The Temperature-Dependent Effectiveness of Platinum-Based Drugs Mitomycin-C and 5-FU during Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in Colorectal Cancer Cell Lines


Abstract

Cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment with curative intent for peritoneal metastasis of colorectal cancer (CRC). Currently, there is no standardized HIPEC protocol: choice of drug, perfusate temperature, and duration of treatment vary per institute. We investigated the temperature-dependent effectiveness of drugs often used in HIPEC.

Methods: The effect of temperature on drug uptake, DNA damage, apoptosis, cell cycle distribution, and cell growth were assessed using the temperature-dependent IC50 and Thermal Enhancement Ratio (TER) values of the chemotherapeutic drugs cisplatin, oxaliplatin, carboplatin, mitomycin-C (MMC), and 5-fluorouracil (5-FU) on 2D and 3D CRC cell cultures at clinically relevant hyperthermic conditions (38-43 °C/60 min).

Results: Hyperthermia alone decreased cell viability and clonogenicity of all cell lines. Treatment with platinum-based drugs and MMC resulted in G2-arrest. Platinum-based drugs display a temperature-dependent synergy with heat, with increased drug uptake, DNA damage, and apoptosis at elevated temperatures. Apoptotic levels increased after treatment with MMC or 5-FU, without a synergy with heat.

Conclusion: Our in vitro results demonstrate that a 60-min exposure of platinum-based drugs and MMC are effective in treating 2D and 3D CRC cell cultures, where platinum-based drugs require hyperthermia (>41 °C) to augment effectivity, suggesting that they are, in principle, suitable for HIPEC.

Keywords: 5-fluorouracil; HIPEC; colorectal cancer; hyperthermia; hyperthermic intraperitoneal chemotherapy; mitomycin-C; platinum-based drugs.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Human colorectal cancer cells are sensitive to hyperthermia. Five different human colorectal cancer cell lines: RKO, HCT116, MDST8, COLO320, and HUTU80, were used to study the sensitivity to hyperthermia. (A) Cell viability assays were performed at 48 h after hyperthermia treatment at 38, 39, 40, 41, 42, and 43 °C. (B) Long-term cell survival after hyperthermia treatment at 41, 42, and 43 °C was assessed using clonogenic survival assays performed at 10 days after treatment. For both experimental setups, cells at 37 °C represent the untreated controls. The mean of 10 independent experiments are presented.
Figure 2
Figure 2
The effect of oxaliplatin is temperature-dependent, mitomycin-C (MMC) is effective at any temperature. To study the temperature-dependent effect of oxaliplatin and MMC, RKO, HCT116, MDST8, COLO320, and HUTU80 cells were exposed to oxaliplatin or MMC under hyperthermic condition for 60 min. Cell viability assays were performed at 48 h after treatment with oxaliplatin (A) or MMC (B). Thermal Enhancement Ratio (TER) values are demonstrated by a color gradient: a darker color indicates higher TER values. Data represent the mean of 3 independent experiments. (C) The cellular uptake of oxaliplatin was assessed by measuring platinum uptake in cells using the CyTOF at 1 h after treatment. Means ± standard error of the mean of 3 independent experiments are presented. (D) DNA double strand breaks were measured by nuclear γ-H2AX staining. Representative pictures of RKO cells stained for γ-H2AX are presented. (E) Quantification of induced foci in RKO, HCT116, MDST8, COLO320, and HUTU80 cells. Data present the number of foci in 150 cells from 3 independent experiments. Apoptosis levels (F) and cell-cycle distribution (G) were studied using the Nicoletti assay, which was performed 48 h after treatment. Means ± standard error of the mean of 3 independent experiments are presented. Representative flow charts of RKO and MDST8 cells are depicted. (H) Live cell imaging was used to study the growth of RKO cells after treatment. Clonogenic survival assays were performed to assess long-term cell survival 10 days after oxaliplatin (I) or MMC (J) treatment. Means ± standard error of the mean of 4 independent experiments are presented. * p < 0.05, *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
The effect of cisplatin and carboplatin is also temperature-dependent in colorectal cancer cells. To study the temperature-dependent effect of other platinum-based drugs, RKO, HCT116, MDST8, COLO320, and HUTU80 cells were exposed to cisplatin or carboplatin, under hyperthermic condition for 60 min. Cell viability assays were performed 48 h after treatment with cisplatin (A) or carboplatin (B). Thermal Enhancement Ratio (TER) values are demonstrated by a color gradient: a darker color indicates higher TER values. Data represent the mean of 3 independent experiments. The cellular uptake of cisplatin (C) and carboplatin (D) was assessed by measuring platinum uptake in cells using the CyTOF at 1 h after treatment. Means ± standard error of the mean of 3 independent experiments are presented. Apoptosis levels (E) and cell cycle distribution (F) was studied using the Nicoletti assay, which was performed 48 h after treatment. Representative flow charts of HCT116 and COLO320 cells are presented. (G) Live cell imaging was used to study the growth of RKO cells after treatment. Clonogenic survival assays were performed to assess long-term cell survival at 10 days after cisplatin (H) or carboplatin (I) treatment. Means ± standard error of the mean of 4 independent experiments are presented. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
5-Fluorouracil (5-FU), as a systemic drug in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) treatment. (A) Thermal Enhancement Ratio (TER) values of 5-FU obtained from cell viability assay are demonstrated by a color gradient: a darker color indicates higher TER values. The mean of 3 independent experiments are presented. Apoptosis levels (B) and cell cycle distribution (C) was studied using the Nicoletti assay, which was performed at 48 h after treatment with 5-FU. (D) Live cell imaging was used to give more insights about the growth of the cells after treatment. (E) Clonogenic survival assays were performed to assess long-term cell survival at 10 days after 5-FU treatment. Means ± standard error of the mean of 4 independent experiments are presented.
Figure 5
Figure 5
P53 status does not affect the sensitivity of colorectal cancer (CRC) to hyperthermia. P53 protein function plays a central role in the arrest of the cell cycle in G1 phase after DNA damage. To study whether p53 status influences the sensitivity for HIPEC treatment, RC10.1, RC10.2, and RKO p53?/? cells with a decrease of p53 levels and function were used. These cells were treated with hyperthermia alone to assess the cell viability (A) 48 h after treatment and clonogenicity (B) was assessed 10 days after treatment by clonogenic survival assays. For both experimental set ups, cells at 37 °C represent the untreated controls. The mean of 10 independent experiments are presented. (C) Thermal Enhancement Ratio (TER) values obtained from cell viability assays are demonstrated by a color gradient: a darker color indicates higher TER values. The mean of 3 independent experiments are presented. (D) The cellular uptake of oxaliplatin, cisplatin, and carboplatin was assessed by measuring the platinum uptake using the CyTOF. Means ± standard error of the mean of 3 independent experiments are presented. (E) Representative flow charts of the Nicoletti assay to assess apoptotic levels and cell cycle distribution. Clonogenic survival assays were performed to assess long-term cell survival 10 days after treatment with oxaliplatin (F), MMC (G), cisplatin (H), carboplatin (I), and 5-FU (J). Means ± standard error of the mean of 4 independent experiments are presented. * p < 0.05.
Figure 6
Figure 6
Graphical conclusion. (A) Platinum-based drugs require hyperthermia to induce significantly more DNA double strand breaks (DSBs), G2 cell cycle arrest, and increased apoptosis in colorectal cancer cells. (B) MMC treatment, even without hyperthermia, induces DNA DSBs, results in G2-arrest and leads to high apoptotic levels in colorectal cancer cells. Created with Biorender.com.
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