HYPERTHERMIA EFFECT ON HUMAN NORMAL BREAST (MCF-10A) AND CANCER (MDA-MB 231 AND MCF-7) CELLS

Objective: In this study, the hyperthermia effect on the viability of human normal breast (MCF-10A) and cancer (MDA-MB 231 and MCF-7) cells was evaluated by MTT assay. Methods: Cells were exposed to heat at 38oC, 39oC, 40oC, 41oC, 42oC, 43oC, and 44oC for five different durations of heat exposure (0.5, 1, 2, 3, and 4 h). Breakpoint temperatures of MCF-10A, MDA-MB 231, and MCF-7 were determined using cumulative equivalent 43°C (CEM43) model. This model was first time used to calculate thermal isoeffect dose (TID) for MCF-10A, MDA-MB 231, and MCF-7. Results: MCF-10A started to die at 42°C for 3 h while MDA-MB 231 and MCF-7 need a temperature of 38°C for 0.5 h; thus, they were identified as the threshold temperatures in CEM43 model. Furthermore, the effect of “43°C incubator 2 h” had similar total thermal dose as “44°C incubator 0.5 h” for MDA-MB 231 and MCF-7. In addition, “43°C incubator 3 h” effect had also almost the same thermal dose as “44°C incubator 1 h” for MDA-MB 231 and MCF-7. Conclusion: A better understanding of the significant correlations between CEM43 and response parameters in clinical trials could be useful to treat breast cancer patients.


INTRODUCTION
Breast cancer is a major global health problem and the most common invasive cancer in women of all ethnic backgrounds. Worldwide, estimated 1.6 million new cases are diagnosed for each year [1,2]. There are many attempts with a multitude of novel therapeutic concepts although the conventional methods based on surgery, chemotherapy, radiotherapy, or their combinations steadily develop [3].
Hyperthermia among them has attracted significant attention and already entered clinical practice as an adjuvant to chemotherapy and radiotherapy [4]. It is used to raise the temperature of a region of the body affected by cancer with minimal or no damaging healthy tissues [5]. Thermal chemosensitization and thermal radiosensitization effects have been observed both in vivo and in in vitro cell culture experiments [6]. At least 18 randomized studies have demonstrated that the synergistic effects of combining hyperthermia with either chemotherapy or radiotherapy or both to achieve better therapeutic effects [5]. This was demonstrated for the breast, cervix, head and neck, pancreas, bladder, esophagus, prostate, lung, vulva/vagina cancers, and for melanoma. Hyperthermia has shown great potential in overcoming multidrug resistant (e.g. doxorubicin) which may result in the accumulation of chemotherapy agents within the target cells [6]. Rolf (2008) observed that synergism as a continuous change with increasing the rate temperatures at which cells are killed by the drug [7]. It is generally accepted that when temperatures are raised from 37ºC to over 40ºC, most alkylating agents (e.g., cyclophosphamide and ifosfamide), platinum compounds, and nitrosoureas (bis-chloroethylnitrosourea, and 1-2-chloroethyl-3-cyclohexyl-1-nitrosourea) are linearly enhanced in their cytotoxic effect. On other the hand, threshold temperatures for the interaction with heat at or near 42.5ºC have a synergistic effect with doxorubicin or bleomycin meanwhile most antimetabolites (e.g. 5-fluorodeoxyuridine and methotrexate), vinca alkaloids, or taxanes show independent action [7,8].
Thus, thermal isoeffect dose (TID) is important because it helps to predict the outcome in vitro for a given heat dose. It can be applied to sensitize phenomena with cytostatic drugs, anticancer agents, and radiation therapy to improve better outcome in breast cancer treatment. The TID for induction of cell death was found to be closely related to the amount of energy required to inactivate proteins and enzymes [9]. Although the Arrhenius analysis could be used to calculate the inactivation energy, it is hard to compare two different time-temperature combinations in that plot. Therefore, Sapareto and Dewey [10] described the term "TID" (meaning two different time-temperature combinations produced the same cell killing effect) for comparing different time-temperature combinations. Calculation of the thermal dose applied in hyperthermia has been successfully integrated into the concept of a TID during a certain duration exposure at a given temperature. Treatment outcome varied greatly between different types of cell lines although the same or different settings of hyperthermia used. For example, the proliferation of human osteosarcoma cells was inhibited by hyperthermia treatment at 42°C whereas heat shock at 44°C inhibited proliferation significantly in normal fibroblasts cells [9]. Therefore, different mechanisms were involved in heat shock-induced cell death among normal cell and cancer cell [5]. According to Omar and Lanks [11] investigated that cancer cells are more susceptible to killing by heat than normal cells after the hyperthermia treatment (43°C-45°C).

Elengoe et al.
In the present study, it was aimed to investigate the percentage viability evaluated by MTT assay with time of heat treatments in breast carcinoma (MDA-MB 231 and MCF-7) and normal breast (MCF-10A) cell lines using seven different temperatures (38°C, 39°C, 40°C, 41°C, 42°C, 43°C, and 44°C) for five different durations of heat exposure (0.5, 1, 2, 3, and 4 h). Moreover, breakpoint temperatures of MCF-10A, MDA-MB 231, and MCF-7 cells were determined using cumulative equivalent 43°C (CEM 43 ) model. This model was first time used to calculate TID for MCF-10A, MDA-MB 231, and MCF-7 cell lines.
Hyperthermic exposure MCF-10A, MDA-MB 231, and MCF-7 cells, 1×10 4 cells/well in 200 µl culture medium, were seeded in each well of 96-well plates and precultured overnight incubation at 37˚C. Then, hyperthermic exposure was performed by placing culture plates in an incubator maintained at 38ºC, 39ºC, 40ºC, 41°C, 42°C, 43°C, and 44°C for studying cytotoxicity of cells. Well temperature was monitored and maintained within 0.1°C during the treatment period. Cultured cells were maintained at 37°C served as controls for all experiments. Cultured cells were subjected to 0.5, 1, 2, 3, or 4 h of hyperthermic exposure for each temperature.

Observation under phase contrast microscope
After hyperthermia treatment, MCF-10A, MDA-MB 231, and MCF-7 cells were observed using a phase contrast microscope (Nikon Ti Eclipse). The photographs were taken at ×10/0.03 magnification.

MTT assay
MTT assay [12,13] is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT, to its insoluble formazan, giving a purple color. The culture medium was removed from the culture plates after appropriate treatment and added with 200 µl of fresh culture medium and 20 μl of MTT media. The well plates were incubated for 4 h at 37°C. The mixture was aspirated slowly from the wells. Then, a volume of 200 μl of isopropanol was added to each well. Well plates were kept in dark for 15 minutes. Finally, absorbance at 570 nm in each well was measured using an ELISA microplate reader (BioTek Instrument). This value was then background subtracted (from media only wells) at 650 nm and compared with controls, which are the values of cells without any treatment for obtaining cell growth. The average absorbance of the control cells exposed to free culture medium was set to represent 100% of viability, and the results were expressed as percentage of these controls.
Cell viability percentage was calculated using the following equation as below: % cell viability = (Optical density of hyperthermia treated cells/Optical density of untreated cells) ×100%.

Statistical analysis
Results were expressed as a mean ± standard error of the mean (SEM) (SEM was within 5% of the average). The mean values were calculated from data taken from one experiment performed in triplicates. Significance testing was performed where indicated using one-factor analysis of variance. The differences were evaluated statistically significant at p<0.05.

Calculation of thermal isoeffect dose
The concept of a TID has been successfully integrated from the calculation of the thermal dose during a certain duration of heat exposure at a given temperature. This relationship can be expressed mathematically by the following isoeffect equation: where T 1 and T 2 are two different temperatures of treatment and t 1 and t 2 are the duration exposure of heat at temperature T 1 & T 2 , respectively. The shape of the dose-response curve is defined by empirical value, R. R is usually assumed as 0.5 above 43 °C and 0.25 below 43°C by Sapareto and Dewey [10]. Heating time periods at different temperatures are converted into equivalent heating minutes at 43°C which can be explained using the TID concept. From equation (1), if set T1 = 43°C and allow T 2 to be varied during heat treatment, get which is the widely used cumulative equivalent minutes 43°C model. Treatment outcome varied hugely between different types of cell lines although the same settings of hyperthermia used. Furthermore, small changes in hyperthermia settings could influence greatly difference in the final treatment outcome. Therefore, the viability of normal and cancer cells is highly influenced by the temperature and duration exposure of heat (hyperthermia settings). For example, induction of apoptosis for human fibrous histiocytoma cells was inhibited hyperthermia treatment at 43°C for 1 h, a temperature lower than that was not able to induce apoptosis although increase the duration of heat exposure. However, Yonezawa et al. observed that necrotic death occurred at temperature of 46°C for 1 h where cell death was passive and involved lysis formation from the damaged cell and the release of its cellular content to the surrounding environment [14]. In contrast, heat shock at 44°C inhibited induction of apoptosis significantly in normal fibroblasts cells [9]. Therefore, different mechanisms were involved in heat shock-induced cell death among normal cell and cancer cells [5].

RESULTS AND DISCUSSION
In this study, MDA-MB 231 and MCF-7 cells were started to die at temperature of 38°C for 0.5 h, whereas MCF-10A cell line was started to show cytotoxicity at 42°C for 3 h of hyperthermic exposure. It was reasonable because MDA-MB 231 and MCF-7 cell lines sensitive to heat; therefore, the facilitating effect of hyperthermia to cancer cell was obvious. This was due to MDA-MB 231 and MCF-7 cells did not have a defense system against heat, as did MCF-10A cells [15]. MCF-10A might express heat shock-induced proteins to reactivate denatured proteins; degrade abnormally structured proteins; and inhibit secretion of abnormal proteins and assisting the transfer of secretory proteins by blockage of folding [16]. In addition, Hsp70 induction by hyperthermia treatment in cancer cells is lower than those in normal cells [17].

Elengoe et al.
Therefore, MCF-10A cells were survived in undesirable circumstances such as at 42°C incubator hyperthermia for 2 h. However, cell death of MCF-10A cells occurred when temperature increased to 42°C (>2 h of heat exposure). MCF-10A, MDA-MB 231, and MCF-7 cells might die exponentially by induction of apoptosis where cell death was active. When the temperature and duration exposure continued to increase, the cellular proteins denature at that high temperature and the cell dies passively (necrosis) before initiation of apoptosis for an extreme condition such as hyperthermia treatment on MDA-MB 231 and MCF-7 cells at a temperature of 44°C for 4 h. In this study, normal cell line might die exponentially by induction of apoptosis but they might not reach until necrosis. Based on Gabai and Kabakov study [18], cytoskeleton damages and impairment of DNA repair system caused by hyperthermia treatment most likely occur during mitosis; therefore, MDA-MB 231 and MCF-7 (cancer) cells were more susceptible to heat than MCF-10A (normal) cell as cancer cells underwent faster cell division than normal cells. According to Watanabe and Suzuki (1989) [19], normal cells have reduced heat sensitivities 5-6 times at growing condition while cancer did not.
Although cancer therapies such as chemotherapy and radiotherapy can induce tumor cell apoptosis, physiological stress conditions such as growth factor, starvation, hypoxia, or heat can also be equally effective to DNA damaging treatment [20]. Abnormalities of these environmental factors affect tumor cell proliferation and response to antitumor therapy.  [22], rat muscle and skin have a greatly enhanced blood flow at temperatures above 42°C while tumors have a greatly reduced blood flow at these temperatures. Reduction of blood flow and blood vessel density, resulting in regions with hypoxia and low pH levels, which is not found in normal tissues under undisturbed conditions. Hypoxia might exhibit anaerobic metabolism with resultant accumulation of lactic acid [23]. Acute acidification pH increases the rate of cell death by decrease heat shock protein levels [24].

CONCLUSION
The temperature of 42°C for 2 h was chosen as the most suitable temperature and duration of heat exposure to kill cancer cells (MDA-MB 231 and MCF-7) without damaging normal cells (MCF-10A).

Calculation of thermal isoeffect dose
Cell death of MCF-10A, MDA-MB 231, and MCF-7 cell lines from 37°C to 44°C for 0.5 h up to 4 h was studied. When exponentially growing cultured cells (MCF-10A, MDA-MB 231, and MCF-7) were exposed to a defined temperature between 37°C and 44°C of heat exposure, a doseeffect curve was defined by plotting the rate of cell viability against the duration of hyperthermia. The survival curves of MDA-MB 231 and MCF-7 cell lines show a linear growth arrest at the beginning of heat exposure which reflecting reversible and nonlethal heat damage and then, followed by exponential cell death. This is typical "shoulder" which reflects a two-step process of cell killing. However, after increasing the temperature to 43°C, more cells were killed during the same period of heat exposure. Therefore, it was concluded that once the hyperthermia treatment starts to show cytotoxicity, the rate of cell death, which is exponential with exposure duration, is dependent on the temperature of exposure [25].
Arrhenius plot is hard to use for compare two different timetemperature combinations. Thus, the term "TID" (meaning two different time-temperature combinations produced the same cell killing effect) was proposed by Sapareto and Dewey for comparing different timetemperature combinations [10]. The thermal dose can be described as the cumulative equivalent minutes at 43°C for any hyperthermia treatment.
In this study, MDA-MB 231 and MCF-7 cells need temperature of 38°C for 0.5 h to induce apoptosis; therefore, it was identified as the threshold temperature in CEM 43 model. For MCF-10A cell line, it was clearly indicated that a 42°C hyperthermia treatment for 3 h started to show very little cytotoxicity (Table 1), so the breakpoint for MCF-10A was at temperature of 42°C for 3 h. The thermal energy dose was required to induce exponential cell death (inactivation energy) which is closely correlated to that required for cellular proteins and enzymes denaturation [10]. It can be combined with cytostatic drugs (e.g. 5-fluorouracil, melphalan, and paclitaxel), anticancer agents, radiation, gene, and immunotherapy to achieve better outcome in breast cancer treatment. For example, the use of simultaneous application of weekly cisplatin and regional hyperthermia resulted in a 50% response rate. However, the response rate was about 15% without regional hyperthermia for patients with locoregional advanced cervix carcinoma in a clinical trial. A better understanding of the significant correlations between CEM 43 and response parameters in clinical trials could useful to treat breast cancer patients.

AUTHOR'S CONTRIBUTIONS
AE designed the research, performed the experiments, analyzed data, and wrote the paper. NJBMA provided the MCF-10A cells. SH contributed critical comments and suggestions. All authors read and approved the final manuscript.