Cytotoxicity of unsaturated fatty acids in fresh human tumor explants: concentration thresholds and implications for clinical efficacy

Abstract

Background: Unsaturated fatty acids (UFAs) exhibit in vitro cytotoxicity against many malignant cell lines and yield decreased cancer incidence and reduced tumor growth in animal models. But clinical and animal studies to date have achieved response using only localized delivery methods such as intratumoral infusion. To explore possibilities for enhanced clinical efficacy, fresh surgical explants of tumors from 22 patients with five malignancies were exposed to gamma-linolenic acid (GLA) and alpha-linolenic acid (ALA) and analyzed with an in vitro chemosensitivity testing system, the Fluorescent Cytoprint Assay (FCA). A total of 282 micro-organ cultures derived from these malignant tumors were exposed to GLA and ALA at different concentrations.

Results: GLA and ALA exhibited greater than 90% cytotoxicity at a sharp concentration threshold between 500 microM and 1 mM against all but two malignant micro-organ cultures tested in 5-10% serum. In tests using 30-40% serum, GLA and ALA killed tumor at concentrations of 2 mM and above.

Conclusions: The concentration threshold of 500 microM to 2 mM exhibited for antitumor activity by GLA and ALA is much higher than that observed in most previously reported cell culture studies but consistent with physiological concentrations found to kill tumor clinically and in animals. A mechanism of antitumor activity by unsaturated fatty acids through selective destabilization of the malignant plasma membrane is considered. An oral regimen is proposed for phase I clinical testing that could push the area under the curve for serum concentration of unbound unsaturated fatty acids over time to much higher levels than previously achieved for systemic administration and into the range that could yield antitumor response.

Figure 1

Cytotoxic activity of UFAs exposed to micro-organs from malignant and normal tissue explants. A: Viability values (0 = sensitive, 1 = resistant) are shown for a metastatic ovarian tumor exposed to GLA (black square) and ALA (Grey square); 26 controls (represented as 0 μM GLA) were all viable. B: Results for a primary ovarian tumor. Viability values of 0 at 2 mM (two each for GLA, ALA) are not shown. Four control values were 1.0 (3) and 0.75 (1). C: Results for a glioblastoma tumor; 6 control were all viable. D: Results for a normal stromal tissue specimen; all 4 controls were viable. A numeric pair (m, n) depicts m and n identical results, respectively, for GLA and ALA represented by the bar below. All four experiments used 10% fetal calf serum.

Figure 2

Aggregate data for UFA exposure to micro-organs from malignant tumor explants in 5-10% fetal calf serum. Viability values are shown for 153 micro-organ cultures from malignant tumors exposed to GLA (Black square) or ALA (Grey square) in 5% or 10% fetal calf serum. Tumor types tested, as detailed under methods, were ovarian adenocarcinoma, breast carcinoma, pancreatic carcinoma, melanoma, and glioblastoma. Parenthesized values above bars here and in the following two figures represent multiple data points as in Fig. 1. For example, at a 50 μM GLA concentration, the viability readings were 1.0 (resistant) for 4 micro-organ cultures, 0.5 (intermediate) for 2, and 0.25 (intermediate-sensitive) for 1.

Figure 3

Aggregate data for UFA exposure to micro-organs from malignant tumor explants in 5-10% human serum. Viability values are shown for 103 micro-organ cultures from malignant tumors exposed to GLA (Black square) or ALA (Grey square) in 5% or 10% human serum. (Two viability readings of 0 for 2.5 mM GLA are not shown.)

Figure 4

Aggregate data for UFA exposure to micro-organs from malignant tumor explants in 30-40% human serum. Viability values are shown for 22 micro-organ cultures from malignant tumors exposed to GLA (Black square) or ALA (Grey square) in 30% or 40% human serum. (Viability readings of 0 for 4 mM GLA and ALA, one each, are not shown.