Magnetic resonance spectroscopy for detection of choline kinase inhibition in the treatment of brain tumors

Abstract

Abnormal choline metabolism is a hallmark of cancer and is associated with oncogenesis and tumor progression. Increased choline is consistently observed in both preclinical tumor models and in human brain tumors by proton magnetic resonance spectroscopy (MRS). Thus, inhibition of choline metabolism using specific choline kinase inhibitors such as MN58b may be a promising new strategy for treatment of brain tumors. We demonstrate the efficacy of MN58b in suppressing phosphocholine production in three brain tumor cell lines. In vivo MRS studies of rats with intracranial F98-derived brain tumors showed a significant decrease in tumor total choline concentration after treatment with MN58b. High-resolution MRS of tissue extracts confirmed that this decrease was due to a significant reduction in phosphocholine. Concomitantly, a significant increase in poly-unsaturated lipid resonances was also observed in treated tumors, indicating apoptotic cell death. MRI-based volume measurements demonstrated a significant growth arrest in the MN58b-treated tumors in comparison with saline-treated controls. Histologically, MN58b-treated tumors showed decreased cell density, as well as increased apoptotic cells. These results suggest that inhibition of choline kinase can be used as an adjuvant to chemotherapy in the treatment of brain tumors and that decreases in total choline observed by MRS can be used as an effective pharmacodynamic biomarker of treatment response.

Figure 1

MTT assay demonstrating inhibition of cellular viability of F98, 9L and 9L-EGFRviii cell lines with MN58b. 24 h MN58b exposure significantly reduced the viability of the F98, 9L and 9L-EGFRviii cells in a dose dependent manner. The data was normalized to untreated control cells and represents the average ± SEM of 3 separate experiments performed in quadruplicate.

Figure 2

Effect of MN58b on ChoK activity in tumor cells. Autoradiography of TLC-separated ¹⁴C-choline containing metabolites (A) allows for the quantification of ¹⁴C-PC and demonstrates a dose-dependent reduction of choline phosphorylation in response to MN58b treatment. The graph (B) shows a dose dependent reduction of ChoK activity in the F98, 9L and 9L-EGFRviii cell lines. Error bars represent ± SEM of 3 separate experiments.

Figure 3

In vitro NMR of F98 cell extracts (A). Untreated cells display high levels of PC (3.22 ppm) and low levels of GPC (3.23 ppm) (top spectrum); 10 μM MN58b reduces the PC resonance relative to GPC (middle spectrum) and 20 μM MN58b further reduces this ratio (bottom spectrum). (B) A bar graph showing the quantitation of PC and GPC from untreated, 10 and 20 μM MN58b-treated cells (C). The PC/GPC ratio demonstrates differences between untreated, 10 and 20 μM MN58b-treated cells. Asterisk (*) represents significant differences with a p-value of <0.05. Error bars represent ± SEM of 3 separate experiments.

Figure 4

Representative in-vivo ¹H MR Spectrum (A) from baseline untreated F98 tumor (bottom line) and MN58b treated (top line) tumor. The MR image demonstrates placement of the voxel for in vivo MRS studies. Bar graph (B) demonstrates the percentage changes in volume in saline-treated (checked) and MN58b-treated (black) tumors. Normal tissue is represented by the white bar. The baseline untreated tumor demonstrates high tCho (horizontal lines, C) and low Lip/lac (D). MN58b treatment (black) resulted in reduced tCho and increased Lip/lac level at 1.3 ppm (D) and polyunsaturated lipids at 2.8 ppm (Lip2.8) (E). Asterisk (*) represents significant differences with a p-value of <0.05. Error bars represent ± SEM.

Figure 5

(A) Representative in vitro ¹H NMR spectrum from perchloric acid extracts of saline-treated (bottom) and MN58b-treated F98 tumors (top). The zoomed region (inset) is the spectral region from the choline-containing metabolites. Bar graph (B) demonstrates changes in PC and GPC levels from contralateral normal tissue, saline-treated tumors and MN58b-treated tumors. Bar graph (C) demonstrates the PC/GPC ratio in the 3 groups. Asterisk (*) represents significant differences with a p-value of <0.05. Error bars represent ± SEM.

Figure 6

Representative histological sections from saline-treated and MN58b-treated F98 rat brain tumors. H&E (A to D, 20 and 40x) and Caspase-3 (E to H, 20 and 40x) immunohistochemistry in tumor sections demonstrating decreased cell density in MN58b-treated tumors (B and D) compared to saline-treated tumors (A and C). Caspase-3 positive cells are increased in MN58b-treated tumors (F and H) compared to saline-treated tumors (E and G) indicating increased apoptosis. Scale bar is 50 μm.