(483h) Targeted Nitric Oxide Pretreatment Alters p53 and O6-Methylguanine-DNA Methyltransferase Activity Resulting in Enhanced Chemosensitivity in Glioma Cells

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 µM) or TMZ (50 µM) 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 µM 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 µM or 2 µM, 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 µM), # compared to
pretreatment with CTX-NO (2 µM) p <0.05,
n = 3