(590b) Treating Glioblastoma Multiforme Using Targeted Nitric Oxide Donors

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in adults[1]. Patients diagnosed with the disease have a median survival of only 12 months, and 5 year survival is less than 5% [2].  The ability of glioma cells to rapidly disperse and invade healthy brain tissue coupled with their high resistance to chemotherapy and radiotherapy have resulted in extremely poor prognosis among patients. In this study we developed an innovative treatment for GBM. We designed nitric oxide (NO) donors which were able to target and deliver NO specifically to tumor cells, hence attacking not only the tumor mass but also the infiltrating cells. NO, in addition to being cytotoxic is also able to increase the permeability of the blood brain barrier and sensitize cells to radiation [3,4].

The first step in developing glioma specific NO-releasing peptides was the identification of glioma targeting peptide sequences.  Two such sequences were identified: a 36-amino acid protein, chlorotoxin (CTX), isolated from scorpion venom and a 12 amino acid peptide sequence VTWTPQAWFQWV (VTW) [5,6]. To convert the targeting sequences into NO donors they were reacted with NO gas in a hypoxic environment for 24 hours [7]. The NO reacted with the free amines on the targeting sequence to form diazeniumdiolates, which in aqueous environments release NO. Measurements from a NO microsensor showed that both biomolecules released NO for over 6 days and over 75% of the NO was released in the first 72 hours.

Tumor targeting abilities of VTW and chlorotoxin subsequent to the reaction with NO were assessed using fluorescence labeling techniques. For this purpose CTX and VTW were labeled with Fluorescein isothiocyanate (FITC) and subsequently reacted with NO. The glioma cells (T98 and U87) and the control cells were incubated with the FITC labeled biomolecules. Fluorescence microscopy showed that both VTW and CTX exhibited high binding to the tumor cells and minimal binding to the control cells. To assess the effect of NO on the proliferation of cells, the glioma cells and normal human astrocytes (NHA) were incubated with varying concentrations of CTX-NO or VTW-NO for 48 hours, after which the cells were counted. The proliferation assays showed that both NO donors were able to decrease the proliferation of tumor cells, while the proliferation of NHAs was not affected (Fig 1).

In this study we were able to couple the targeting ability of the biomolecules with their NO releasing capabilities to provide a more efficient NO delivery method, thereby developing an improved treatment for GBM.

REFERENCES

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4.     Yin, D., et al., Clin Cancer Res, 2008. 14(12): p. 4002-4009.

5.     DeBin, J.A., J.E. Maggio, and G.R. Strichartz, Am J Physiol Cell Physiol, 1993. 264(2): p. C361-369.

6.     Wu, C., et al.,. Journal of Controlled Release, 2008. 130(2): p. 140-145.

7.     Jun, H.-W., L.J. Taite, and J.L. West, Biomacromolecules, 2005. 6(2): p. 838-844.