Alteration of Drug Sensitivity in Human Colon Cancer Cells after Exposure to Heat: Implications for Liver Metastasis Therapy using RFA and Chemotherapy

Abstract

Radiofrequency ablation (RFA) is gaining popularity for treating colorectal liver metastases by inducing image guided tumor hyperthermia. In order to reduce tumor recurrence, adjuvant therapies have been administered post-RFA. We hypothesized that tumor cells escaping RFA cytotoxicity by being in the sublethal zones of tumor might develop differential behavior toward cytotoxic drugs. Here, we used cultured human colorectal cancer cells to evaluate the interaction between heat treatment and chemotherapeutic agents. Human colon cancer cell lines HT29 and HCT116 were subjected to temperatures of 42 degrees to 50 degrees C for 15 min, in combination with 5-fluorouracil, oxaliplatin, or irinotecan at different sequences. Cytotoxicity was determined by MTT assay. The cell cycle progression was analyzed by flow cytometry with propidium iodide staining. The expression of several genes associated with drug sensitivity was quantitated by real-time RT-PCR before and after heat treatment. Either heat treatment at 45 degrees C by simultaneous or pre-treatment with three different chemotherapeutic agents didn't affect the cytotoxicity of the combined treatment to HT29 and HCT116 cells, except for irinotecan treatment in HCT116 cells. However, when pre-exposure to 45 degrees C, HCT116 cells, but not HT29 cells, developed resistance to these three drugs. In an analysis of cell cycle profile after the drug followed heat treatment, a longer delay in cell cycle progression in HCT116 cells was observed in comparison to HT29 cells. Furthermore, HCT116 and HT29 cells exhibited different expression profiles of several drug-related genes in response to heat treatment at 45 degrees C. An observation of a differential response to the drug and heat treatment sequences between two human colon cancer cell lines suggests that tumor heterogeneity and selection of chemotherapeutic agents need to be under consideration in the clinical setting.

Keywords: Hyperthermia; cell cycle; chemotherapy; colon cancer; drug resistance; gene expression.

Figure 1

Effect of different temperatures on cell viability. HT29 (•) and HCT116 (□) cells were subjected to water bath at an indicated temperature for 15 min and then returned to a 37 °C incubator. Cell viability was determined by MTT assay 72 h after heat treatment.

Figure 2

Effect of immediate heart treatment on the sensitivity to 5-FU, oxalipalatin and irinotecan. HT29 (A) and HCT116 (B) cells were subjected to various concentrations of a drug, immediately followed by heat treatment at 37° (○) 42° (•) and 45°C (▾) for 15, min, Drugs were removed from medium after 24 h. Cell viability was determined by MTT assay 72 h after heat treatment. Data are shown as mean ± SD. Cell survival for each temperature was corrected for heat cytotoxicity.

Figure 3

Effect of pre-exposure to heat on the sensitivity to 5-FU, oxaliplatin and irinotecan. HT29 (A) and HCT116 (B) cells were exposed to heat at 37° (○), 42° (•) and 45°C (▾) for 15 min. Twenty-four hours after heat treatment, cells were subjected to various concentrations of a drug for 24 h. Cell viability was determined by MTT assay 72 h after heat treatment. Data are shown as mean ± SD. Cell survival for each temperature was corrected for heat cytotoxicity.

Figure 4

Effect of pre-treatment of 5-FU, oxaliplatin and irinotecan on heat-sensitivity at different temperatures, HT29 (A) and HCT116 (B) cells were pretreated with a drug for 24 h before heat treatment at 37° (□), 42°() and 45°C (▪) for 15 min. Drugs were removed form medium before heat treatment. Cell viability was determined by MTT assay 72 h after heat treatment. Data are shown as mean ± SD. *p < 0.05, compared to cells at 37°C by the Student's test. Cell survival for each termperature was corrected for heat cytotoxicity.

Figure 5

Effect of heat treatment and chemotherapeutic agents on cell cycle distribution. HT29 and HCT116 cells were subjected to 5-FU (5 uM), oxaliplatin (1 uM), or irinotecan (5 uM) with or without heat treatment at 45°C. In the single treatment, cells were harvested 24 h after treatment and subjected to cell cycle analysis. In the combined treatment, cells were pre-exposed to heat treatment at 45°C for 15 min. Twenty-four hours after heat treatment, cells were subjected to a drug for another 24 h. Flow cytometry with propidium iodide staining was used for cell cycle analysis. The numbers at the right top corner of each histogram indicate the percentage of cell phase distribution in the order of sub-Gl, Gl, S, and G2/M from top to bottom. The data represent three independent experiments.

Figure 6

Effect of heat treatment on the mRNA levels of several genes associated with drug sensitivity. Total RNAs were isolated from HT29 and HCT116 cells at 6 h and 24 h after heat treatment (45°C for 15 min). Cells subjected to 37°C were used as controls. The mRNA level of each gene was measured by real-time RT-PCR and its expression level was normalized to GAPDH expression. Ratio of gene expression is given as log of normalized target gene expression after heat treatment versus controls. For clarity, SD is not shown and all are less than 10% of each value.