Targeting the oncogenic protein beta-catenin to enhance chemotherapy outcome against solid human cancers

Abstract

Background: Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of β-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3β) through phosphorylation. Cell growth and proliferation is associated with β-catenin translocation from the cytoplasm into the nucleus. This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on β-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of β-catenin, (2) investigate the kinetics and the mechanism of β-catenin inhibition, and (3) confirm that inhibition of β-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs.

Results: In six human cancer cell lines, the inhibition of total and nuclear expression of β-catenin by MSeA was dose and time dependent. The involvement of GSK-3β in the degradation of β-catenin was cell type dependent (GSK-3β-dependent in HT-29, whereas GSK-3β-independent in HCT-8). However, the pronounced inhibition of β-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.Knockout of β-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.Collectively, the generated data demonstrate that β-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs.

Conclusions: This study demonstrates that β-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of β-catenin by GSK-3β is not a general mechanism but is cell type dependent.

Figure 1

MSeA effect on intracellular expression of β-catenin. Six human cancer cell lines including 2 colorectal (HT-29 and HCT-8), 2 head and neck (FaDu and A253) and 2 prostate (PC3 and C42) were treated with various doses of MSeA (panel 1A) and various times (panel 1B). Western blot analyses show that MSeA inhibits intracellular level of β-catenin in dose and time dependent manners.

Figure 2

MSeA effect on the nuclear expression of β-catenin. Colorectal cancer cells express a high-level of nuclear β-catenin indicating activation (panel 2A). Treatment with MSeA results in inhibition of the nuclear expression of β-catenin indicating suppression of the active form of β-catenin (panel 2B).

Figure 3

MSeA inhibition of β-catenin is due to degradation. Colorectal cancer cells were treated with a protein synthesis inhibitor, cycloheximide (CHX) and MSeA alone or in combination for various times up to 30 minutes (panel 3A) and for 24 h (panel 3B). No early changes in the β-catenin level were observed (panel 3A). However, 24 h combination treatment with CHX/MSeA resulted in same inhibition after MSeA alone (panel 3B).

Figure 4

The role of GSK-3β in degradation of β-catenin. Colorectal cancer cells were treated with various doses of MSeA alone (panel 4A) or in combination with GSK-3β inhibitor, Lithium Chloride (LiCl, panel 4B). The level of total GSK-3β, p-GSK-3β and β-catenin were determined using western blots. Treatment with MSeA alone had no effect on total GSK-3β but decreased the level of p-GSK-3β only in HT-29 (panel 4A). Treatments with LiCl increased the level of p-GSK-3β (inactive form of GSK-3β) but had no effect on β-catenin expression (panel 4B). Combination treatment of LiCl/MSeA continued to inhibit expression of β-catenin only in HCT-8 (panel 4A)

Figure 5

Combination treatment effect on the expression of β-catenin. Cancer cells (panel 5A) and nuclear extraction of colorectal cancer cells (panel 5B) were treated with MSeA, SN-38 and docetaxel alone or in combination. Expression of β-catenin was determined using western blots. Combination treatments of MSeA/SN-38 continued to decrease expression of β-catenin when compared with all other groups. Combination treatments of MSeA/docetaxel did not reverse the inhibitory effect of MSeA alone on expression of β-catenin (panel 5A). The active form (nuclear expression) of β-catenin was inhibited by the combination treatment of MSeA/SN-38 when compared with other group (panel 5B).

Figure 6

The effect of various drug treatments on cell growth and proliferation in β-catenin knockout cells. Beta-catenin was silenced in colorectal cancer cells (HCT-8, panel 6A). HCT-8 wild type cells (HCT-8WT), scramble controls (HCT-8SC) and 2 β-catenin knockout clones (HCT-8RH7 and HCT-8RF4) were tested. Expression of β-catenin was significantly lower in HCT-8RH1 and HCT-8RF4 when compared with all other groups (panel 6A). Cancer cell growth and proliferation were determined in HCT-8WT, HCT-8SC, HCT-8RH7 and HCT-8RF4 after treatment with various doses of SN-38, docetaxel, paclitaxel, oxaliplatin, 5-FU and topotecan using SRB assay. HCT-8RH7 and HCT-RF4 cells were significantly more sensitive to various drug treatments when compared with HCT-8WT and HCT-8SC (panel 6B). * denotes a p value of less than 0.05 when compared with all other groups.