The data presented in this work demonstrate that the TPP derivatives drug A and, to a lesser extent, drug B selectively inhibit growth of a cell line derived from a malignant breast metastasis (DU4475) compared to a transformed cell line derived from normal human breast epithelial cells (HBL-100). Combinations of A and B also display selective growth inhibition, although in contrast to previous results the combinations do not act synergistically in this cell model. Drugs A and B, used alone at high concentrations and in combination, were shown to cause increases in mobile or NMR-visible lipid as measured by 1D 1H NMR. These increases were confirmed by 2D NMR, which also revealed changes in phospholipid metabolites similar to the actions of the parent compound TPP. The significance is that congeners of the cytotoxic compound TPP cause changes in lipid metabolism similar to that of the parent compound at comparable effective doses. Moreover, 5-FU, a structurally and functionally dissimilar cytotoxic agent in current clinical use, also causes similar changes in lipid metabolism. However, the fact that increased NMR-visible lipid does not occur with MTX treatment indicates that these lipid changes are not unique to CLPS and are not characteristic of all cytotoxic agents.
The selective cytotoxicity of A and B against DU4475 relative to HBL-100 cells by factors of 13.6 and 3.6, respectively, is consistent with our previous research showing that the parent compound TPP has a selectivity ratio of 42.3 in the same cell model (14, 15). This selectivity may reflect higher transmembrane potentials in the DU4475 breast carcinoma cell line compared to the transformed breast cell line HBL-100, which is often characteristic of neoplastic cells (1, 2, 9). The growth inhibitory effects of these compounds (and selectivity ratios) occur at doses comparable to those demonstrated in PaCa-2 human pancreatic carcinoma cells and CV-1 untransformed monkey kidney epithelial cells, and similarly show compound B to be effective at lower absolute doses than compound A, albeit only marginally for DU4475 cells (2). MTX and, to a lesser extent, 5-FU were shown to be relatively nonselective against DU4475 compared to HBL-100 cells. Although many antineoplastic agents in current clinical use are effective against proliferating cells in vivo, they do not demonstrate remarkable selectivity against tumor-derived compared to untransformed cells in vitro where both cells types are actively proliferating (2, 11).
Compounds A and B are potentially capable of covalently combining more effectively in malignant cells than normal cells in situ to form an adduct more active than the precursors, hence resulting in target-selective synergism (2, 3). Combinations of A and B have been shown to exhibit target-selective synergism against Ehrlich Lettre ascites (ELA) carcinoma cells relative to untransformed CV-1 cells in vitro (3). However, cytotoxic synergism of combinations of compounds A and B was not demonstrated in either HBL-100 or DU4475 cells, with the combinations acting antagonistically or, at best, additive. This indicates either that the two individual drugs do not act synergistically or that the two drugs do not form a more active combination product in this cell model. This could be due to a number of factors. Compounds A and B could be taken up at different rates by different cells, which can be affected by differences in drug lipophilicity or cellular plasma membrane potential (2). Moreover, the difference in mitochondrial relative to plasma membrane potential between cell types may cause the drugs to localize in different parts of the cell (2). The structural similarities of A and B may result in competitive inhibition of drug uptake or action. Alternatively, compound C may self-assemble within these cells, but its cytotoxic effects may be curtailed by rapid elimination or metabolism. It is also possible that compound C is simply not as effective as either of its precursors against these cell lines, or that treatment with a different concentration or schedule may affect the synergism of these two compounds. Since the synergistic action of drug C is based on enhanced toxicity subsequent to combination of the drug precursors, further experiments are needed to assess the cytotoxicity of compound C and determine whether or not these criteria are met in HBL-100 and DU4475 cells.
Increased NMR-Visible Lipid
Proton NMR demonstrated that the major detectable change induced by treatment of two human breast cell lines with high concentrations (IC50) of the cationic lipophilic compounds A, B, and A+B (1:1) is large increases in resonances from NMR-visible lipid. This increase was also observed in HBL-100 cells following treatment with a combination of A+B at lower concentrations (IC10). The increased signal intensity at 1.3 ppm reflects predominantly an increase in the concentration of mobile lipid acyl chains that give rise to NMR-visible lipid signals, with smaller contributions from lactate and threonine methyl groups. The contribution from lactate was minimized by washing of the cells during harvest. The observed lactate levels in 1D and 2D spectra were not consistent under any of the conditions employed. Increases in mobile lipid can be due to either an increased production of fatty acyl chains or an increased mobility of existing fatty acyl chains, or both. HBL-100 cells treated with 5-FU, but not MTX, also developed increased resonances from mobile lipid acyl chains and glycerol backbone, indicating that these changes are not unique to CLPS, nor are they general for all anticancer agents. Moreover, the observation that high levels of mobile lipids could occur at a concentration that is not cytotoxic (IC10 A:B combination) indicates that the production of mobile lipid is not directly related to the degree of toxicity of CLPS and, by implication, not directly related to cell death. Furthermore, the decrease in mobile lipids observed at higher A+B combinations does demonstrate that mobile lipid production is concentration dependent. This confirms our previous study of TPP-treated HBL-100 cells, in which the maximum in lipid acyl chain resonances in 1D spectra and lipid cross-peak volumes in 2D spectra occurred at a concentration approximately 10-fold less than the IC50 (15). Increases in NMR-visible lipid have been observed in cultured cells following treatment with a number of cytotoxic agents (17–23) in response to adverse culture conditions (16, 23) and can be modulated by such factors as the degree of drug resistance (24, 25). The relationship between these diverse stimuli and increased NMR-visible lipids is not currently understood, and it is unclear whether the similar phenotype changes arise from the same or different cellular pathways.
In 2D spectra, both HBL-100 and DU4475 cell lines display a CLPS-induced increase in the G′ (G1′ and G3′) cross peak arising from the geminal protons on carbons 1 and 3 of the glycerol backbone, indicating that increased NMR-visible lipid arises from triglycerides, phosphoglycerides, or their derivatives (26–28). Spectra from drug-treated HBL-100 and DU4475 cells also showed a significant increase in the volumes for the two cross peaks GA and GB arising from the vicinal coupling between HA and HB on carbons 1 and 3, and HC on C-2 on the glyceride backbone. This suggests a contribution from triglycerides, although the intensity of these cross peaks was often weak—especially in HBL-100 cells. Restriction of head group mobility means that the vicinal cross peaks GA and GB (GA > GB) are often visible only in 2D COSY spectra of cells or lipoproteins containing large amounts of triglyceride (27, 29), and cells containing a large number of cytoplasmic lipid droplets, e.g., oleate-treated myeloma cells (30). No cross peaks specific to the glyceride backbone of phospholipids were observed in the spectra of HBL-100 and DU4475 cells, which suggests that the NMR-visible lipid acyl chains are not derived from phospholipids. The asymmetry of protons in the glycerol backbone of phospholipids causes a shift in GA and GB to 4.10, 5.20 and 4.37, 5.20 ppm (with a corresponding shift in G′ to 4.10, 4.37 ppm) and the appearance an additional cross peak at 3.95, 5.20 ppm arising from the vicinal coupling of HC on C-2 to the equivalent protons on C-3 adjacent to the phosphate group (28).
The drug-induced increase in lipid acyl chains observed using 1D and 2D NMR is consistent with changes observed in HBL-100 and DU4475 cells treated with the structurally related compound, TPP. TPP treatment also caused a concentration-dependent increase in GPC and decreases in PC at low TPP concentrations (15, 16). In HBL-100 cells, PC recovered at higher TPP concentrations, and an increase in choline was also observed. In the present study, increases in choline were observed in HBL-100 cells or DU4475 cells following high-dose treatment with drug A, and in HBL-100 cells treated with 5-FU or the drug A+B combination at the IC50. PC was observed to decrease in all high-concentration treatments in HBL-100 cells. Increases in GPC were only observed for high-dose (IC50) A+B treatment. We have previously proposed that the induction of mobile lipid and the accompanying changes in phospholipid metabolites with TPP treatment can be explained by the action of phospholipases on existing cellular phospholipids (15, 16). The increased fatty acyl chain resonances and GA and GB cross peaks observed here indicate accumulation of neutral glycerides, such as triglycerides, which could be formed from the fatty acids liberated by phospholipase A1 and A2 activity. While an increase in GPC may be expected to accompany this activity, this may not be observable in all situations. In TPP-treated HBL-100 cells we only observed significant increases in GPC at concentrations above the IC50 (15).
In proliferating tumor cells, relatively high levels of PC and/or PE are detected by both in vitro and in vivo by 31P NMR, and these metabolites generally decrease significantly after effective chemotherapy (31, 32). HBL-100 cells (but not DU4475 cells) treated with A, B, or A+B showed a decrease in phosphocholine at doses that inhibit cell proliferation by 50%. In addition, a decrease in phosphocholine has been observed in HBL-100 and DU4475 breast carcinoma cells treated with TPP (14, 15), as well as in murine cells cultured under adverse conditions (16). Since PC and PE are soluble membrane precursors, they are more likely to be freely mobile, and therefore changes in cross-peak volumes are more likely an indicator of concentration changes. Inhibition of phospholipase C and/or decreased choline kinase activity could lead to a reduced concentration of PC. Decreases in PC have been correlated with reduced membrane synthesis arising from decreased activity of choline kinase (33, 34). Some investigators have suggested it could be due to reduced phospholipid breakdown associated with signal transduction pathways that stimulate cell division (35).
Further insight into the drug-induced processes that may give rise to such alterations in lipid metabolism can be obtained from other studies investigating the toxic effects of CLPS. The consistent observation that TPP and other cationic lipophilic compounds cause substantial mitochondrial damage (4, 7, 36) suggests that this may be the source of NMR-visible lipid. The damage of mitochondria could result in the release or buildup of a number of metabolites, including fatty acids and GPC. Generally, these catabolites would be used for recycling into phospholipids, production of triglycerides, or for mitochondrial β-oxidation. However the utilization of fatty acids would be restricted by the amount of available ATP, already depleted by cationic lipophilic treatment (4, 37). ATP is required to convert fatty acids to their acyl-S-CoA derivatives for further lipid synthesis. Moreover, mitochondrial damage would inhibit the process of β-oxidation. The observed NMR-visible lipid accumulations could reflect such events, and would also depend on the drug type, cytotoxicity of the drug, dose and treatment schedule, and on the cell system examined. Lipid accumulation is involved in cellular stress and cell death following a variety of insults, including treatment with CLPS, although the actual role of this process remains unclear. Elucidation of the cytotoxicity mechanism of CLPS could identify various stages in the lipid metabolism of cells, which could be used for the design of new anticancer therapies.