Cancer Res Treat.  2012 Sep;44(3):195-201.

Constitutive Expression of MAP Kinase Phosphatase-1 Confers Multi-drug Resistance in Human Glioblastoma Cells

• Yu H
• Park J
• Lee J
• Choi K
• Choi C

Abstract

PURPOSE
Current treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide. However, it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues. Moreover, combination therapy is not always successful because glioblastoma has diverse resistances. To overcome these limitations, we examined the combined effects of chemotherapy and knockdown of mitogen-activated protein kinase phosphatase-1 (MKP-1).
MATERIALS AND METHODS
We used ten different anti-cancer drugs (cisplatin, cyclophosphoamide, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, mitomycin C, and vincristine) to treat glioblastoma multiforme (GBM) cells. Knockdown of MKP-1 was performed using siRNA and lipofectamine. The basal level of MKP-1 in GBM was analyzed based on cDNA microarray data obtained from the Gene Expression Omnibus (GEO) databases.
RESULTS
Anti-cancer drug-induced cell death was significantly enhanced by knockdown of MKP-1, and this effect was most prominent in cells treated with irinotecan and etoposide. Treatment with these two drugs led to significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in a time-dependent manner, while pharmacological inhibition of JNK partially inhibited drug-induced cell death. Knockdown of MKP-1 also enhanced drug-induced phosphorylation of JNK.
CONCLUSION
Increased MKP-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human GBM. Therefore, MKP-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy.

Keyword

Dual specificity phosphatase 1; Glioblastoma; JNK mitogen-activated protein kinases; Apoptosis; Chemotherpy; Anti-cencer drug resistance

MeSH Terms

Apoptosis
Camptothecin
Cell Death
Dacarbazine
Deoxycytidine
Doxorubicin
Drug Resistance
Drug Resistance, Multiple
Dual Specificity Phosphatase 1
Epirubicin
Etoposide
Fluorouracil
Gene Expression
Glioblastoma
Humans
JNK Mitogen-Activated Protein Kinases
Lipids
Mitomycin
Oligonucleotide Array Sequence Analysis
Phosphorylation
Phosphotransferases
Protein Kinases
RNA, Small Interfering
Camptothecin
Dacarbazine
Deoxycytidine
Doxorubicin
Dual Specificity Phosphatase 1
Epirubicin
Etoposide
Fluorouracil
JNK Mitogen-Activated Protein Kinases
Lipids
Mitomycin
Phosphotransferases
Protein Kinases
RNA, Small Interfering

Figure

• 

  Fig. 1 MAP kinase phosphatase-1 (MKP-1) is highly expressed in human glioblastomas. (A) The expression of MKP-1 was evaluated using the microarray data of brain cancer patients provided by the Gene Expression Omnibus (GEO) database (GSE ID GSE2223). The results are presented as the means±SEM, and Tukey's post-hoc test was applied to significant group effects in ANOVA, ***p<0.001. (B) The expression of MKP-1 was measured by western blot analysis in human primary astrocytes, human glioblastomas (U251MG, LN215 and U373MG), human astroglioma (CRT-MG), human hepatomas (HepG2, Huh7), and human cervical adenocarcinoma (HeLa) cell lines. (C) The expression of MKP-2 was measured by western blot analysis in the same cell lines as in (B). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hPA, human primary culturesd astrocytes.

• 

  Fig. 2 The reduction of MAP kinase phosphatase-1 (MKP-1) expression increases sensitivity to anti-cancer drugs. (A) U251MG cells were transfected with control or MKP-1 specific siRNA (siMKP-1). After 48 hr of transfection, the transfection efficiency was determined by quantitative real-time polymerase chain reaction and normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. (B) MKP-1 protein expression was determined by western blot analysis. (C) Cells were treated with 10 different anti-cancer drugs 24 hr after siRNA transfection at the concentrations listed in Table 1. Cell viability was then evaluated by a WST-1 assay. The results are presented as the mean±SEM (n=4). Asterisks indicate a significant difference as determined by a Student's t-test, **p<0.01, ***p<0.001, and NS, not significant.

• 

  Fig. 3 The effect of MAP kinase phosphatase-1 (MKP-1) knockdown on irinotecan- and etoposide-induced cell death. Cells were transfected with control or MKP-1 specific siRNA (siMKP-1) for 48 hr, after which they were incubated with varying doses of irinotecan (A) or etoposide (B) for an additional 24 hr. Cell viability was evaluated by WST-1 assay. The results are presented as the mean±SEM (n=4). Asterisks indicate a significant difference as determined by a Student's t-test, *p < 0.05 and ***p < 0.001.

• 

  Fig. 4 The involvement of c-Jun N-terminal kinase (JNK) in irinotecan and etoposide-induced cell death. (A) Cells were transiently transfected with control or MAP kinase phosphatase-1 (MKP-1) specific siRNA (siMKP-1) for 48 hr, after which they were incubated in the presence or absence of irinotecan or etoposide (10 mg/L) for 15 min. Western blot analysis was used to determine the protein expression of phospho-JNK, total JNK, caspase-3, and cleaved caspase-3 in total cell lysates. (B) Cells were transiently transfected with control or siMKP-1 for 48 hr, after which they were incubated in the presence or absence of SP600125 (JNK inhibitor, 10 µmol/L) for 1 hr, and then treated in the presence or absence of irinotecan (10 mg/L) for an additional 24 hr. The cell viability was then evaluated by a WST-1 assay. The results are presented as the mean±SEM (n=4). Tukey's post-hoc test was applied to significant group effects in ANOVA, **p<0.01, ***p<0.001. (C) Cell viability was measured by flow cytometry after tetramethylrhodamine ethyl ester (TMRE; 100 nmol/L) staining. The labeled numbers indicate live cells. Images are representative of three independent experiments. p-JNK, phosphorylated JNK; t-JNK, total JNK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Reference

1. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008; 359:492–507. PMID: 18669428.

2. Meyer MA. Malignant gliomas in adults. N Engl J Med. 2008; 359:1850. PMID: 18946076.

3. Krakstad C, Chekenya M. Survival signalling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Mol Cancer. 2010; 9:135. PMID: 20515495.

4. Reijneveld JC, Voest EE, Taphoorn MJ. Angiogenesis in malignant primary and metastatic brain tumors. J Neurol. 2000; 247:597–608. PMID: 11041327.

5. Bredel M, Zentner J. Brain-tumour drug resistance: the bare essentials. Lancet Oncol. 2002; 3:397–406. PMID: 12142169.

6. Wick W, Weller M, Weiler M, Batchelor T, Yung AW, Platten M. Pathway inhibition: emerging molecular targets for treating glioblastoma. Neuro Oncol. 2011; 13:566–579. PMID: 21636705.

7. Riemenschneider MJ, Hegi ME, Reifenberger G. MGMT promoter methylation in malignant gliomas. Target Oncol. 2010; 5:161–165. PMID: 20725792.

8. Bredel M. Anticancer drug resistance in primary human brain tumors. Brain Res Brain Res Rev. 2001; 35:161–204. PMID: 11336781.

9. Patterson KI, Brummer T, O'Brien PM, Daly RJ. Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochem J. 2009; 418:475–489. PMID: 19228121.

10. Bermudez O, Pages G, Gimond C. The dual-specificity MAP kinase phosphatases: critical roles in development and cancer. Am J Physiol Cell Physiol. 2010; 299:C189–C202. PMID: 20463170.

11. Keyse SM. Dual-specificity MAP kinase phosphatases (MKPs) and cancer. Cancer Metastasis Rev. 2008; 27:253–261. PMID: 18330678.

12. Small GW, Somasundaram S, Moore DT, Shi YY, Orlowski RZ. Repression of mitogen-activated protein kinase (MAPK) phosphatase-1 by anthracyclines contributes to their antiapoptotic activation of p44/42-MAPK. J Pharmacol Exp Ther. 2003; 307:861–869. PMID: 14557375.

13. Small GW, Shi YY, Edmund NA, Somasundaram S, Moore DT, Orlowski RZ. Evidence that mitogen-activated protein kinase phosphatase-1 induction by proteasome inhibitors plays an antiapoptotic role. Mol Pharmacol. 2004; 66:1478–1490. PMID: 15448190.

14. Wang J, Zhou JY, Wu GS. ERK-dependent MKP-1-mediated cisplatin resistance in human ovarian cancer cells. Cancer Res. 2007; 67:11933–11941. PMID: 18089824.

15. Loda M, Capodieci P, Mishra R, Yao H, Corless C, Grigioni W, et al. Expression of mitogen-activated protein kinase phosphatase-1 in the early phases of human epithelial carcinogenesis. Am J Pathol. 1996; 149:1553–1564. PMID: 8909245.

16. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN, et al. Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. Nat Genet. 2008; 40:600–608. PMID: 18372904.

17. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer. Nat Rev Drug Discov. 2006; 5:219–234. PMID: 16518375.

18. Wang Z, Xu J, Zhou JY, Liu Y, Wu GS. Mitogen-activated protein kinase phosphatase-1 is required for cisplatin resistance. Cancer Res. 2006; 66:8870–8877. PMID: 16951204.

19. Small GW, Shi YY, Higgins LS, Orlowski RZ. Mitogen-activated protein kinase phosphatase-1 is a mediator of breast cancer chemoresistance. Cancer Res. 2007; 67:4459–4466. PMID: 17483361.

20. Chamberlain MC. Bevacizumab for the treatment of recurrent glioblastoma. Clin Med Insights Oncol. 2011; 5:117–129. PMID: 21603247.

21. Jakobsen JN, Hasselbalch B, Stockhausen MT, Lassen U, Poulsen HS. Irinotecan and bevacizumab in recurrent glioblastoma multiforme. Expert Opin Pharmacother. 2011; 12:825–833. PMID: 21385110.

22. Watanabe K, Kanaya H, Fujiyama Y, Kim P. Combination chemotherapy using carboplatin (JM-8) and etoposide (JET therapy) for recurrent malignant gliomas: a phase II study. Acta Neurochir (Wien). 2002; 144:1265–1270. PMID: 12478337.