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. 2007 Sep;98(9):1388-93.
doi: 10.1111/j.1349-7006.2007.00545.x. Epub 2007 Jul 19.

Inhibition of GSK-3 beta activity attenuates proliferation of human colon cancer cells in rodents

Abbas Shakoori  1 , Wei Mai, Katsuyoshi Miyashita, Kazuo Yasumoto, Yutaka Takahashi, Akishi Ooi, Kazuyuki Kawakami, Toshinari Minamoto
Affiliations

Inhibition of GSK-3 beta activity attenuates proliferation of human colon cancer cells in rodents

Abbas Shakoori et al. Cancer Sci. 2007 Sep.

Abstract

The authors' recent discovery that glycogen synthase kinase-3beta (GSK-3beta) participates in colon cancer cells' survival and proliferation prompted us to investigate whether GSK-3beta inhibition alters proliferation of colon cancer cells in vivo. Groups of four or five athymic mice (Balb/c, nu/nu) with subcutaneous xenografts of SW480 human colon cancer cells were treated with dimethyl sulfoxide (DMSO) or different doses (1, 2 and 5 mg/kg body weight) of either small-molecule GSK-3beta inhibitor (SB-216763 and AR-A014418) by intraperitoneal injection three times per week for 5 weeks. Compared with DMSO (a diluent of the GSK-3beta inhibitors) as a control, either GSK-3beta inhibitor significantly inhibited proliferation of cancer cell xenografts in the rodents in a dose-dependent manner. Histochemical and immunohistochemical analysis of tumor xenografts demonstrated a significant, dose-dependent decrease in fractions of proliferating cells and an increase in the incidence of apoptosis of cancer cells in mice treated with either GSK-3beta inhibitor. No adverse events or effects were observed in the rodents during the course of treatment, except for rare lethal accidents due to intraperitoneal injection. Morphological examination showed no apparent pathologic changes in major organs including the lungs, liver, pancreas, kidneys, spleen and large bowel of rodents treated with DMSO and the GSK-3beta inhibitors. The results indicate that the GSK-3beta inhibitors would be a novel class of therapeutic agent for colon cancer.

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Figures

Figure 1
Figure 1
Design and protocol of the animal experiment. At week 0, 1 × 106 SW480 human colon cancer cells were subcutaneously inoculated into each mouse. Two weeks later subcutaneous tumors that were formed in all mice were size‐matched and the mice were randomly assigned to seven groups for treatment with dimethyl sulfoxide (DMSO, a diluent of the GSK3β inhibitors) or either of two small‐molecule GSK‐3β inhibitors, SB‐216763 and AR‐A014418. DMSO and indicated doses of the GSK‐3β inhibitors were given to mice by intraperitoneal injections three times per week. After 5 weeks’ treatment, all mice were euthanized for necropsy. At the beginning of treatment, one mouse in each of the groups treated with the GSK‐3β inhibitors died because of a lethal accident (i.e. bleeding in the peritoneal cavity) due to the intraperitoneal injection.
Figure 2
Figure 2
Representative rodents in groups of mice treated with dimethyl sulfoxide (DMSO) or indicated doses of GSK‐3β inhibitors, SB‐216763 and AR‐A014418. After 5 weeks’ treatment with either inhibitor, there was significant dose‐dependent decrease in tumor size in mice treated with different doses (1 mg/kg, 2 mg/kg, and 5 mg/kg body weight, shown at the top of the panels) of the inhibitors, in comparison with those treated with DMSO. Arrows point to subcutaneous tumors formed by inoculated SW480 colon cancer cells.
Figure 3
Figure 3
(a) Effect of intraperitoneal injection of GSK‐3β inhibitors on proliferation of SW480 xenografts and (b) comparison of xenograft weights in rodents treated with dimethyl sulfoxide (DMSO) and indicated doses of the kinase inhibitors. (a) Tumor sizes were measured weekly and their volumes calculated. Administered agents and their doses are indicated on the right together with corresponding symbols. There was a dose‐dependent decrease in tumor volume both with SB‐216763 and AR‐A014418 from week 5 and beyond (P < 0.05). (b) Mean weights ± SD of the tumors removed at necropsy from the respective groups of mice with indicated treatments. Statistically similar differences were found in tumor weights between mice with the respective treatments. (a,b) There were no statistically significant differences in tumor volume or weight between mice treated with any dose of SB‐216763 and AR‐A014418, or between mice treated with 2 mg/kg and 5 mg/kg of the inhibitors, respectively.
Figure 4
Figure 4
Scores of terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labeling (TUNEL)‐ and proliferating cell nuclear antigen (PCNA)‐positive cells, respectively, in tumor tissues removed from mice at necropsy after 5 weeks’ treatment with dimethyl sulfoxide (DMSO) and indicated doses (1 mg/kg, 2 mg/kg, and 5 mg/kg body weight) of GSK‐3β inhibitors. Representative sections of histochemical (TUNEL) and immunohistochemical (PCNA) staining are shown in the upper panels. There were (a) dose‐dependent increases in TUNEL‐positive cell rates and (b) decreases in PCNA‐positive cell rates in tumors treated with SB‐216763 or AR‐A014418 (P < 0.05), compared with those treated with DMSO.
Figure 5
Figure 5
Expression and subcellular localization of GSK‐3β and β‐catenin in xenografts of SW480 human colon cancer cells in mice treated with dimethyl sulfoxide (DMSO) and 5 mg/kg of AR‐A014418, respectively. GSK‐3β expression was found in the cytoplasm of cancer cells. Cytoplasmic and nuclear accumulation of β‐catenin was observed in most cancer cells. No differences were found in intensity of expression or subcellular localization of these molecules between the tumor cell xenografts in mice treated with DMSO and AR‐A014418. Nuclei were counterstained with hematoxylin. The scale bar in each panel indicates 100 µm.
Figure 6
Figure 6
Effect of intraperitoneal injection of GSK‐3β inhibitors on body weights of animals during the course of treatment. Body weights of the mice were measured weekly for 3 weeks before inoculation of SW480 colon cancer cells, as indicated with a minus number on the week axis, and over the treatment. The results are displayed for respective groups of mice with indicated doses (1 mg/kg, 2 mg/kg, and 5 mg/kg body weight) of GSK‐3β inhibitors. All panels include the data of body weights of mice treated with dimethyl sulfoxide (DMSO; indicated by closed circles) as a control. No significant differences in body weight were observed between the groups.
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