proliferation in both SKOV3 and OVCA432 (3). In this study, the novel inhibitor of GSK3β, 9ING41, induced apoptosis. Cleaved PARP and activated caspase-3 were upregulated, indicating that GSK3βi induce apoptosis in two serous ovarian cancer cell lines. The absence of cleaved PARP and activated caspase-3 with LiCl and SB216763 could be dose and potency related. The downstream target of GSK3β, glycogen synthase, was phosphorylated less in response to 9ING41 compared to solvent control suggesting that the drug was capable of blocking the target kinase intracellularly. Cyclin D1 expression was different between the cell lines. Some have reported that cyclin D1 expression is decreased with inhibition of GSK3β (3, 22) while others have reported an increase in TCF-dependent activities, which correlates with an increase in apoptosis when GSK3β is inhibited in two different IOSE cell lines (14). We have observed an increase in cyclin D1 expression in SKOV3 cells treated with 9ING41, but there is no increase in cellular proliferation. In fact there was a significant increase in apoptosis in SKOV3 cells when treated with 9ING41. Our data coincides with the previous observation in which inhibition of GSK3β increases cytosolic β–catenin levels, that upon nuclear translocation activates TCF transcription, which causes apoptosis of OSE (14). Overall, the growth inhibition observed is not associated with changes in cell cycle. Although both LiCl and SB216763 are known GSK3β inhibitors, the compounds had different effects on OVCA432 and SKOV3 ovarian cancer cells. LiCl is a noncompetitive inhibitor that binds many different targets in the cell. While both 9ING41 and SB216763 inactivate GSK3β kinase activity by competitively binding to the active ATP site, 9ING41 also enhanced GSK3β ^ser9 inhibitory phosphorylation (3, 15).

Overexpression of GSK3β, as demonstrated by independent studies in ovarian cancer, conveys a growth advantage that when blocked allows for apoptosis and slower tumor growth (3, 9). In vivo, 9ING41, slowed tumor growth after 7 and 14 days. While tumor volume did not remain significantly lower for the entire xenograft, metabolic clearance suggests that modification to the chemical structure to improve bioavailability or dosing animals with a higher concentration of drug might be able to improve the in vivo anti-tumor activity. Although the maximum tolerance study was conducted on male rats, the lack of toxicity detected in nude female mice, based on body weight, implies that 9ING41 would not be toxic if tested in higher concentrations and suggests that further studies should be done to confirm the use of GSK3βi for serous ovarian cancers. These novel GSK3βi were initially synthesized to potentially treat a variety of conditions including neurodegeneration and cancer. The maximum tolerated dose studies used the oral route of administration in order to account for first-pass liver metabolism effects on serum concentration. For the in vivo analysis of tumor xenografts, the dose and i.p. administration was chosen based on previous reports evaluating subcutaneously grafted thyroid cancers (15). Toxicity was not detected from either route of administration. Because the non-selective inhibitor LiCl has been used for decades to treat neurological conditions, the likelihood that giving a GSK3β inhibitor would increase cancer risk has not been substantiated (23). Interestingly, LiCl was reported to significantly slow SKOV3 tumor growth (3). However, the previous study mixed the drugs and cells together before subcutaneous injection and therefore might have selectively induced apoptosis before the xenograft could properly form. This method could mimic a model for early stage ovarian cancer or post-cytoreductive surgery since the tumor was established. In the current study, tumors were allowed to grow until palpable before the animals were systemically injected i.p. with the drugs. LiCl was not able to significantly reduce tumor growth in vivo indicating the necessity for a novel and more selective GSK3βi, like 9ING41.

The function of the GSK3β, APC, axin, and β-catenin complex in the human ovary and in human ovarian cancer is not fully understood (24). In normal OSE, β-catenin seems to be stabilizing cell junctions and is mostly located at the cell surface (14, 25). Expression of mutant β-catenin or introduction of siRNA against β-catenin was previously demonstrated to reduce apoptosis in ovarian cancer cells (14). A significant increase in β-catenin and GSK3β is detected in ovarian cancer as compared to normal cells without β-catenin nuclear localization (20, 21, 26). Inhibition of GSK3β causes a reduction in N-cadherin and this may provide a mechanism for reduced cellular proliferation and slower tumor growth in vivo (12, 27).

The GSK3β chemical inhibitor 9ING41 is a novel drug capable of slowing proliferation by inducing apoptosis and reducing tumor volume in vivo. While deregulation of many members of the Wnt pathway have been demonstrated in a variety of tumors, blocking GSK3β slowed tumor growth in vitro and in vivo by inducing apoptosis in ovarian cancer cells. Due to the low toxicity associated with currently approved GSK3βi like LiCl, combined with lack of body weight changes in the current xenograft, these compounds merit additional studies to determine if they might be a reasonable therapeutic approach for serous cancers. In summary, novel GSK3βi are more potent cytotoxic compounds for ovarian cancer cells as compared to commercially available inhibitors and may function to slow tumor growth by inducing apoptosis.