Abstract
Background/Aim: Despite improvement in current therapies, the 5-year overall survival rate of colorectal carcinoma is still low especially in its metastatic form. On the other hand, hyperthermia has been utilized as a cancer treatment approach to improve overall therapeutic efficacy. In the present study, we have aimed to develop an optimized hyperthermic protocol against an in vitro model of human colon carcinoma, as a single and/or adjuvant treatment approach. Materials and Methods: We have utilized an in vitro model of human colorectal carcinoma consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cell lines. Cells were exposed to 45°C, over 120 min, in the presence or absence of chemotherapeutic (5-Fluorouracil, Capecitabine) and targeted (Bevacizumab, Cetuximab) drugs. Cell viability levels were determined by the Alamar-blue assay while determination of cell death, reactive oxygen species (ROS) production, mitochondrial membrane depolarization (ΔΨμ) levels and cell cycle progression were performed by flow cytometry. Results: CaCo2 and HT29 cells showed a differential response towards i) cell viability, ii) cell death, iii) ROS and ΔΨμ levels as well as iv) cell cycle distribution, in the presence of hyperthermia alone (monotherapy) or in combination with the above-mentioned drugs (adjuvant therapy). Finally, normal colon epithelial (CCD841CoN) cells remained minimally affected. Conclusion: We have developed an optimized experimental hyperthermic protocol, as a promising monotherapy and/or adjuvant therapy approach, with the capacity to potentiate chemotherapeutic as well as targeted drug-induced cytotoxicity against a model of colorectal carcinoma, to a variable degree.
Colorectal cancer (CRC) is one of the most frequent types of cancer and among the deadliest. It is currently considered to be the third-leading cause of cancer-related deaths worldwide (1). The severity of the disease is reflected by the fact that although the 5-year survival rate is approximately 70-90% in patients with localized tumor, in cases of metastatic CRC, there is a dramatic decrease of relative rates to 10-12% (2). Currently, surgical resection of CRC is the main therapeutic approach for localized tumors, followed by radiotherapy with/without chemotherapy as single or combined therapeutic strategies. On the other hand, in patients with mCRC a combination of radiotherapy/chemotherapeutic drugs [5-fluorouracil (5-FU), irinotecan and oxaliplatin] along with targeted therapeutic agents (bevacizumab, cetuximab, panitumumab and regorafenib) is the most common treatment approach (3-5). However, the presence of high heterogenicity among patients with CRC, regarding different genetic backgrounds and the presence of thousands of somatic mutations in both oncogenes and tumor-suppressor genes, also reflects the high variability in individual responses of patients to different CRC treatment modalities. Thus, there is an urgent need for the development of more efficient therapeutic protocols leading to improved clinical outcomes (6, 7).
Hyperthermia is currently considered the fourth type of anticancer therapy following surgery, radiotherapy and chemotherapy (8). Generally, hyperthermia is defined as an abnormal increase of tissue/body temperatures through an external thermal source. Specifically in therapy terms, it is defined as a modest elevation of temperature in the range of 39-45°C with the aim of directly killing cancer cells (9, 10). The basic property underlying the use of hyperthermia in the clinical setting is the observed high sensitivity of cancer cells to temperature oscillations, ultimately resulting in reduced proliferative rates, as evidenced by various in vitro and in vivo cancer models (11, 12). In this context, hyperthermia has been widely utilized as an apoptotic inducer (13, 14) as well as a sensitizer to both radiotherapy and chemotherapy, thereby enhancing their therapeutic efficacy in a variety of cancer types (15, 16). Overall, hyperthermia is characterized by significant biological effects including: i) reduction of cell survival, ii) induction of a cellular stress response, iii) immunomodulatory activity, iv) evasion of DNA-repair mechanisms, vi) alterations of tumor microenvironment and vii) sensitization to radiotherapy and chemotherapy, further justifying the importance of such a therapeutic approach in the clinical setting (17, 18). However, it is widely accepted that the efficacy of hyperthermia as a single or in combinatorial anticancer therapy is associated with various challenging factors including temperature range and duration of application thus suggesting the need for developing optimized protocol(s) towards an efficient therapeutic outcome (16).
In the present study, we utilized an in vitro model of CRC consisting of colorectal carcinoma (HT29, CaCo2) and normal colon epithelial (CCD841CoN) cells in an attempt to optimize an experimental platform of hyperthermic exposure as a monotherapy or adjuvant therapy against CRC. To this end, we explored the potential of hyperthermia as an adjuvant therapeutic approach by potentiating the effectiveness of chemotherapeutic (5-FU, capecitabine) as well as targeted drugs (bevacizumab, cetuximab) all of which are widely used in the clinical setting.
Materials and Methods
Cell lines and reagents. The human colorectal carcinoma (CaCo2, HT29) cell lines were purchased from Sigma-Aldrich (St. Louis, MO, USA) while human normal colon (CCD841CoN) cells were obtained from LGC Standards (Teddington, UK). CaCo2 and CCD 841CoN cells were maintained in minimal essential medium while HT29 cells were cultured in McCoy’s 5A medium. All media were supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin). Cells were grown in a humidified incubator at 37°Cwith5% CO2, grown as monolayers, and sub-cultured when confluency reached 80-90%. All tested cell lines were cultured for a maximum of 15-20 passages before the use of new stocks. The cell culture media as well as additional culture reagents (fetal bovine serum, phosphate buffer saline (PBS), trypsin and antibiotics) were purchased from Labtech (Heathfield, UK) and Biosera (Kansas City, MO, USA). Resazurin sodium salt used for the estimation of cell viability levels was purchased from Fluorochem (Hadfield, UK). Finally, all cell culture plasticware was obtained from Greiner Bio One (Stonehouse, UK). For the determination of apoptosis, CellEvent Caspase 3/7 Green flow cytometry assay kit was purchased from Thermo Fisher Scientific (Medisell, Nicosia, Cyprus) while JC-1 assay used for the determination of mitochondria membrane depolarization was from Invitrogen, Thermo Fisher Scientific. Finally, dihydrorhodamine-123 (DHR) assay from Sigma-Aldrich (St. Louis, MO, USA) was used for the determination of reactive oxygen species (ROS) levels.
Hyperthermic exposures. HT29, CaCo2 and CCD841CoN cell lines were exposed to 45°C for different times in a humidified incubator (with 5% CO2). Specifically, an appropriate number of cells were plated into 96-well cell culture plates and incubated at 37°C overnight. In all experimental protocols, and before exposure to different hyperthermic treatments, the culture medium was changed and cell culture plates were transferred into an incubator set at 45°C with 5% CO2 and then incubated for 120, 180 and 240 min. At the end of hyperthermia, plates were returned to an incubator at 37°C for an additional incubation of 24 h (post-exposure) prior to all analyses.
Determination of cell viability. Determination of cell viability was performed using the Alamar-blue assay. Briefly, cells were seeded into 96-well plates, in 100 μl/well, and incubated overnight prior to exposure to different temperatures and time-courses. In a first series of experiments, cell viability was determined immediately after starting hyperthermic incubation (0 h) as well as 24 h after exposure. In all monotherapy protocols, 20,000, 22,000 and 15,000 CaCo2, HT29 and CCD841CoN cells/well, respectively, were plated before cell viability was measured immediately whereas 10,000, 11,000 and 12,000 of the same cells/well, respectively, were plated for the determination of cell viability levels at 24 h post-exposure. Similarly, in all adjuvant protocols, CaCo2, HT29 and CCD841CoN cells were plated at a density of 3,000, 4,500 and 5,000 cells/well when cell viability was determined at 72 h post-exposure in all drug combinations. In both protocols, cells under untreated (control) conditions were incubated with complete medium at 37°C. At each endpoint, fresh medium (containing 0.1 mg/ml resazurin) was added to each well of a 96-well plate and plates were incubated for 2-6 h (depending on the cell line) at 37°C. Afterwards, cell culture plates were centrifuged and absorbance was recorded at 570 nm and 600 nm (reference wavelength), using a Tecan Spark multi-mode plate reader (Mannedorf, Switzerland). Cell viability was calculated and expressed as percentage of that of untreated (control) cells.
Determination of apoptosis. Determination of apoptosis was performed with the use of CellEvent Caspase 3/7 Green flow cytometry assay kit, according to the manufacturer’s instructions. Briefly, cells were plated into 60 mm dishes and allowed to adhere overnight. On the next day, cells were exposed to 45°C for 120 min in the absence (monotherapy) or presence (adjuvant therapy) of different compounds including 5-FU (3.25 μg/ml), capecitabine (360 μg/ml), bevacizumab (250 μg/ml) and cetuximab (250 μg/ml). All plates were then returned to 37°C for a further 24 h (monotherapy conditions) or 72 h post-exposure incubation period (adjuvant therapy conditions), respectively. Next, cells were harvested, washed twice with PBS and a single-cell suspension of 106 cells/ml was prepared. Then, 0.5 μl of CellEvent Caspase 3/7 Green detection reagent was added to 0.5 ml of cell suspension and samples were incubated at 37°C for 30 min. Five minutes prior to the end of the incubation period, 1 μM of 4’,6-diamidino-2-phenylindole (DAPI) was added in order to detect necrotic cells. Caspase-3/7-positive and DAPI-positive cells were identified as apoptotic and necrotic cells, respectively. Data were acquired by flow cytometry.
Determination of intracellular ROS levels. Following hyperthermic exposure, cells were harvested and washed twice with PBS. A single-cell suspension of 106 cells/ml was prepared and incubated for 15 min at 37°C with DHR (5 μΜ for CaCo2 cells, 20 μM for HT29 cells and 10 μM for CCD841CoN cells). DAPI (1 μM) was also added to each sample which were then incubated for 5 min in order to stain dead cells. Data were then acquired by flow cytometry. Dead (DAPI-positive) cells were excluded from further data analysis.
Determination of mitochondrial membrane depolarization. After the end of hyperthermic exposure, cells were harvested and washed twice with PBS. A single-cell suspension of 106 cells/ml was used for this series of experiments. JC-1 staining solution was prepared according to the manufacturer’s instructions. JC-1 was added at a final concentration of 1 μg/ml into 0.3 ml of PBS-suspended cells and samples were incubated at 37°C for 30 min. Afterwards, cell suspensions were washed twice with PBS and centrifuged at 154 × g for 5 min. Data were then acquired by flow cytometry.
Cell-cycle analysis. FxCycle PI/RNase staining solution was used according to the manufacturer’s instructions and was purchased from Thermo Fisher Scientifics (Medisell). Following hyperthermia, cells were harvested and washed twice with PBS. Approximately 0.5×106 cells were fixed in cold 70% ethanol, for 1 h or longer at 4°C until being further processed. Afterwards, in order to remove ethanol, cells were washed twice with PBS and finally suspended in FxCycle PI/RNase staining solution for 30 min at room temperature in the dark.
Data acquisition and analysis. For each sample analyzed by flow cytometry, data acquisition and analysis of 20,000 events was performed using a FACS Canto II flow cytometer and Flow Jo V10 software (BD Biosciences, San Jose, CA, USA).
Data are expressed as mean values±standard error of the mean, while comparisons were performed between untreated (control) and treated groups. All data analyses and calculations were performed by using the Microsoft Office Excel 2016 software. In addition, means were compared by one-way analysis of variance with Tukey’s test for multiple comparisons. Graph Pad Prism (GraphPad Software, San Diego, CA, USA) was used for all statistical analyses. A value of p<0.05 was considered statistically significant.
Results
Based on our previous findings, exposure of cells to different temperatures (41°C to 47°C) over different time-courses (1-4 h) caused a reduction of cell viability levels in CaCo2 and HT29 colorectal cancer cells, of variable degree, according to the intensity of the exposure (data not shown). Overall, HT29 cells were more sensitive to these hyperthermic conditions than CaCo2 cells. However, a significant decrease was only observed at 45°C and 47°C (both of which caused a significant decline in cell viability at 24 h post-treatment) with 47°C being substantially more cytotoxic than 45°C, in both cell lines (data not shown). In this context, we included normal colon epithelial (CCD841CoN) cells to determine the non-cytotoxic profile of the 45°C exposure protocol. To this end, CaCo2, HT29 and CCD841CoN cells were exposed to 45°C for different times (120, 180 and 240 min) and viability was assessed immediately after as well as 24 h post-treatment.
Our results indicated that at 45°C, CaCo2 cells were more resistant (Figure 1A) than HT29 cells (Figure 1B) under all exposures and regardless of being assessed either immediately or 24 h-post exposure. On the contrary, CCD841CoN cells were significantly more resistant 24 h post exposure (when compared to HT29 and CaCo2 cells), thus indicating their thermotolerance even at prolonged duration (240 min) (Figure 1C). Given the minimal cytotoxicity observed at 180 and 240 min, in CCD841CoN cells, we concluded that an optimized hyperthermic protocol would include the experimental conditions of 45°C for 120 min, with measurement at 24 h post exposure.
The effect of hyperthermia-induced cytotoxicity on HT29 (A), CaCo2 (B) and CCD841CoN (C) cells. Cells were exposed to 45°C for 120, 180 and 240 min then cell viability was determined immediately after as well as at 24 h after exposure. Data are expressed as means±standard error of the mean and are representative of three independent experiments. Significantly different at p<0.05 from the control at 37°C #immediately after exposure and *24 h post exposure.
Next, we sought to evaluate different endpoints of cytotoxicity as a biological response to hyperthermia against CaCo2, HT29 and CCD841CoN cells. Specifically, we determined the generation of ROS (Table I), extent of apoptotic and necrotic cell death (Figure 2), mitochondrial membrane depolarization (ΔΨμ) (Table II) and cell-cycle arrest (Figure 3) by various flow cytometry-based methodologies.
The effect of hyperthermia on the production of reactive oxygen species (ROS). CaCo2, HT29 and CCD841CoN cells were exposed to 45°C for120 min and ROS levels were determined by flow cytometry at 24 h post exposure. Data shown represent the fold change compared to untreated (control) cells expressed as mean values±standard error of the mean and are representative of two independent experiments.
The effect of hyperthermia on cell death in CaCo2, HT29 and CCD841CoN cells. Cells were exposed to 45°C for 120 min and monitored by means of flow cytometry. Cytograms show caspase 3/7 activation in CaCo2, HT29 and CCD841CoN cells at 24 h post treatment, where L, A and N indicate live, apoptotic and necrotic cell subpopulations, respectively.
The effect of hyperthermia on cell-cycle distribution in CaCo2, HT29 and CCD841CoN cells. Cells were exposed to 45°C for 120 min and monitored by means of flow cytometry. Histograms show distribution of cells in sub-G1, G1, S and G2/M cell-cycle phases in CaCo2, HT29 and CCD841CoN cells at 24 h post treatment.
Firstly, we investigated the potential involvement of ROS in mediating hyperthermia-induced cytotoxicity. For this reason, we utilized a DHR fluorescent probe to detect intracellular ROS by flow cytometry. Our data revealed that hyperthermia did not cause a statistically significant alteration of total intracellular ROS in any cell line tested, indicating that ROS were not involved in modulating its cytotoxic effect (Table I). Secondly, we utilized a commercially available CellEvent Caspase 3/7 Green flow cytometry assay kit, together with DAPI, in order to determine the levels of apoptotic and necrotic cells, respectively. Overall, there was no indication of apoptotic nor necrotic cell death being involved. In fact, the rates of these two modes of cell death were comparable to those observed in untreated cells (at 37°C) (Figure 2 and Supplementary Table I). To this end, we also evaluated ΔΨμ as an indicator of the capacity of hyperthermia to induce the intrinsic apoptotic cascade. For this reason, we utilized JC-1 dye as an indicator of mitochondrial dysfunction in a flow cytometry-based approach. Our results revealed that in CaCo2 cells, there was no depolarization of mitochondrial membrane (as the ratio of JC-1 aggregates/JC-1 monomers was comparable to that of untreated cells) induced by hyperthermia. However, this was not the case in HT29 cells (in which hyperthermia induced mitochondrial membrane depolarization shown as a reduced ratio of JC-1 aggregates/JC-1 monomers), although this was not shown to be statistically significant. Moreover, CCD841CoN cells remained largely unaffected, thus suggesting an effect of our hyperthermia protocol specifically against CRC cells only (Table II). Thirdly, we examined (by flow cytometry) the effect of hyperthermia in inducing cell-cycle arrest. Briefly, in CaCo2 cells, hyperthermia caused a statistically significant induction of growth arrest indicated by increased cell subpopulations at the G2/M and S cell-cycle phases. Alternatively, in HT29 cells, hyperthermia caused a statistically significant increase in the G2/M cell cycle phase only while the S phase remained relatively unaffected. Finally, hyperthermia also induced very minimal and non statistically significant changes in the G2/M phase of the cell cycle only, in CCD841CoN cells, indicative of their overall unresponsiveness to these hyperthermic exposure conditions (Figure 3 and Supplementary Table II).
The effect of hyperthermia on mitochondrial membrane depolarization. CaCo2, HT29 and CCD841CoN cells were exposed to 45°C for 120 min and mitochondrial membrane depolarization was determined by flow cytometry as the ratio of JC-1 aggregates to JC-1 monomers at 24 h post exposure. Data shown are mean values±standard error of the mean and are representative of two independent experiments.
In the next series of experiments, we evaluated the cytotoxic potential of chemotherapeutic (5-FU and capecitabine) as well as targeted drugs (bevacizumab and cetuximab) in the presence or absence of hyperthermia. Specifically, CaCo2, HT29 and CCD841CoN cells were exposed to each drug at 37°C or 45°C with cell viability being determined at 72 h post treatment. According to our data, 5-FU caused a significant decrease of cell viability in CaCo2 and HT29 cells, whilst hyperthermia did not induce a potentiation of its cytotoxic effect (Figure 4). In addition, exposure to capecitabine alone resulted in significant reduction of cell viability in both CaCo2 and HT29 cells, the magnitude of which was substantially potentiated by hyperthermia. This was especially the case in HT29 cells, suggesting their increased thermo-sensitivity when compared to CaCo2 cells (Figure 4A and B). On the other hand, response profiles comparable with that for capecitabine were observed with both bevacizumab and cetuximab, in that there was a slight decrease of cell viability when used alone and a much more profound effect in the presence of hyperthermia. Once again, the magnitude of this potentiation was more evident in HT29 than in CaCo2 cells, an observation in agreement with previous results confirming an increased thermosensitivity of these cells (Figure 4A and B). Finally, all tested drugs (whether as single agents or in combination with hyperthermia) were shown to minimally affect CCD841CoN cells as evident by cell viability (above 80% in all tested conditions) only being slightly reduced (Figure 4C).
The effect of hyperthermia on drug-induced cytotoxicity in CaCo2, HT29 and CCD841CoN cells. Cells were exposed to 3.25 μg/ml 5-fluorouracil (5-FU), 360 μg/ml capecitabine, 250 μg/ml bevacizumab or 250 μg/ml cetuximab either as single agents at 37°C or in combination with hyperthermia (45°C) for 120 min, and cell viability was determined. Data are expressed as means±standard error of the mean and are representative of three independent experiments. Significantly different at p<0.05 from the control at #37°C and at *45°C.
Next, we determined the mode of cell death induced by each of these drugs (as single agents and in combination with hyperthermia) in CaCo2 and HT29 cells only (given our observation that CCD841CoN cells remained largely unaffected). Exposure of CaCo2 cells to each drug as a single agent caused no significant changes in their apoptotic or necrotic subpopulations. However, in the presence of hyperthermia all drugs were capable of potentiating the effect of cell death (apoptotic/necrotic) to a variable degree (Figure 5 and Supplementary Table III). In the case of HT29 cells, at 37°C, a similar pattern of cell death was observed to that of CaCo2 cells, with the exception of 5-FU. At 45°C apoptotic cell death was more profound for all drugs tested (Figure 5 and Supplementary Table III). Finally, we assessed the capacity of these drugs to regulate cell-cycle progression. Specifically, in CaCo2 cells, at 37°C, all drugs reduced the subpopulation of cells in the S and G2/M phases of the cell cycle. At 45°C, all drugs appeared to follow the same pattern that is, to minimally reduce the subpopulation of cells in the S phase while that of the G2/M phase was slightly increased (Figure 6 and Supplementary Table IV). On the other hand, in HT29 cells, at 37°C, all tested drugs induced a minimal increase of the subpopulation of cells in the sub-G1 phase. At 45°C, there appeared to be a minimal growth arrest indicated by a slight decrease in cells at the S and G2/M phases of the cell cycle (Figure 6 and Supplementary Table IV).
The combinational effect of hyperthermia with drugs on the activation of cell death in CaCo2 and HT29 cells. Cells were exposed to 3.25 μg/ml 5-fluorouracil (5-FU), 360 μg/ml capecitabine, 250 μg/ml bevacizumab or 250 μg/ml cetuximab, either as single agents at 37°C or in combination with hyperthermia (45°C) for 120 min, and monitored by means of flow cytometry. Cytograms show caspase-3/7 activation in CaCo2 and HT29 cells at 72 h post treatment, where L, A and N indicate live, apoptotic and necrotic cell subpopulations, respectively.
The combinational effect of hyperthermia with drugs on cell-cycle distribution in CaCo2 and HT29 cells. Cells were exposed to 3.25 μg/ml 5-fluorouracil (5-FU), 360 μg/ml capecitabine, 250 μg/ml bevacizumab or 250 μg/ml cetuximab either as single agents at 37°C or in combination with hyperthermia (45°C) for 120 min, and monitored by means of flow cytometry. Histograms show distribution of cells in sub-G1, G1, S and G2/M cell-cycle phases in CaCo2 and HT29 cells at 72 h post treatment.
Discussion
CRC is associated with high rates of incidence and mortality among both men and women, worldwide. Currently, treatments against CRC include surgery, radiotherapy and chemotherapy. However, the clinical benefit is limited to patients with early and localized tumors. Moreover, due to the acquired chemo- and radioresistance to conventional therapeutics, along with the occurrence of systemic toxicity and several side-effects, the overall survival rate among patients with CRC is still low (2). In order to overcome such limited response rates, the use of more efficient therapeutic protocols is more than urgent. Recent scientific data provide evidence of the therapeutic safety and efficacy of various combinational treatments in the context of reducing systemic toxicity (thereby improving therapeutic outcomes) and consequently improve overall survival in patients with cancer. In this context, hyperthermia is considered an important anticancer and non-invasive therapeutic procedure (19). It is capable of inducing cell death thereby leading to the activation of senescence, induction of mitotic catastrophe and activation of necrosis and apoptosis (11, 12). Moreover, its pleiotropic effects also involve the immune system, the tumor microenvironment and the vascular system (12, 20). In addition, hyperthermia is efficient in targeting hypoxic and low-nutrient areas of tumors that remain less sensitive to both radiation and chemotherapy-based treatments (12, 20). However, in the clinical setting, hyperthermia has been restricted to being a sensitizing factor which can potentiate therapeutic outcome(s) through combinational treatment protocols. It has been reported that such combinations improve local tumor control and survival rates among patients with brain, pancreatic, lung, liver, bladder, gastric and colorectal cancer (16, 21-24). Furthermore, it appears that the therapeutic potential of hyperthermia is subject to the temperature applied, duration of treatment, cancer cell type and treatment protocol design (single or combinational), thereby affecting cancer cell response as well as therapeutic outcome (16). Thus, the development of an optimized hyperthermia protocol against a valid cancer cell model is of paramount importance in determining the underlying molecular mechanisms of the mode of action of hyperthermia.
In this context, initially, we aimed to develop an optimized hyperthermia protocol as a potential anticancer approach in an in vitro model of CRC [consisting of human colon cancer (HT29 and CaCo2) cells] and further determine its safety against human normal colon epithelial (CCD841CoN) cells. Overall, our data at 24 h post exposure indicated that conditions of 45°C for 120 min were optimum. To the best of our knowledge, such an experimental approach is novel in that we have developed a hyperthermia-based therapeutic protocol that is cytotoxic to CRC cells only, while normal colon epithelium cells remained largely unaffected. The inclusion of normal cells in experimental studies of cancer therapeutics is of crucial importance in documenting cancer-related specificity and safety (by minimizing cytotoxicity to normal cells that surround the tumor site). Finally, another important finding was the increased sensitivity of HT29 cells to hyperthermia compared with CaCo2 cells. In general, HT29 cells are considered to be drug-resistant when compared to other colorectal cell lines (including CaCo2) (25, 26) while they are also characterized by having mutated/deleted p53 alleles (27, 28). In addition, the HT29 cell line is derived from an aggressive type of CRC as opposed to the origin of CaCo2 cells (25). Taking all this into consideration, it would be reasonable to expect a differential response profile between these two cell lines, following hyperthermic exposure. However, the effect of other factors, including the role of heat-shock proteins and cell-cycle progression regulators should also not be excluded. Another study has shown that although HT29 cells were more resistant to oxaliplatin, hyperthermia resulted in increased drug sensitivity, superior to that observed in CaCo2cells (25). Similarly, in our study, it may be speculated that hyperthermia can act as a chemosensitizing agent (by reversing HT29 resistance) and, as such, this may be a rational explanation for the observed thermosensitivity of these cells.
Moreover, we observed that our hyperthermic conditions did not result in a statistically significant alteration of apoptotic or necrotic subpopulations of CaCo2 and HT29 cells, at 24 h post exposure, a response also observed in normal epithelial CCD841CoN cells. However, other studies have shown the occurrence of necrotic, apoptotic or both modes of cell death following hyperthermia. For instance, exposure of mastocytoma cells to 42°C resulted in a slight increase of apoptotic cell death, a response further enhanced at 43-44°C. In addition, the same study also reported that exposure to 45°C resulted in the activation of both apoptosis and necrosis, while at 46-47°C there was an increase in necrosis only (29). These findings indicate that the cellular response to hyperthermia is temperature-dependent and that it is more than likely that necrotic cell death occurs at elevated temperatures (30). In support of this, necrotic lesions following focal hyperthermia were also reported in a murine model of colorectal liver metastasis (31). Furthermore, several studies indicate that hyperthermia alone can trigger apoptotic cell death in various types of tumors including melanoma (13), colon carcinoma (32), glioma (33), osteosarcoma (34) and cervical cancer (35). On the other hand, hyperthermia has been reported to induce ROS production, thereby leading to mitochondrial-induced activation of apoptotic cell death in various models of colonic, lymphoma and prostatic carcinomas (36-38). In this context, we evaluated the involvement of an oxidative stress response as a potential anticancer mechanism following hyperthermia in CaCo2, HT29 and CCD841CoN cells. Our data revealed that hyperthermia did not cause alterations in the redox state of colorectal nor normal epithelial cells, thus suggesting that ROS are not involved in modulating hyperthermia-induced cytotoxicity. Furthermore, when assessing ΔΨμ following hyperthermia, we observed a slight decrease of the ratio of JC-1 aggregates/JC-1 monomers (indicative of apoptotic activation) in HT29 but not in CaCo2 and CCD841CoN cells. The work of others has indicated the involvement of ROS production and alterations in mitochondrial membrane potential as underlying mechanisms of hyperthermia-induced apoptosis. For instance, short exposure of osteosarcoma U-2OS cells to 43°C caused an increase in intracellular ROS levels and mitochondrial dysfunction, leading to activation of caspase-3 (34). Exposure to sub-lethal hyperthermia was shown to also activate apoptosis through ROS formation, an effect that caused enhancement of the anticancer activity in doxorubicin-treated hepatoma (HepG2) cells (39). Moreover, when assessing the cell-cycle distribution, significant cell-cycle growth arrest was observed (especially at the G2/M phase of the cell cycle) in CaCo2 and HT29 cells exposed to hyperthermia. On the contrary, normal epithelial (CCD841CoN) cells exhibited a normal distribution of cell-cycle phases similarly to that of cells under control (untreated) conditions. Our findings are consistent with those of others reporting alterations in cell-cycle progression of cancer cells as a response to hyperthermia. In particular, exposure of HCT116 colorectal carcinoma cells to 42°C was shown to induce apoptotic cell death through increased transcriptional activation of p53 while at the same time causing growth arrest at the G1/G0 cell-cycle phase (32). Finally, exposure to the same hyperthermic conditions also resulted in the potentiation of oxaliplatin-induced growth arrest at the G1/S phase of the cell cycle in CaCo2 and HT29 cells (25).
Various drugs are currently used in the clinical setting as first-line treatment options in patients with metastatic CRC such as 5-FU and capecitabine (both chemotherapeutic agents) in addition to other targeted drugs such as bevacizumab (an anti-vascular endothelial growth factor agent) and cetuximab (an anti-EGFR agent) (3-5, 40-43). In this context, we aimed to determine if our hyperthermia protocol was capable of potentiating the therapeutic action of these drugs in our in vitro model of CRC. In fact, hyperthermia did potentiate the cytotoxic effects of these drugs to a variable extent in both CaCo2 and HT29 cells. Specifically, HT29 cells were shown to be more sensitive than CaCo2 cells to the combined action of hyperthermia by reducing cell viability, inducing cell death and increasing cell-cycle growth arrest. Once again, CCD841CoN cells were minimally affected by the action of these drugs as single agents or in combination with hyperthermia. Previous studies by other groups have also evaluated the effect of hyperthermia as an adjuvant approach against CRC cell lines. For instance, two recent studies provided evidence that a combinatorial protocol of hyperthermia with nanoparticles containing 5-FU resulted in increased levels of DNA damage against HT29 cells (44, 45). In addition, others have shown the synergistic effect of a triple combinational protocol (i.e. hyperthermia, tumor necrosis factor-related apoptosis-inducing ligand and mitomycin) in inducing apoptosis in various CRC cell lines (LS174T, LS180, HCT116, CX-1) (46). Finally, in another study, hyperthermia was shown to enhance the cytotoxic effect of cisplatin against CaCo2 cells by inducing apoptosis (47).
Overall, the findings of our study provide evidence that our hyperthermia protocol was capable of inducing significant cytotoxicity against CaCo2 and HT29 cells without affecting CCD841CoN cells. Although hyperthermia-induced cytotoxicity has been the subject of intense research in CRC cells, to our knowledge, there has not been a study where the effect of hyperthermia was examined in normal colon epithelial cells as a means of determining a safety profile for a range of hyperthermic temperatures. This has allowed us to optimize the temperature range of hyperthermia-induced cytotoxicity to being specific for CRC cells only and not for normal colonic epithelial cells (surrounding the tumor site). To our knowledge, this is a unique and novel feature of our protocol development as a monotherapy or adjuvant therapy. In this context, the development, optimization and application of our hyperthermia protocol might potentially lead to reduction of the acquired/effective dose of any therapeutic agent, thereby reducing its systemic toxicity to healthy cells and ultimately its side-effects. Finally, we documented an effect of hyperthermia (to a variable degree) in potentiating the therapeutic action of chemotherapeutic as well as targeted drugs used in the clinical setting. Although such observations have been made before, in the case of chemotherapeutic drugs, as far as we are aware, there has never been a study demonstrating such a potentiating effect with targeted drugs. Consequently, we aim to delineate the underlying major cellular pathways (e.g. oxidative stress, cell death and cell-cycle growth arrest) involved in the potentiation of such drug action in order to develop adjuvant therapies, in line with the concept of personalized medicine, so that they can greatly benefit patients with CRC.
Acknowledgements
This work was partially supported by i) a grant provided by the Cyprus Institute of Neurology and Genetics (Telethon Cyprus), Nicosia, Cyprus (MIP) and ii) a scholarship provided by the Hellenic Surgical Society (GP).
Footnotes
Authors’ Contributions
SB and MIP developed the concept and designed the current study; GP and TM conducted the experiments, collected and processed the data; GP, TM, MIK, SB and MIP drafted and revised the article. All Authors have read and approved the final submitted version of the article.
Supplementary Material
Supplementary research data can be accessed at: <https://www.cing.ac.cy/en/about-us/biomedical-sciences-/cgtup/supplementary-research-data>
Conflicts of Interest
The Authors have no conflicts of interest to disclose.
- Received February 23, 2022.
- Revision received March 18, 2022.
- Accepted March 24, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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