Synergistic interaction between lipid-loading and doxorubicin exposure in Huh7 hepatoma cells results in enhanced cytotoxicity and cellular oxidative stress: implications for acute and chronic care of obese cancer patients

Abstract

There has been a dramatic increase in the number of clinically obese individuals in the last twenty years. This has resulted in an increasingly common scenario where obese individuals are treated for other diseases, including cancer. Here, we examine interactions between lipid-induced steatosis and doxorubicin treatment in the human hepatoma cell line Huh7. The response of cells to either doxorubicin, lipid-loading or a combination were examined at the global level by DNA microarray, and for specific endpoints of cytotoxicity, lipid-loading, reactive oxygen species, anti-oxidant response systems, and apoptosis. Both doxorubicin and lipid-loading caused a significant accumulation of lipid within Huh7 cells, with the combination resulting in an additive accumulation. In contrast, cytotoxicity was synergistic for the combination compared to the individual components, suggesting an enhanced sensitivity of lipid-loaded cells to the acute hepatotoxic effects of doxorubicin. We demonstrate that a synergistic increase in reactive oxygen species and deregulation of protective anti-oxidant systems, most notably metallothionein expression, underlies this effect. Transcriptome analysis confirms synergistic changes at the global level, and is consistent with enhanced pro-inflammatory signalling in steatotic cells challenged with doxorubicin. Such effects are consistent with a potentiation of progression along the fatty liver disease spectrum. This suggests that treatment of obese individuals with doxorubicin may increase the risk of both acute (i.e. hepatotoxicity) and chronic (i.e. progress of fatty liver disease) adverse effects. This work highlights the need for more study in the growing therapeutic area to develop risk mitigation strategies.

Fig. 1. Synergistic cytotoxicity between doxorubicin and lipid-loading in Huh7 cells. (A) Huh7 cells were exposed to varying concentrations of fatty acids or a 300 μM FFA mixture (2 : 1 v/v oleate : palimitate) for 24 h and then cell viability measured by MTT assay. Huh7 cells, either naïve (black bars) or pre-loaded with 300 μM FFA mixture for 24 h (open bars), were exposed to varying concentrations of fatty acids, or doxorubicin for 24 h, and then cell viability (B) or (C) caspase 3/7 activity was measured. A combination index was calculated to examine potential interactions between the FFA mixture and doxorubicin. Each data point represents the mean of three independent experiments. Error bars represent the standard error of the mean (SEM). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle control or the indicated comparison.

Fig. 2. Additive lipid-accumulation between doxorubicin and lipid-loading in Huh7 cells. Huh7 cells, either naïve or pre-loaded with 300 μM FFA mixture (2 : 1 v/v oleate : palimitate) for 24 h, were exposed to 3.6 μM doxorubicin for 24 h. Intracellular lipid accumulation was detected by Nile Red and (A) quantified or (B) visualised by fluorescence microscopy. For lipid quantitation, each data point represents the mean of three independent experiments. Error bars represent the standard error of the mean (SEM). * = p < 0.05.

Fig. 3. Transcriptomic analysis of Huh7 cells exposed to doxorubicin and/or lipids reveals marked commonality in response. Huh7 cells, either naïve or pre-loaded with 300 μM FFA mixture (2 : 1 v/v oleate : palimitate) for 24 h, were exposed to 3.6 μM doxorubicin for 4 h or 12 h, and then transcriptomes analysed by Illumina bead array. Differentially expressed genes (DEGs) are presented as Hive plots: each axis represents and individual comparison, with DEGs presented as grey nodes, with position along the axis determined by fold-change; where DEGs are common between two axes, they are connected by an arc. DEGs were determined from three independent treatments for each condition, with an adjusted p-value <0.05 deemed significant.

Fig. 4. Synergistic oxidative stress between doxorubicin and lipid-loading in Huh7 cells. Huh7 cells, either naïve or pre-loaded with 300 μM FFA mixture (2 : 1 v/v oleate : palimitate) for 24 h, were exposed to the indicated concentration of doxorubicin for various times and then various parameters measured: (A) ROS, (B) transcripts associated with oxidative stress response, and (C) metallothionein protein levels. Each data point represents the mean of three independent experiments. Error bars represent the standard error of the mean (SEM) * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Fig. 5. Synergistic interactions between doxorubicin and lipid-loading in hepatocytes. In naïve hepatocytes (left panel), exposure to either doxorubicin or free fatty acids (FFA) results in steatosis and oxidative stress. GPX/MT-mediated adaptation acts to reduce ROS levels, minimising the necessity for cell death through apoptosis. In steatotic hepatocytes (right panel), the combination of doxorubicin and FFA exposure leads to additive lipid accumulation, and synergistic intracellular ROS levels. This overwhelms the GPX/MT adaptive system, resulting in increased cell death, release of pro-inflammatory signals and potentiation through the fatty liver disease spectrum.