Overexpression of inosine 5'-monophosphate dehydrogenase type II mediates chemoresistance to human osteosarcoma cells

Abstract

Background: Chemoresistance is the principal reason for poor survival and disease recurrence in osteosarcoma patients. Inosine 5'-monophosphate dehydrogenase type II (IMPDH2) encodes the rate-limiting enzyme in the de novo guanine nucleotide biosynthesis and has been linked to cell growth, differentiation, and malignant transformation. In a previous study we identified IMPDH2 as an independent prognostic factor and observed frequent IMPDH2 overexpression in osteosarcoma patients with poor response to chemotherapy. The aim of this study was to provide evidence for direct involvement of IMPDH2 in the development of chemoresistance.

Methodology/principal findings: Stable cell lines overexpressing IMPDH2 and IMPDH2 knock-down cells were generated using the osteosarcoma cell line Saos-2 as parental cell line. Chemosensitivity, proliferation, and the expression of apoptosis-related proteins were analyzed by flow cytometry, WST-1-assay, and western blot analysis. Overexpression of IMPDH2 in Saos-2 cells induced strong chemoresistance against cisplatin and methotrexate. The observed chemoresistance was mediated at least in part by increased expression of the anti-apoptotic proteins Bcl-2, Mcl-1, and XIAP, reduced activation of caspase-9, and, consequently, reduced cleavage of the caspase substrate PARP. Pharmacological inhibition of IMPDH induced a moderate reduction of cell viability and a strong decrease of cell proliferation, but no increase in chemosensitivity. However, chemoresistant IMPDH2-overexpressing cells could be resensitized by RNA interference-mediated downregulation of IMPDH2.

Conclusions: IMPDH2 is directly involved in the development of chemoresistance in osteosarcoma cells, suggesting that targeting of IMPDH2 by RNAi or more effective pharmacological inhibitors in combination with chemotherapy might be a promising means of overcoming chemoresistance in osteosarcomas with high IMPDH2 expression.

Figure 1. Chemoresistance in IMPDH2-overexpressing Saos-2 cells.

A: Western blot analysis of IMPDH2 expression in Saos-2 wild-type cells, Saos-2 cells transfected with the empty vector (Saos-2 pCMS), Saos-2 cells overexpressing IMPDH2 (Saos-2 cdsIMPDH2), and Saos-2 cells transfected with a shRNA construct directed against IMPDH2 (Saos-2 shIMPDH2). B: Cell viability of different Saos-2 cell lines after treatment with cisplatin at the indicated concentrations for 72 h. Analyses were performed in triplicate and the results are presented as mean ± SD (** p<0.01 compared to Saos-2 wild-type cells). C: Cell viability of different Saos-2 cell lines after treatment with methotrexate (MTX) at the indicated concentrations for 96 h. Analyses were performed in triplicate and the results are presented as mean ± SD (** p<0.01 compared to Saos-2 wild-type cells).

Figure 2. Western blot analysis of different apoptosis-related proteins in Saos-2 wild-type cells and Saos-2 cdsIMPDH2 cells after treatment with cisplatin (2.5 µg/ml) at the indicated time points.

Figure 3. Chemosensitivity of Saos-2 wild-type, Saos-2 pCMS, Saos-2 shIMPDH2, and Saos-2 cdsIMPDH2 cells treated with the IMPDH inhibitor mycophenolic acid (MPA) at the indicated concentrations for 72 h with or without cisplatin (2.5 µg/ml).

Analyses were performed in triplicate and the results are presented as mean ± SD.

Figure 4. Influence of the IMPDH inhibitor mycophenolic acid (MPA) on cell proliferation of different Saos-2 cell lines.

A: MPA was added to the culture medium at the indicated concentrations and cell proliferation was determined using WST-1 assay at 0, 24, 48, and 72 h. B: Saos-2 wild-type cells were treated with the indicated concentrations of MPA for 48 h with or without the addition of 10 µM guanine. Cell proliferation was analyzed by WST-1 assay at 450 nm. Analyses were performed in triplicate and the results are presented as mean ± SD.

Figure 5. Chemosensitivity of Saos-2 cdsIMPDH2 cells cotransfected with an IMPDH2-specific shRNA construct.

A: Stable Saos-2 cdsIMPDH2 cells were cotransfected with three different shRNA constructs directed against IMPDH2. Stable cell lines were generated, and IMPDH2 mRNA expression was analyzed by quantitative PCR and compared to that in Saos-2 wild-type, Saos-2 pCMS, and Saos-2 cdsIMPDH2 cells. B: Western blot analysis of IMPDH2 expression in Saos2 cdsIMPDH2 cells cotransfected with an IMPDH2 specific shRNA construct. C: Analysis of chemosensitivity of Saos-2 wild-type, Saos-2 pCMS, Saos-2 cdsIMPDH2, and Saos-2 cdsIMPDH2 cells stable cotransfected with shRNA construct specific for IMPDH2. Cells were treated with cisplatin at the indicated concentrations for 72 h before cell viability was analyzed by propidiumiodide staining. Analyses were performed in triplicate and the results are presented as mean ± SD. (** p<0.01).