272510 Targeted Nitric Oxide Pretreatment Alters p53 and O6-Methylguanine-DNA Methyltransferase Activity Resulting in Enhanced Chemosensitivity in Glioma Cells

Wednesday, October 31, 2012: 2:36 PM
Somerset West (Westin )
Shahana Safdar, Chemical and Biomolecular Engineering, Georgia Tech, Atlanta, GA, Courtney A. Payne, Spelman College, Atlanta, GA, Nam H. Tu, Georgia Institute of Technology, Atlanta, GA and Lakeshia Taite, Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA

Introduction: Carmustine, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ) are chemotherapeutics commonly used to treat glioblastoma multiforme. However a significant number of patients are resistant to these therapeutics resulting in poor patient survival. In the past, high doses of nitric oxide (NO) have been used to induce chemosensitivity in glioma cells. However the high doses coupled with the non-specific delivery of NO result in toxic effects to healthy tissue. Thus, to limit these side effects we have developed a targeted NO donor to enhance the efficacy of TMZ and BCNU on human malignant glioma cells.

Materials and Methods: To synthesize glioma-targeting NO donor, Chlorotoxin (CTX), a glioma targeting protein isolated from the venom of the Deathstalker scorpion, was reacted with NO gas for 24 hours in a hypoxic environment transforming CTX in to CTX-NO. Two malignant human glioblastoma cell lines T98 and U-87MG, normal human astrocytes (NHA) and human brain and microvascular endothelial cells (HBMEC) were used for in vivo studies. The cells were incubated for 2 hours with CTX-NO, after which they were incubated with BCNU (75 ÁM) or TMZ (50 ÁM) for 48 hours. At the end of the incubation period, cell viability, intercellular MGMT levels, or p53 expression was quantified.

Results and Discussion: TMZ and BCNU did not significantly change the viability of T98G cells. However, pretreatment with 2 ÁM of NO followed by TMZ or BCNU significantly decreased T98G cell viability to 76.8 ▒ 8.5% and 67.1 ▒ 1.8% of the control. Conversely, U-87MGs were more sensitive to both chemotherapeutics and NO pretreatment further enhanced this chemosensitivity (Fig. 1). CTX-NO resulted in no significant changes in the chemosensitivity of non-tumor control cells (Fig. 1). The resistance of tumor cells TMZ and BCNU is partially due to the presence of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) therefore we investigated the effect of CTX-NO on MGMT levels in glioma cells. When T98G cells were treated with NO at concentrations of 1 ÁM or 2 ÁM, MGMT levels were decreased to 73.1 ▒ 5.9% and 60.1 ▒ 9.9% of the control. Another biomolecule that plays an important role in determining chemosensitivity is the protein p53 which a pivotal role in maintaining the balance between DNA repair mechanisms and induction of apoptosis. In T98G cells, which express mutant p53, CTX-NO treatment was able to significantly decrease in a dose dependant manner. Conversely, CTX-NO treatment did not affect p53 levels in U-87MG cells which express wild-type p53.

Conclusion: In this study we used small doses of a targeted NO donor, CTX-NO, to chemosensitize malignant glioma cells to chemotherapeutic agents, BCNU and TMZ. Furthermore the data showed that low levels of NO were able to decrease MGMT and p53 levels, both of which contribute to chemoresistance in glioma cells. Using targeted NO to sensitize cells towards chemotherapeutics holds great potential as part of a treatment plan for patients with various tumors that are normally unresponsive to chemotherapy.

Figure 1: Cell viability of (A)T98G cells, (B)U-87MG, (C)NHA and (D)HBMEC. Cells counted are presented as a percentage of the number of cells that received no treatment. * compared to no pretreatment, ** compared to pretreatment with CTX-NO (1 ÁM), # compared to pretreatment with CTX-NO (2 ÁM) p <0.05, n = 3

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