EFFECT OF HYPERTHEMIA IN
TUMORCELLS WITH AN
ISOCITRATE DEHYDROGENASE 1
MUTATION
J.T.S. Schlösser
Department: Biomedical Sciences Supervisor: M. Khurshed Professor: C. J. F. van Noorden
11th august 2018
Abstract:
Isocitrate dehydrogenase 1(IDH1) mutations occurs in various cancer types. IDH1 mutations alters the metabolic environment of the cell by producing 2-hydroxyglutarate(2-HG) thereby consuming NADPH. This results in oncogenesis and decreased detoxification of the IDH1-mutated cells. However, it also increases the treatment sensitivity towards radiotherapy(RT) and chemotherapy as in cisplatin due to changes in reactive oxygen species(ROS)-levels, epigenetics and homologous recombinant(HR) system activity. Hyperthermia(HT) also changes the activity of the HR system and is being used in combination with radio/chemotherapy. In this research, we will investigate the effects of HT in IDH1 mutated(IDH1mut) cancer cells, suggesting that HT will be a valuable addition. Clonogenic
survival assay, cell cycle distribution and immunofluorescence experiments were performed with wild-type IDH1(IDH1wt) and IDH1mut HCT116 cell lines. Thereby adding HT to the combined treatments of RT and HT/cisplatin and HT. In these experiments, we found a higher treatment sensitivity in the IDH1mut cancer cells comparatively to the IDH1mut. Moreover, we
found an increased amount of DSBs in the IDH1mut cancer cells for al treatments comparatively to the IDH1wt cancer cells. Thus, in this research we proclaim that HT is indeed a valuable addition to the treatments of RT and cisplatin for IDH1mut cancer cells.
Introduction
IDH1 is an enzyme that reversibly converts isocitrate to α-ketoglutarate(αKG) with a concomitant reduction of NADP+ to NADPH (1–4). This metabolic reaction is an important aspect of the detoxification of the cytoplasm by using NADPH as detoxification agent (3,5). The heterozygous mutation of IDH1(IDH1mut) causes loss of wild type-IDH1(IDH1wt) enzymatic function via an additional metabolic manifestation: converting αKG to 2-HG with a concomitant oxidation of NADPH to NADP+ (1,2,4,6). The oxidation of NADPH decreases the IDH1-facilitated. NADPH production by 38%. The IDH1mut is prevalent in various cancers, such as
secondary glioma 95%, chondrosarcoma 95%, cholangiocarcinoma 80%, ALL 50%, AML 30-50%(5). On the other hand, 2-HG competitively inhibits the αKG-dependent dioxygenases because of its chemical similarity (2,6,7). 2-HG is an oncometabolite and levels up to 50 mmol/L in the cytoplasm of IDH1mut cell, whereas IDH1wt shows trace amounts of 2-HG in the cytoplasm (2,4,6). These metabolic changes cause alterations in the epigenetics (5,8,9), deficiency of the HR system(1,9) and induces hypoxia by increasing the ROS-levels in the IDH1mut (2,4,5,10). This results in the
oncogenesis of cells with an IDH1 heterozygous mutation (1,2,4,5).
However, recent studies have shown a prolonged survival of glioma patients with an IDH1mut cancer type
versus the IDH1wt cancer type (1,5,11). This could be
caused by intrinsically reduced malignancy and/or the higher sensitivity towards anti-cancer treatments. The aims in the field of this treatment sensitivity are the epigenetics, ROS-production and HR system (1,5,9). The HR system is part of the DNA repair system and is the most important system for the repair of double strand breaks(DSBs). The HR system maintains the integrity of the DNA by using the sister chromatid (1,12,13). DSBs are induced directly by radiation, indirectly via chemotherapy and random replication errors, if not repaired the cell continues to go in apoptosis (12,13). Inhibition of the HR system will lead to sensitization towards the treatment of RT and/or chemotherapy caused by unrepaired DNA. Khurshed et al. (14) described a higher amount of DSBs towards cisplatin in IDH1mut cancer cells. Moreover, recent studies show a decreased activity of the HR system in the IDH1mut cancer cells in comparison to the IDH1wt
cancer cells (1,9). This is interesting since several
studies state that HT also decreases the activity of the HR system (12,15–20).
HT is induced by elevation of the temperature to 40-45oC of the cancer cells with heatwaves or a warmth
bath (12,16,21). This causes a temporal (2-4 hr) degradation of the BRCA2 protein and the dislocation of Rad51 protein, which are essential proteins for functioning of the HR system, thereby impairing the repair of the DSBs. When treated with HT of 42oC, the
cells will be temporarily radio- and chemo- sensitised and is therefore an additional treatment instead of a monotreatment (12,22). Moreover, HT below 42oC
tends to have minimal to none side-effects, which confirms its clinical relevance towards anti-cancer therapies (23).
In this research, the effects of HT combined with radio- and chemotherapy as in cisplatin will be investigated in the IDH1wt and IDH1mut cancer cells. We hypothesize
that IDH1mut cancer cells are more sensitive to HT.
Furthermore, we hypothesize that HT will be a valuable addition to the combined treatment for a higher treatment sensitivity due to its effect on the HR system.
Materials and Methods
Cell culture
The experiments were performed using the homozygous HCT116 IDH1wt/wt cell line (Horizon Discovery) and the heterozygous mutated knock-in HCT116 IDH1wt/R132H cell line (Horizon Discovery). The cells
were cultured in DMEM medium (Gibco; Life Technologie; Thermo Fisher Scientific) supplemented with 10% Fetal bovine serum(FBS) (Thermo Fisher Scientific), 100 units/mL penicillin (Thermo Fisher Scientific) and 100 mg/mL streptomycin (Thermo Fisher Scientific). The cells have been incubated in a 5%CO2 and 37oC incubator. When needed the cells
were splitted by trypsinizing them with 0.25% trypsin in EDTA solution.
Therapies and Reagents
The radiation inflicted on the plated cells were supplied by a radioactive Cs137-source with variable quantities of 1, 2 and 4 Gray(Gy), depending on the experiment. HT treatment was realised by a warm bath with 5% CO2. The plated cells were placed in the warm bath for
The chemo-reagent used for chemotherapy is cis-diamminedichloridoplatin(cisplatin) that is provided by [Pharmachemie B.V.]. The variable concentrations of 2.5 µM and 5 µM were incubated for 48 hours in the medium, after incubation the medium was refreshed. The inhibition of the IDH1mut in the cell is realised with
an addition of the reagent AGI-5198 (Medchem Express). The AGI-5198 is dissolved in DMSO and diluted to 1 µM in the medium of the cell culture and incubated for at least 7 days prior to experimenting with the cell lines.
Clonogenic survival assay
The clonogenic survival assays are performed to investigate the survival rate of the cell lines with different treatments. The cells are plated in different quantities, depending on the harmfulness of the treatment, in a 6/12-well. After plating and treated cells are kept at 37oC and 5%CO2 in an incubator until
sufficiently large colonies were formed (8-10 days). Afterwards, the cells were fixated with a fixation solution of 6% glutaraldehyde (Merck) plus 0.01% crystal violet solution (Merck), incubated for 4-24 hours. After fixating the fixation solution was recycled and the wells were washed 1 time with water and 2 times with demi-water. After washing the well-plates were left to dry overnight and manually counted under a light microscope (Leica MZ6, Leica Microsystems; ref. 33).
The combined treatment of RT and HT was performed minimal 4 hours after plating the cells. First RT was applied with 2 Gy, and 4 Gy. After RT, the cells were treated with HT and placed in the incubator (37oC and
5% CO2). The different quantities cells/mL in this
clonogenic survival assay were, 100 cells/mL for the control (0 Gy) without HT and control with HT, 500 cells/mL for RT of 2 Gy with or without HT, 4000 cells/mL for RT of 4 Gy without HT and 8000 cells/mL for RT of 4 Gy with HT.
The combined treatment of cisplatin and HT was performed minimal 4 hours after plating the cells. ChT was applied with concentrations of 2.5 µM and 5 µM 5 minutes before treated with HT. After the combined treatment, the cells were placed in the incubator (37oC and 5% CO2). The different quantities cells/mL in this
clonogenic survival assay were, 100 cells/mL for the control (0 µM) without HT and control (0 µM) with HT, 1000 cells/mL for 2.5 µM without HT, 3000
cells/mL for 2.5 µM with HT, 6000 cells/mL for 5 µM without HT and 10000 cells/mL for 5 µM with HT
EdU incorporation for Cell cycle analysis
The cell cycle analysis was provided by incorporation of the thymide analogue 5-ethynyl-2’deoxiuridine (EdU) in the DNA of the cell using the Click-iTâ EdU Imaging Kit (InvitrogenTM). EdU has an Alexa FluorTM
647 picolyl azide attached, which allows fluorescent measurement of the amount of DNA in the single cells by a flow cytometer. The amount of DNA represents the cell cycle phases and with that the cell cycle distribution and cell cycle arrest of the cells. The cell cycle was analysed between the control and monotreatment of HT for both cell lines to check if there emerged a cell cycle arrest for HT in both cell lines.
500000 cells/mL were plated in 6-well plates for the control and 4 hours before treated with HT in both cell lines. 16 hours after HT, 10 µM of EdU was added and incubated for 1 hour. After incubation, the cells were harvested and fixated as described by the manufacturers’ protocol. Next, the Click-iT Plus reaction cocktail was added to the fixated cells and incubated for 30 minutes. Finally, the cells were washed as suggested and measured with the flow cytometer (BD FACS Canto II, 633/635nm excitation filter; BD Biosciences).
Immunofluorescence with g-H2AX staining
Prior to the staining with g-H2AX, 1 mL of 100000 cells were plated in the 6-well plates. The 6-well plates included coated cover glasses (coating: 0.01% poly-D-lysine, Merck) that were placed at the bottom of the wells. For the combined treatment of cisplatin and HT, medium (included the dead cells) was removed after 3 hours incubation (37oC and %CO
2) of the cells after
plating. 1 mL of 5 µM cDDP was added 5 minutes before HT treatment depending on the groups and controls, 1 mL of medium without cisplatin was added to the groups without treatment of cisplatin. After treatment, the cells were incubated (37oC and %CO
2)
overnight and stained as described below. For combined treatment of RT and HT, medium was refreshed after 3 hours incubation (37oC and %CO
2) of the cells after
plating and incubated overnight (37oC and %CO 2). The
groups with monotreatment of HT were treated 24 hours after plating. Directly after HT groups with RT were irradiated with 1 Gy what is equal to 20 DSBs, as stated in recent studies literature(?). After RT, the cells
were incubated (37oC and %CO
2) for 30 min and
stained as described below.
After treatment cells were fixed with 2% paraformaldehyde (Merck). After fixation, cells were washed with PBS (Lonza) and permeabilized with 0.1% triton X-100 in TNBS (PBS + 1% FCS) for 30 minutes. Next, the cells were incubated at room temperature with the first antibody of monoclonal mouse g-H2AX (diluted 1:100 in TNBS; MilliporeSigma) for 90 minutes. After the first antibody and washing with PBS, the secondary antibody with goat a-mouse-Cy3 (diluted 1:100 in TNBS; MIlliporeSigma) was incubated for 60 minutes at room temperature protected from light. Next, the cells were washed and DAPI-mounting gel (Vector Labaroties) was added. After DAPI staining the cover glasses were sealed at coverslips and imaged with fluorescence using a wield-field microscope (Leica DM6 FS fixed stage fluorescence microscope), at least 10 cells per group were imaged.
Statistical analysis
The data of the clonogenic survival assay after counting was normalized using Excelâ. The statistical analysis and graphs of the normalized data was performed with Graphpad Prism 7.0â with a statistical significance level of a = 0.05.
The flow cytometry data of the cell cycle analysis was received by FACSDivaTM. The graphs of the cell cycle
distribution were constructed and analysed using Flowjoâ software. The graphs were statistically compared manually.
After imaging the images with at least 10 cells per group, the images were edited with LAS Xâ software. The most representative cell was selected of every group of imaged cells. The respective statistical analysis was performed manually.
Results
Both cell lines show sensitivity and cell cycle arrest when treated with HT
To investigate the treatment sensitivity of HT at the IDH1wt and IDH1mut HCT116 cell lines, we performed a
clonogenic survival assay with monotreatment of HT. As shown in figure 1A, both cell lines show higher treatment sensitivity when treated with HT in comparison to the untreated control. Besides the treatment sensitivity, we investigated the cell cycle
phase distribution of the cell lines treated with HT, in order to investigate whether cancer cell lines show a cell cycle arrest when treated with HT. As shown in figure 1B, both cell lines go into a cell cycle arrest when treated with HT. The controls of the cell lines don’t show a cell cycle arrest. These results suggest that both cell lines are sensitive to HT as monotreatment.
Combined treatments of HT and RT/cisplatin shows
increased treatment sensitivity in IDH1MUT
Clinically, HT is being used in combination with other radio- or chemotherapy for clinical effect. Thus, we performed a clonogenic survival assay in the IDH1wt
and IDH1mut HCT116 cell lines in order to determine
the effect of HT in combination with RT or cisplatin. The cisplatin is used as chemo-reagent for chemotherapy, because of the high impact in respect to IDH1mut cancer cells. The IDH1mut cell line showed an
increased sensitivity in comparison to the IDH1wt in
both combined treatments (RT and HT/cisplatin and HT). The treatment of RT and HT was performed with 2 Gy and 4 Gy. Both Gray quantities showed a lower survival rate of the IDH1mut in comparison to the
IDH1wt (figure 2A). In addition, the combined treatment
of cisplatin and HT was performed with a cisplatin concentration of 2.5 µM. The combination of cisplatin and HT also showed a lower survival rate in the IDH1mut cell line compared to the IDH1wt cell line
(figure 2B). These results suggest that the IDH1mut cell
line is more sensitive to the combined treatments (RT and HT/cisplatin and HT) in comparison to the IDH1wt
cell line.
IDH1MUT inhibition restores sensitivity to treatment
In order to confirm the relationship between IDH1mut and increased sensitivity to RT and cisplatin, we treated the cancer cells with an IDH1 mutation inhibitor, AGI-5198. We repeated clonogenic survival assays with the combined treatments and pre-treated both cell lines AGI-5198 inhibitor. As shown in the figures 3A and 3B, the addition of AGI-5198 showed lower treatment sensitivity in the IDH1mut cell line without the AGI-5198 when treated with the combined treatments (RT and HT/cisplatin and HT). This states that the IDH1 mutation in the IDH1mut cell line is responsible for the
enhanced treatment sensitivity towards the combined treatments. To further empower this result, we found no effect of AGI-5198 on IDH1wt cancer cells (figures 3C
Increased amount of DSBs in the IDH1MUT when
treated with combined treatments of RT/cisplatin and HT
The lower survival rate in the IDH1mut cell line
compared to the IDH1wt cell line induced by the
combined treatments of RT/cisplatin and HT could be caused by an enhanced DNA damage or unrepaired DNA damage of the DSBs. As described in the introduction, RT and cisplatin induces DSBs and HT inhibits the DNA repair HR system of the DSBs. When the amount of DSBs increases in the replicating cells and repair is not possible, the cells go in apoptosis and lowers the survival rate when treated with the combined treatments of RT/ChT and HT. In order to investigate the amount of DSBs in both cell lines when treated with the combined treatments, we performed immunofluorescence experiment with g-H2AX staining in both cell lines with the same variable treatments as performed with the clonogenic survival assays. g-H2AX is a histone-protein that is active at the site of a DSB, so if this protein is stained and imaged with a fluorescent microscope the excited fluorescence light is an indication of a DSB. As shown in the figures 4A and 4B there is an increased amount of DSBs in the IDH1mut when treated with HT, RT, cisplatin, RT-HT and cisplatin-HT in comparison to the IDH1wt. This result
could be responsible for the increased treatment sensitivity in the IDH1mut in comparison to the IDH1wt
when treated with the combined treatments.
Discussion
We showed that both cell lines are sensitive towards monotreatment of HT. Moreover, both cell lines show a cell cycle arrest when treated with monotreatment of HT. HT in combination with RT or cisplatin increases sensitivity of the IDH1mut cancer cells comparatively to
the IDH1wt cancer cells. Inhibition of the IDH1 mutation in IDH1mut cancer cells by AGI-5198, restores the sensitivity caused by the combined treatments, which confirms the responsibility of the IDH1 mutation for the sensitivity. Besides, we found that the amount of DSBs in the IDH1mut cancer cells is higher when treated with mono- and combined treatment in comparison to the amount of DSBs in the IDH1wt cancer cells. So,
addition of HT to the treatment of RT and cisplatin shows a higher sensitivity in IDH1mut cancer cells
caused by the IDH1 mutation.
Previous studies showed a sensitivity and cell cycle arrest when treated with monotreatment of HT in cells without IDH1 mutation(12,17–19). We showed the
same results in the IDH1mut cancer cells, in order to rule
out the cell cycle distribution as underlying factor for the higher sensitivity.
Prior studies demonstrated the sensitivity towards RT and cisplatin in IDH1mut cancer cells. Molenaar et
al.(11) showed a radiosentization in IDH1mut cancer
cells and Khurshed et al.(14) showed a chemosentization towards cisplatin. We showed similar results (figure 2A and 2B) with the same cell lines. However, our most interesting finding and first in the field to show is the higher sensitivity in IDH1mut cancer
cells towards combined treatment of RT and HT/cisplatin and HT in comparison to IDH1wt.
Additionally, we also demonstrated that addition of the IDH1 inhibitor AGI-5198 restores this sensitivity in the IDH1mut HCT116 cell lines for both treatments, which
confirms the responsibility of the IDH1 mutation. In order to increase the robustness of these results, more cell lines should be integrated in these experiments. These findings supplements the previous research (4,5,11,14) and settle a promising entrance for further research.
After the found sensitivity, we did the first step to investigate the mechanism behind the higher sensitivity. The increase of DSBs in the IDH1mut cancer cells
comparatively to the IDH1wt after treatment with monotreatment of HT, monotreatment of RT, monotreatment of cisplatin, combined treatment of RT and HT and combined treatment of cisplatin and HT. The monotreatments of HT(21), RT(11) and cisplatin has already been described in previous research(11,14,18,21). But, we are the first to demonstrate that addition of HT to RT/cisplatin creates even more DSBs in the IDH1mut cancer cells comparatively to the IDH1wt and the monotreatments.
This result gives the right direction towards the mechanism of the discovered sensitivity.
As described in the Method, we treated the HCT116 cell lines with cisplatin concentrations of 2,5µM and 5µM for the clonogenic survival assays. However, the results with the cisplatin concentration of 5µM was excluded, since its impact at the HCT116 cell lines in combination with HT was too big to give convenient results. Moreover, we also used a cisplatin concentration of 5µM for the immunofluorescence experiments, which shows necrotic characteristics in combination with HT in IDH1mut cancer cells (Figure 4B). So methodologically, we recommend excluding the cisplatin concentration of 5µM in combination with
HT or revisit the method protocol for adding HT to the 5µM chemo-reagent cisplatin
The higher sensitivity and increased amount of DSBs in IDH1mut cancer cells could have various explanations. An increased ROS-level in the HCT116 cells is one possible mechanism underlying these finding. The increased ROS-levels elevates both, the sensitivity as the amount of DSBs (4). Additionally, HT and the metabolism of the IDH1mut cancer cells increases the ROS-levels (5,11,15,24). For HT, the ROS-levels increase due to the changed microenvironment and denaturised proteins that are essential for detoxification. The elevated ROS-levels in the IDH1mut cancer cells is
due to the accumulation of 2-HG in the cytoplasm. Further research to ROS-levels and its mechanism should clarify this possible explanation for the results in this research.
A second possible explanation for the higher sensitivity and increased amount of DSBs in IDH1mut cancer cells
is a deficient HR system. An HR deficiency causes difficulty in repairing DSBs properly and thereby increasing the amount of DSBs in the HCT116 cells. As stated in the introduction, HT causes temporarily inactivation of the HR system by temporarily degrading BRCA2 and dislocating RAD51(21). Moreover, Sulkowski et al.(1) showed a lower activity of the HR system in IDH1mut HCT116 cell lines. On the other
hand, Ohba et al.(25) has shown a higher activity of the HR system when treated with the chemo-reagent temozolomide in the IDH1mut U87MG and IDH1mut
T47P. This leaves the question whether this is caused by the treatment of temozolomide or the characteristics of the used cell lines. To clarify these contradictory results and to point out the exact mechanism of our findings, again more cell lines should be included. Furthermore, experiments relating the mechanism of the HR system in IDH1mut cancer cells treated with RT and
HT/cisplatin and HT should be performed to elucidate this explanation for our results. Possible experiments could be the localization of the RAD51 at DSB-sites or a HR efficiency test.
In this research, we showed that addition of HT to the treatments of RT and cisplatin increases the treatment sensitivity for IDH1mut cancer cells. With this result, we
proclaim that HT is a valuable addition to the treatments of RT and cisplatin for IDH1mut cancer cells, which confirms our hypothesis. More knowledge about its mechanism is needed to deliver a full explanation for the effects of adding HT to the treatments in IDH1mut
cancer cells. When the effects and mechanisms of the
addition of HT to radio/chemotherapy in IDH1wt and
IDH1mut cancer cells in vitro are clarified. The next step is to continue to perform these experiments in vivo to make it clinically relevant. If the results in vivo are similar to the results in vitro, the dose of the violent treatments radiotherapy and chemotherapy could be decreased. This should make treatment for patients with an IDH1mut cancer more bearable with less side-effects and preservation of the effectiveness of the treatment. This would be a major step in knowledge and clinical treatment of IDH1mut cancers.
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A B Control HT IDH1wt IDH1mut
Figure 1. Clonogenic survival assay and cell cycle distribution analysis treated with HT of the IDH1wt and IDH1mut cell
lines.
A, clonogenic survival assay of IDH1wt and IDH1mut HCT116 cells treated with monotreatment of HT. The clonogenic
fractions(y-axis) represents the counted colonies per group(x-axis), control(37oC) and HT(42oC). the clonogenic fraction is normalized to the control within the cell line starting with a normalized clonogenic fraction of 1 with a linear scale. B, Cell cycle analysis of IDH1wt and IDH1mut with monotreatment of HT. The y-axis are the counted cells by the flow
cytometer and x-axis represents the amount of DNA by means of the measured incorporated fluorescent nucleotide EdU by the flow cytometer. G1(first peak), S(valley) and G2(second peak) represents the cell cycle phases in the cell cycle
distribution.
G2
G1
0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 ID H 1w t H T ID H 1m u t - + - + 0 G y 2 G y 4 G y C lo n o g e n ic f r a c t io n - + - + - + - + * * * * 0 .0 0 0 1 0 .0 0 1 0 .0 1 0 .1 1 H T - + 0 u M 2 '5 u M C lo n o g e n ic f r a c t io n ID H 1w t ID H 1m u t - + - + - + *** A B
Figure 2. Clonogenic survival assays of the IDH1wt and IDH1mut treated with the combined treatments of RT and
HT/cisplatin and HT.
A, clonogenic survival assay of IDH1wt and IDH1mut HCT116 cells treated with the combined treatment of RT and HT.
The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 1A (control (37oC) = - and HT (42oC) = +), but within the variable inflicted radiation of 0, 2 and 4 Gy. the clonogenic fraction is
normalized to the control (37oC and 0 Gy) within the cell line starting with a clonogenic fraction of 1 with a logarithmic scale. B, clonogenic survival assay of IDH1wt and IDH1mut HCT116 cells with the combined treatment of cisplatin and HT. The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 1A, but within the variable cDDP concentration of 0 and 2,5 µM. the clonogenic fraction is normalized to the control (37oC and
0 .0 1 0 .1 1 H T C lo n o g e n ic f r a c ti o n ID H 1w t ID H 1m u t ID H 1m u t+ A G I-5 1 9 8 - + 0 G y 2 G y 4 G y - + - + - + - + - + - + - + - + * * * * * 0 .0 0 1 0 .0 1 0 .1 1 H T C lo n o g e n ic f r a c ti o n - + 0µ M 2 '5µ M - + - + - + - + - + * * W T ID H 1M U T ID H 1M U T+ A G I-5 1 9 8 * * 0 .0 0 1 0 .0 1 0 .1 1 H T C lo n o g e n ic f ra c ti o n ID H 1w t - + 0 G y 2 G y 4 G y ID H 1w t + A G I-5 1 9 8 - + - + - + - + - + 0 .0 0 1 0 .0 1 0 .1 1 H T C lo n o g e n ic f ra c ti o n 0µ M 2 ,5µ M ID H 1w t ID H 1m u t + A G I-5 1 9 8 - + - + - + - + A B C D
Figure 3. Clonogenic survival assays of the IDH1wt and IDH1mut included the additional IDH1mut inhibitor AGI-5198
treated with the combined treatments of RT and HT/cisplatin and HT.
A, clonogenic survival assay of IDH1wt, IDH1mut HCT116 cells and IDH1mut HCT116 cells and the IDH1mut inhibitor
AGI-5198 treated with the combined treatment of RT and HT. The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 2A. the clonogenic fraction is normalized to the control of every variable inflicted radiation within the cell line starting with a clonogenic fraction of 1 with a logarithmic scale. B, clonogenic survival assay of IDH1wt, IDH1mut HCT116 cells and IDH1mut HCT116 cells and the IDH1mut inhibitor
AGI-5198 treated with the combined treatment of cisplatin and HT. The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 2B. the clonogenic fraction is normalized to the control of every variable concentration of cDDP within the cell line starting with a clonogenic fraction of 1 with a logarithmic scale. C, clonogenic survival assay of IDH1wt and IDH1wt HCT116 cells and the IDH1mut inhibitor AGI-5198 treated with the
combined treatment of RT and HT. The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 3A. the clonogenic fraction is normalized to the control of every variable inflicted radiation within the cell line starting with a clonogenic fraction of 1 with a logarithmic scale. D, clonogenic survival assay of IDH1wt and IDH1wt HCT116 cells and the IDH1mut inhibitor AGI-5198 treated with the combined treatment of cisplatin
and HT. The clonogenic fractions(y-axis) represents the counted colonies per group(x-axis), same groups as in figure 3B. the clonogenic fraction is normalized to the control of every variable concentration of cDDP within the cell line starting with a clonogenic fraction of 1 with a logarithmic scale
A IDH1wt IDH1mut B IDH1wt IDH1mut
Control Control HT RT RT/HT HT cisplatin cisplatin/HT
Figure 4. immunofluorescence of DSBs by staining the y-2HAX of the IDH1wt and IDH1mut cells when treated with
the combined treatments of RT and HT/cisplatin and HT.
A, immunofluorescence y-2HAX staining experiment with the combined treatment of RT and HT to the IDH1wt and
IDH1mut HCT116 cells. The groups are indicated on the left, control (42oC and 0 Gy), monotreatment of HT (42oC and 0
Gy), monotreatment of RT (37oC and 1 Gy) and the combined treatment of RT and HT (42oC and 1 Gy). The cell lines
are indicated at the top, IDH1wt and IDH1mut. The red fluorescent dots are the stained y-2HAX foci representing the
DSBs. B, immunofluorescence y-2HAX staining experiment with the combined treatment of cisplatin and HT to the IDH1wt and IDH1mut HCT116 cells. The groups are indicated on the left, control (42oC and 0 µM), HT monotreatment
(42oC and 0 µM), monotreatment of cisplatin (37oC and 1 µM) and the combined treatment of cisplatin and HT (42oC
and 1 µM). The cell lines are indicated at the top, IDH1wt and IDH1mut. The red fluorescent dots are the stained y-2HAX
