Elsevier

Neurobiology of Disease

Volume 33, Issue 2, February 2009, Pages 243-249
Neurobiology of Disease

17-AAG sensitized malignant glioma cells to death-receptor mediated apoptosis

https://doi.org/10.1016/j.nbd.2008.10.005Get rights and content

Abstract

17-AAG is a selective HSP90-inhibitor that exhibited therapeutic activity in cancer. In this study three glioblastoma cell lines (U87, LN229 and U251) were treated with 17-AAG, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the combination of both. Treatment with subtoxic doses of 17-AAG in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, suggesting that this combined treatment may offer an attractive strategy for treating gliomas. 17-AAG treatment down-regulated survivin through proteasomal degradation. In addition, over-expression of survivin attenuated cytotoxicity induced by the combination of 17-AAG and TRAIL. In summary, survivin is a key regulator of TRAIL–17-AAG mediated cell death in malignant glioma.

Introduction

Malignant gliomas are the most commonly diagnosed malignant adult primary brain tumors. Median survival for glioblastoma is 12 to 15 months. Surgical resection or diagnostic biopsy is usually the first step in therapy, followed by adjuvant radiation and chemotherapy (Stupp et al., 2006).

Heat-shock-proteins (HSPs), especially HSP90, are up regulated in various human tumors, and targeting their function might provide new therapeutic opportunities (Altieri, 2004). The protein holding and protein folding functions of the HSPs are critical not only in normal unstressed cells, but also in transformed cells, in which the normal functions of HSPs are used to facilitate cell growth and cell cycle progression (Altieri, 2004). As an example, HSP90 stabilizes Akt and oncogenic forms of mutant epidermal growth factor receptor, both of which contribute to the growth of a variety of cancers including gliomas (Basso, 2002, Lavictoire, 2003). 17-allylaminogeldanamycin (17-AAG) is a potent HSP90 inhibitor that inhibited cellular proliferation and induced apoptosis in colon cancer cells (Vasilevskaya and O'Dwyer, 2005).

Recently, a novel molecular and functional interaction between HSP90 and survivin, a member of the Inhibitor of Apoptosis protein family, has been identified (Fortugno et al., 2003). Survivin has attracted attention because of three intriguing features: i: an over-expression in nearly every tumor, including malignant glioma, ii: a cell cycle-regulated expression at mitosis and localization to the mitotic apparatus, iii: a dual function in inhibiting apoptosis and regulating mitosis (Fortugno et al., 2003). The Inhibitor of Apoptosis Proteins (IAPs) such as survivin are expressed at high levels in many tumors including glioblastoma and have been associated with refractory disease and poor prognosis (Blum, 2006, Chakravarti, 2002). They block apoptosis at the core of the apoptotic machinery by inhibiting effector-caspases (Fulda et al., 2002).

Targeting death receptors to trigger apoptosis in tumor cells is an attractive concept for cancer therapy. To this end, TRAIL appears to be a relatively safe and promising death ligand for clinical application since TRAIL induces almost selectively apoptosis in cancer cells. However, many tumors remain resistant to treatment with TRAIL, and this resistance may be caused by deregulated expression of anti-apoptotic molecules (Siddiqui et al., 2008). IAPs including survivin are also capable of inhibiting TRAIL-mediated apoptosis in malignant glioma cells.

In the present study we investigated the effects of 17-AAG on the expression of survivin in three human glioma cell lines, U87, U251 and LN229 and demonstrated that the down-regulation of survivin is mediated by the proteasome. In addition we provided evidence that the suppression of survivin by 17-AAG enhanced TRAIL-mediated apoptosis.

Section snippets

Cell culture, reagents, plasmids and transfections

Human glioblastoma cell line U87 (p53-wild type), U251 and LN229 (p53 mutant) were purchased from the Amercian Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM Glutamax-I 4500 g/l glucose (Invitrogen, Karlsruhe, Germany) with 10% FBS and 1% Penicillin/Streptomycin (Invitrogen, Karlsruhe, Germany) and was incubated at 37 °C in a humidified atmosphere containing 10% carbon dioxide.

17-AAG was obtained from Axxora (Loerrach, Germany) and malignant glioma cell lines

HSP90 is over-expressed in human glioblastoma tissues compared to normal human brain tissue

Although there are a few data of HSP90 in human malignant brain tumors in vivo (Hermisson et al., 2000), there is no information about the HSP90 expression level in glioblastoma compared to normal brain tissue. Immunohistochemistry demonstrated strong HSP90 expression in human glioblastomas compared to normal brain tissue (Fig. 1A). On the subcellular level, HSP90 immunoreactivity was mainly seen in the cytoplasm in a diffuse pattern, with some antibody reactivity in the nucleus and in the

Discussion

Despite aggressive treatment strategies, patients with glioblastoma still have a dismal prognosis, which highlights the need for novel treatment approaches (Fulda et al., 2002). HSP90 is typically upregulated in various human cancers, and targeting their function may provide novel therapeutic opportunities (Altieri, 2004). In our immunohistochemical analysis HSP90 was over-expressed in glioblastoma compared to normal human brain tissue and reactive astrocytes. This is also in line with previous

Acknowledgments

This work was supported by grants from the Post-Doc Program of the University of Heidelberg to Dr. Markus David Siegelin. The plasmid pcDNA3-survivin was kindly provided by Dr. Dario Altieri.

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