Targeted Nitric Oxide Donor Enhances Efficacy of Chemotherapeutics Used to Treat Glioblastoma Multiforme

Nitric oxide (NO) plays a ubiquitous role in human physiology. Research has shown that at sufficient concentrations NO can induce apoptosis as well as increase chemosensitization in tumor cells. However, thus far NO role in cancer therapy has been limited because of its short half-life and the variety of effects it can have on numerous biological function.  Previously we have shown that by derivatizing chlorotoxin (CTX), a protein specific to Glioblastoma multiforme (GBM) cells, we can synthesize targeted NO donors, which deliver NO only to the therapeutic target, thereby significantly decreasing their viability. GBM is the most common malignant central nervous system tumors and patients diagnosed with the disease have very poor prognoses. The two most popular chemotherapeutics used in treatment are temozolomide (TMZ) and carmustine (BCNU), which have both shown only modest improvements in patient survival. In this study we investigated the effect of the targeted NO donor, CTX-NO on the chemosensitivity of GBM cells to the chemotherapeutics BCNU and TMZ.

To assess the effect of NO on cell viability, T98G and U-87MG human glioblastoma cells were incubated with varying concentration of CTX-NO, with and without BCNU for 48 hours, trypsinized, and counted. At a NO concentration of 2 µM, CTX-NO alone reduced glioma cell viability to 81.4 ± 5.5% of the control cell number. When cells were incubated with only BCNU, glioma cell viability was not significantly reduced. However when cells were incubated with a combination of CTX-NO and BCNU, the glioma cell viability was decreased to 45.3 ± 1.5% (Fig 1A). When these cells were reseeded in fresh media their proliferation was hindered by the prior exposure to the combination therapy whereas the proliferation of cells previously exposed to only CTX-NO or only BCNU was not significantly affected (Fig 1B). In contract BCNU decreased the viability of U-87MG to 33.8 ± 2.1% and whereas the combination therapy of BCNU and the NO donor decreased cell viability to 31.2 ± 2.7%.  Thus the NO donor does not affect the sensitivity of U-87MG cells to BCNU.

We have shown that the targeted NO donor, CTX-NO, can enhance the effect of carmustine on T98G cells but not in U-87MG cells. Additionally the combination therapy affected the proliferation ability of glioma cells even after the therapeutic agents had been removed. To further elucidate the mechanism of this NO induced chemosensitivity, the affect of NO on O6-methylguanine DNA methyltransferase activity will be examined.

Figure 1:  (A) Cell viability of T98G cells incubated with varying concentration of CTX-NO, with and without carmustine. Tumor cell viability is decreased dramatically when cells are exposed to a combination of CTX-NO and carmustine. (B) Cell proliferation of cells that were exposed to CTX-NO, with and without carmustine, for 48 hours, trysinized, incubated with fresh media for 48 hours and then counted. The ability to proliferate was severely hindered by the combination therapy.

Error bars indicate standard deviation, *p <0.05