VX-984 | Dinaciclib

DNA double-strand breaks repair inhibitors potentiates the combined effect of VP-16 and CDDP in human colorectal adenocarcinoma (LoVo) cells

Paulina Kopa1 · Anna Macieja2 · Elzbieta Pastwa3 · Ireneusz Majsterek4 · Tomasz Poplawski2

Abstract

I. Background: A combination of etoposide (VP-16) and cisplatin (CDDP) is the standard treatment for certain colon can-cers. These drugs promote the death of cancer cells via direct and indirect induction of the most lethal DNA lesions – DNA double-stand breaks. However, cancer cells can reverse the DNA damaging effect of anticancer drugs by triggering DNA repair processes. In eukaryotic cells, the main DNA repair pathway responsible for DNA double-stand breaks repair is nonhomologous end-joining (NHEJ). Inhibitors of DNA repair are of special interest in cancer research as they could break the cellular resistance to DNA-damaging agents and increase the efficiency of standard cancer treatments. In this study, we investigated the effect of two NHEJ inhibitors, SCR7 and NU7441, on the cytotoxic mechanism of VP-16/CDDP in a LoVo human colorectal adenocarcinoma cell line. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding, whereas NU7441 is a highly potent and selective DNA-PK inhibitor.
II. Methods and Results: Both inhibitors synergistically increased the cytotoxicity of CDDP and VP-16 when combined, but the effect of SCR7 was more pronounced. SCR7 and NU7441 also significantly increased VP-16; CDDP induced DNA double-stand breaks level and delayed drug-induced DSB repair, as seen on the comet assay and measured using H2AX foci. We also observed changes in cell cycle distribution and enhanced apoptosis ratio in colorectal adenocarcinoma cells treated with DNA repair inhibitors and VP-16/CDDP.
III. Conclusions: Our data support the hypothesis that NHEJ inhibitors could be used in conjunction with standard therapy to provide effective clinical improvement and allow reduction in drug doses.

Keywords Cisplatin · Etoposide · DNA damage and repair · NHEJ · SCR7 · NU7441

Introduction

Colorectal cancer (CRC) is the third most common type of tumor worldwide, which was cause of death of almost 800,000 of people in 2015 according to statistical data provided by the World Health Organization (WHO) and American Cancer Society [1]. The probability of developing CRC increases with age. Although CRC primarily occurs in the elderly population (over 45 years), an increasing number of CRC cases are being detected in people younger than 45 years. CRC occurring in younger patients shows characteristics similar to those seen in older patients. However, some reports suggest that it has aggressive histopathological characteristics and is usually diagnosed at an advanced stage [2, 3].
CRC treatment usually depends on the severity of the disease and metastases; it includes surgery and adjuvant chemotherapy or radiotherapy. Chemotherapy can include a combination of anticancer drugs including antimetabolites, platinum derivatives, and topoisomerase inhibitors (5-fluorouracil, leucovorin, capecitabine, irinotecan, oxaliplatin, cisplatin (CDDP), and etoposide (VP-16)). Platinum derivatives and topoisomerase inhibitors directly and indirectly induce DNA double-strand breaks (DSBs) in the CRC cells, which are mainly repaired via non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) [4, 5].
NHEJ is considered the primary DSB repair pathway. It is much faster than HRR, but is error prone. The NHEJ mechanism involves three main steps: joining two DNA ends together, DNA end processing to make them ligatable, and the final ligation step. The key NHEJ proteins are DNAPKcs, Ku proteins, XLF/Cernuunos, Artemis, and Ligase IV. NHEJ repairs most of the damage that anticancer agents (platinum compounds and topoisomerase II inhibitors) and ionizing radiation (IR) induce [6, 7].
NHEJ seems to be intact in most cases of CRC; it contributes to the intrinsic cellular resistance to DSB-inducing agents. Lowering NHEJ activity seems to be a promising approach to sensitizing cancer cells to DSBs inducers with simultaneous reduction of the total dose of these agents [8–13] including CDDP and VP-16 combination treatment [13–15]. Among the NHEJ inhibitors in development, two are the most promising: NU7441 and SCR7. The first is potent inhibitor of the DNA-PKcs, the second one target Ligase IV [16, 17]. The aim of this study was to analyze the effects of ligase IV and DNA-PKcs inhibition using SCR7 and NU7441 on VP-16/CDDP-induced DSBs in a human colorectal adenocarcinoma cell line (LoVo). LoVo cell line is well characterized and commonly used colorectal cancer cell line. It has mutations in one of major CRC driver genes – KRAS. Moreover, LoVo has functional DSB repair pathway as no gain and lost function mutations have been identified in key NHEJ genes like ATM, TP3, PRKDC, LIG4, XRCC5 and DCLRE1C according to Cancer Cell Line Encyclopedia (https: //portals.broadi nst itute.org/ccl e). It has also no mutator phenotype (no MSI present) [18–21]. Taken together, this cell line is good model for DNA repair studies.

Materials & methods

Chemicals

NU7441 (Selleckchem, Munich, Germany), SCR7 (SigmaAldrich, Saint Louis, MO, USA), and VP-16 (SigmaAldrich, Saint Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, MO, USA) as 10 mmol/L stocks and stored at −20 °C. CDDP (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in distilled water to a 1 mg/mL solution (3.3 mM) final stock and stored at room temperature in a dark environment. The final concentration of DMSO during experiments was 0.5%. The control samples were also incubated with 0.5% DMSO. All other chemicals were from Sigma-Aldrich (Saint Louis, MO, USA).

Cell line and culture

A human colorectal adenocarcinoma cell line (LoVo) was obtained from the HPA Culture Collections (Sigma-Aldrich, Saint Louis, MO, USA). LoVo was cultivated in DMEM/ F12 medium (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% v/v of fetal bovine serum (FBS) (Lonza, Basel, Swiss) and 1% v/v penicillin/streptomycin solution (Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C in 5% CO2. Cells were free of Mycoplasma and the doubling time was estimated as ~24 h. Accutase (Sigma-Aldrich, Saint Louis, MO, USA) was used to gently harvest cells prior to the experiments.

Cytotoxicity studies

The cytotoxic effect of studied anticancer agents was measured with a CCK-8 assay (Sigma-Aldrich, Saint Louis, MO, USA) according to manufacturer instructions. Briefly, cells were seeded into 96-well plates at a density of 5 × 103 cells per well and cultivated 24 h prior to the experiment. Next, cells were exposed to the anticancer agents for 24 h. VP-16 and CDDP were added alone or in combination together. During experiments with DNA DSBs inhibitors, NU7441 and SCR7 were added at a final concentration of 10 μM to the medium 1 h before the anticancer agents. CCK-8 reagent was added to the fresh culture medium at 10% concentration and incubation was continued for 3 h. The CCK-8 assay is based on dehydrogenase activity detected in viable cells. The absorbance at 450 nm was quantified with the Bio-Rad 550 plate reader (Hercules, CA, USA). Cytotoxicity was calculated as a percentage of vehicle control cells. The IC50 value represented the concentration of the tested agent that is required for 50% inhibition of the cell viability. This was estimated with BioDataFit 1.02 software (chang biosc ience .com/stat/ec50.html). The reduction factor (Rf) was calculated from the ratio of the results from I C50 for drugs alone to the results of IC50 of the combination drugs with inhibitors (IC50 drug/ IC50 drug + inhibitor). All experiments were repeated at least three times.

Combination index analysis

The combination index (CI) was calculated according to Chou’s CI model with CompuSyn software (ComboSyn, Inc., Paramus, USA). The CI value informs us about the interaction between drugs. CI > 1, denotes antagonism; CI = 1, denotes additive effects; CI < 1, indicates synergism [22].

Comet assay for DNA damage and repair

The alkaline version of comet assay was used for DNA damage and repair studies, as described previously with minor modifications [23]. A freshly prepared suspension of LoVo cells in 0.75% low-melting-point agarose dissolved in PBS was spread onto microscope slides that were precoated with 0.5% normal-melting-point agarose. The cells were then lysed in buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris and Triton X-100) at 4 °C for 1 h. After lysis, the slides were unwound for 20 min with a buffer (300 mM NaOH, 1 mM EDTA), and electrophoresis was performed in the buffer (30 mM NaOH, 1 mM EDTA) at 17 V, 32 mA for 20 min. After electrophoresis, the slides were dried and stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Saint Louis, MO, USA; 2 μg/mL in PBS) and covered with cover slips. Samples were incubated with DAPI for at least 60 min and analyzed under a microscope ((Eclipse fluorescence microscope – Nikon, Tokyo, Japan coupled with the COHU 4910 video camera (San Diego, USA) and UV filter block)) with, Lucia-Comet v. 4.51 software. To prevent additional DNA damage, all the steps described above were conducted under dimmed light or in the dark. For each sample, 50 random images were chosen in two slides so each sample was analyzed to the final 100 of random comets images. The percentage of DNA in the tail (% tail DNA) was analyzed. It was seen to be positively correlated with DNA strand breaks. For each image, the content of DNA on the head and tail was measured. Percentage of DNA in a tail corresponded to the level of DNA damage.
For analysis of DNA repair efficiency, the differences between time points of repair incubation was taken as an index of DNA repair efficiency. The cells (including control samples) were washed and resuspended in fresh, drug-free DMEM medium preheated to 37 °C, after the treatment with studied drugs. The DNA repair was run without or in the presence of double-strand breaks inhibitors (1 h incubation with 10 μM NU7441 or SCR7).

H2AX histone phosphorylation

We used commercially available kit Human H2A.X (phospho S139) In-Cell ELISA Kit (IR) (ab131382) (Abcam, UK). It allows to detect the phosphorylated Ser 193 on the H2AX, using A rabbit monoclonal antibody specific to H2A.X phospho S139. Experiments were carried-out according to the manufacturer instructions.

Cell cycle distribution analysis

Cells were seeded into culture dishes and cultivated in DMEM medium at 37 °C for 24 h. Subsequently, cells were treated with studied drug combinations with/without preincubation with double strand inhibitors NU7441 and SCR7 (10 μM final concentration) for at least 24 h. Next, cells were harvested, washed with PBS, fixed with 70% ethanol, and stained with propidium iodide (40 μg/mL) and DNase-free RNase (200 μg/mL) for 30 min at 37 °C prior to analysis. DNA content was analyzed with a Beckman Counter flow cytometer (Beckman Counter Inc., Fullerton, CA, USA). Cell cycle distribution was expressed as the percentage of cells in each phase of the cycle. The final results are the mean of three independent measurements.

Cell apoptosis assay

For the analysis of apoptosis, FITC Annexin V kit (BD Biosciences, San Jose, CA, USA) was used. The first steps were similar to those in the cell cycle distribution analysis. After 4 h of incubation, each sample was harvested and washed with PBS. Next, cells were resuspended in 1X Binding Buffer at a final concentration of 1 × 106 cells/mL. Then cells were stained with 5 μL of FITC Annexin V and 5 μL of PI and analyzed with flow cytometry after 15 min of incubation at room temperature in the dark. Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine to identify apoptotic cells. In healthy cells, phosphatidylserine is predominantly located along the cytosolic side of the plasma membrane. Phosphatidylserine loses its asymmetric distribution in the plasma membrane and translocates to the extracellular membrane upon initiation of apoptosis. This event can be detect with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes PI, therefore green cells are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic phosphatidylserine, as well as cell uptake of PI, therefore these cells are green/red. However green/red cells also include small fraction of cells underwent rapid necrosis nor apoptosis.

Statistical analysis

The Shapiro-Wilk test was used to test normality. Results from cytotoxicity, CI, and flow cytometry analysis were expressed as means ± SD. Results from alkaline comet assay were expressed as median ± range. The differences between samples with the normal distribution were evaluated with the t-Student statistical test. For those without normal distribution, U-Mann Whitney test was used. ROC curve analysis was used to determine the NHEJ inhibitors effect on DNA repair efficiency. The data were analyzed using data analysis software system (STATISTICA version 13.3 software; TIBCO Software Inc. 2017).

Results

NHEJ inhibitors sensitized human colorectal adenocarcinoma (LoVo) cells to VP‑16, CDDP and VP‑16/CDDP combination

CCK-8 assay was used to determine cytotoxicity of LoVo cells treated with DSBs inhibitors NU7441 and SCR7. Both inhibitors did not evoke cytotoxic effect in LoVo cells at 10 μM dose (Fig. 1f), therefore, we chose this dose to study the potential sensitization of LoVo cells to VP-16 and CDDP. This observation was in agreement with others [24]. Next, we determined the viability of LoVo cells treated with anticancer drugs alone, in combination before and after DSBs inhibitors NU7441 and SCR7 supplementation for 24 h. As indicated in Supplementary Table 1 and Fig. 1, decrease in cell survival was observed after incubation with anticancer drugs alone (Fig. 1a–e) or in combination (Fig. 1c). The calculated IC50 values for VP-16, CDDP and in combination were lowered when cells where pretreated with DSBs inhibitors at 10 μM dose (Supplementary Table 1), so we concluded that NU7441 and SCR7 enhance chemo-sensitivity of LoVo cells. The maximum reduction of LoVo cells viability was observed for SCR7 – the calculated reduction factor (Rf) for CDDP/VP-16 in combination was 2.81. NU7441 also significantly enhanced the chemo - sensitivity of LoVo, but the observed effect was less pronounced (Rf = 1.69). To address the nature of observed effect LoVo cells were incubated with VP-16/CDDP at the constant ratio [(IC50)VP-16/ (IC50)CDDP ratio, 1:1.17] with or without DSBs inhibitors (Fig. 1c). This allowed us to calculate the combination index and determine the effects of a combination of drugs. As presented in Supplementary Table 2 and the isobologram (Fig. 1d), the synergism was observed for VP-16/CDDP in combination pretreated with both SCR7 or NU7441 at 52 and 104 μM total dose (0.5 IC50 and IC50 respectively). The maximum effect was obtained for a total dose of 52 μM (0.5 IC50; 24 μM VP-16 + 28 μM CDDP); we therefore have chosen this dose in further experiments aimed to understand the molecular mechanism of chemo-sensitization.

NHEJ inhibitors increased DNA damage in human colorectal adenocarcinoma (LoVo) cells exposed to VP‑16/CDDP combination by reducing DNA repair efficiency

To assess the level of DNA damage induced by VP-16/CDDP combination without or with double-strand breaks inhibitors, an alkaline version of comet assay was performed. The level of DNA damage was significantly higher for the cells incubated with drugs combination as compared to the control (3% vs. 35%). Both DSBs inhibitors increased the total level of DNA damage (NU7441 38.3%, SCR7 40.2%). To confirm the results obtained in comet assay, we also analyzed the phosphorylation of H2AX histone, which occurs in the response to DSBs. Figure 2 summarizes the results of DNA damage analysis. We also analyzed DNA repair efficiency after incubation of LoVo cells with CDDP/ VP-16. Figure 3 shows the level of DNA damage for the VP-16/CDDP combination alone or in the pre-incubation with DSBs inhibitors (NU7441 or SCR7) at the time points: 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 4 h, and 6 h. LoVo cells were able to recover nearly all DNA within 4 h after VP-16/CDDP treatment, whereas DSBs inhibitors decreased DNA repair efficiency and only 60% of DNA was recovered after 6 h. ROC curves analysis demonstrated statistically significant (p < 0.001) effect of NU7441 and SCR7 on repair efficiency reduction of LoVo cells exposed to VP-16/CDDP combination.

NHEJ inhibitors changed, exposed to VP‑16/CDDP combination LoVo cell cycle distribution

The analysis of distribution of cells among different cell cycle phases after treatment of drugs combination alone or in the presence of double-strand breaks inhibitors, NU7441 or SCR7, was performed using flow cytometry (Fig. 4). For the samples with VP-16/CDDP combination, accumulation in S phase (72.6% vs. 39.3% for control sample) and reduction for G1 (from 44.1% to 13.8%) and G2 phase (from 14.7% to 9.4%) was observed. Pre-incubation with both inhibitors lead to the cells accumulation in the S (62.1% for NU7441 and 66.7% for SCR7) phase of cell cycles. Moreover, for both of them, increase in G2 phase (9.7% and 10.1% for NU7441 and SCR7, respectively) compared to the drug combination samples was observed. Application of drug combination and both inhibitors was also connected with increases of cell content in the subG1 phase that include inter alia cells underwent apoptosis (compared to the control sample). These results are summarized in Fig. 4.

NU7441 stimulated apoptosis in human colorectal adenocarcinoma (LoVo) cells exposed to VP‑16/ CDDP combination

The analysis of cell percentage that underwent death by apoptosis or necrosis after incubation with presented cytostatic and inhibitor combinations, using flow cytometry analysis was also performed. For the sample with VP-16/ CDDP combination alone, the percentage of the living cells reached 77.3%. Early apoptotic and necrotic cells were 7% 96-well plate (5 × 103 cells/well) and (a) treated only with increased concentration of NU7441 (squares) or SCR7 (circles) and with increased concentration of VP-16 (b, grey) or CDDP (c, dark grey) or VP-16/CDDP combination (d, black) alone (triangles) or with 1 h pre-incubation with 10 μM NU7441 (squares) or 10 μM SCR7 (circles) for 24 h. After this time, cell viability test was performed using CCK-8 assay. Combination index plot (CompuSyn software) (e) for VP-16/CDDP combination in the absence (triangle) or presence of 10 μM NU7441 (square) or 10 μM SCR7 (circle). (f) Enlarged part of graph for effect of DNA repair inhibitors on the viability of LoVo cells treated with CDDP (for dose range 0–1 μM). VP-16, NU7441 and SCR7 stocks were prepared in DMSO and CDDP stock in distilled water and further diluted in culture medium. The final DMSO concentration was kept at <5 μL/mL. Results were calculated as a percentage of vehicle control cells. Data are presented as means ± SD of three separate experiments. Statistically significant difference: *P < 0.05. CI = combination index, Fa = fraction affected, VC = VP-16/CDDP combination alone, VC + NU = VP-16/CDDP combination with NU7441 1 h pre-incubation, VC + SCR = VP-16/CDDP combination with SCR7 1 h pre-incubation and 5.7% of the population, respectively plus 9.9% cells underwent late apoptosis and necrosis (indistinguishable). Pre-incubation of the LoVo cell line with NU7441 inhibitor led to reduction of living cells (from 85.3% for control to 63.3%) and increased level of early apoptotic (7% vs. 17.1%) and necrotic cells (5.7% vs. 5.3%). We also observed increase level of cell fraction with late apoptosis/necrosis (9.9% vs 14.3%). The discrepancy is statistically significant (p < 0.001). In the samples pre-treated with SCR7 inhibitor, there is no increase in the level of cells undergoing early apoptosis (7.0% vs 6.1%) and necrosis (5.7% vs 6.7%), along with a corresponding reduction in the percentage of living cells (75.8%), compared to the control sample which is the result of small increase of late apoptosis/necrosis. However, these results only slightly differ from samples without inhibitor (compared with major differences for samples with NU7441) (Fig. 5).

Discussion

The resistance of cancer cells to standard therapy initiated a chapter of novel therapeutics investigations aimed at eliminating this phenomenon and increasing the effectiveness of standard therapy. Several trials have focused on this issue, especially regarding searching new therapeutic molecules that act to inhibit DNA damage response (DDR), since PARP inhibitors have proven their usefulness and strength. Inhibition of DDR in the cancer cells should correspond with elevated sensitivity of these cells to standard therapy. Keeping up with this trend of scientific research, the main problem is to find the precise combination of inhibitor and standard chemo- or radiotherapeutics that will be characterized by high effectiveness with a relatively low dose. [25, 26]
Here, we focused on the effect of DNA double-strand breaks repair inhibitors, NU7441 and SCR7, on the sensitization of human colorectal adenocarcinoma cell line to combination of VP-16 and CDDP. We provide data for the effects of both NU7441 and SCR7 on sensitization of the LoVo cell line to VP-16 and CDDP combination. During our study, we used a concentration of 10 μM for both inhibitors, which corresponded to effective dose analysis by many authors. For NU7441, the effective dose used in different publications ranged between 3 and 10 μM [27, 28]; for SCR7, the dose ranged from 1 to 20 μM [16, 29].
Our results strongly indicated that usage of both inhibitors led to sensitization of the human colorectal adenocarcinoma cell line to VP-16 and CDDP. Furthermore, NU7441 and SCR7 allowed significant reduction in the dose of VP-16 and cisplatin required for 50% inhibition of cell growth ( IC50 value). More pronounced sensitization was obtained for both inhibitors for CDDP (2-fold for NU7441 and 2.5-fold for SCR7) compared to VP-16 (1.7-fold for NU7441 and 2-fold for SCR7). Moreover, both inhibitors also increased the sensitivity of the LoVo cell line to a combination of VP-16 and CDDP (1.7-fold for NU7441 and 2.8-fold for SCR7). Furthermore, both inhibitors increased the range of total concentration of VP-16 and cisplatin at which they could act synergistically. These data are in agreement with results of other studies. Zhao et al. indicated a significant effect of NU7441 on sensitization of cancer cell line to topoisomerase II inhibitors: VP-16 and doxorubicin and IR. Moreover, the authors suggest that this effect is connected with specific DNA-PK inhibition, which was confirmed by comparison of non-efficient (V3) and proefficient (V3-YAC) cell lines. They also investigated the effect of this inhibitor on the p53 pathway [24]. Alikarami et al. obtained similar results of increased sensitization to another topoisomerase II inhibitor – doxorubicin (25 μM) after incubation with 10 μM NU7441 (cytotoxicity for 24 and 48 h) for B cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines: Nalm6 and SUP-B15 [27]. NU7441 also possessed significant chemosensitivity activity to AMR and CPT-11 (topoisomerase inhibitors) on non-small cell lung carcinoma (A549 cell line), according to Yanai et al.’s investigation [23]. The effect of NU7441 was also confirmed by Dai et al. in the trials of Akt inhibitor (TCN- 10 μM) in combination with NU7441 (10 μM) and cisplatin (25 μM) on CDDP resistant epithelial ovarian cancer (PEO4 – with resistance vs. PEO1 with normal sensitization to CDDP) [28]. A combination of these three compounds leads to reduced resistance to CDDP for PEO4 cells. However, here, the authors discussed combinatory effect of NHEJ inhibitor and Akt inhibitor on sensitivity to CDDP [28]. Mice models presented by Srivastava et al. provided information about the effect of SCR7 on increased sensitivity to VP-16 and radiation [29].
Pre-incubation of LoVo cell line with NU7441 and SCR7 significantly increases level of DNA damage after exposure to VP-16/CDDP combination. Both the analyzed inhibitors provide similar level of DNA repair efficiency reduction (about 70%), which contributed to cancer cell death. VP-16 acts as a topoisomerase II inhibitor that increased the level of double-strand breaks. The CDDP mechanism of action is associated with intrastrand and adducts formation that are converted to DSBs during DNA replication or transcription if unrepaired [30, 31]. Our data are in agreement with the results of other studies. NU7441 inhibitor increases tumor cell lines sensitivity to standard treatment which is correlated with increasing level of DNA damage and reduction in DNA repair capacity [17, 32]. This mechanism is described as an effect of NHEJ inhibition and aggregation of DNA damage introduced by standard therapeutics. Tavecchio et al. confirmed these results in V3-YAC and MO59-Fus-1 (with normal activity of DNA-PK) cell lines, compare to cell line defective for DNA-PK (V3 and MO59J) [33]. Similar results were obtained by Sunada et al. and Yu et al. for a combination of NU7441 and X-ray on A549, H460, and H1299 cell lines [34, 35]. Moreover, Yang et al. demonstrated that combination of IR and NU7441 leads to HepG2 cell line decreased viability and elevated level of DNA DSBs, which correlates with reduced expression of DNA-PK [36]. Furthermore, Gkotzamanidou et al. described a similar effect on increase in DSBs formation and reduction of DNA repair efficiency for SCR7 and melphalan combination for RPMI18226 and LR5 (sensitive and melphalan-resistant) cell lines and BMPCs from myeloma patients. Moreover, the authors also obtained significant sensitization of each cell line to melphalan [16].
Application of both NHEJ inhibitors leads to accumulation of LoVo cells in the S phase of the cell cycle and reduction of the number of cells in the G2/M phase. For VP-16 and CDDP combination alone, we also observed a reduced number of cells in the G0/G1 phase (compared to control samples), which was not observed for the samples with NU7441 and SCR7 inhibitors. This result suggested impact of both inhibitors on G2/M checkpoint arrest. Moreover, a combination of both therapeutics leads to reduction in the living cells and increase in the number of apoptotic and necrotic cells. The obtained results suggested a more pronounced effect of apoptosis induction by NU7441, because of the significantly reduced number of living cells and the significantly increased number of apoptotic cells. For SCR7, this effect was limited to a minor reduction in living cells and increase in the level of apoptotic and necrotic cells. This suggested a different mechanism of action of both inhibitors associated with apoptosis induction.
The observed effect of NU7441 on cell cycle and apoptosis induction is similar to others. Van Oorschot et al. described a combined effect of radiation, hyperthermia, and NU7441 on breast cancer cell lines; the growth inhibition correlated with DSB induction, G2/M cell cycle phase arrest, and 2–2.5-fold increased apoptosis (with elevated level of caspase-3) [37]. Similar results were obtained by Sunada et al., where NU7441 increased lung tumor cells to IR with DNA fragmentation, G2/M phase arrest and apoptosis induction in p53-dependent manner [34]. However, Vavrova et al. indicated accumulation of HeLa cells in G2/M phase after combined treatment with 15 Gy radiation and NU7441 (with increased phosphorylation of Chk1 and Chk2) [38]. Sw620 and LoVo cell accumulation in G2/M phase also observed Zhao et al. for treatment with doxorubicin, VP-16 and IR in combination with NU7441 [24]. Moreover, co-treatment of lung tumor cell lines with irradiation, PARP1 (AG014699), and NU7441 inhibitors reduced cell viability, increased level of DNA damage and p21 expression, and arrested the cell cycle at the G2/M phase [39]. Gkotzamanidou et al. established that pre-incubation of multiple myeloma cell lines with SCR7 increases γH2AX foci and induces apoptosis [16].
Recently, it was shown that SCR7 is not only a DNA ligase IV inhibitor but also targets other ligases: DNA ligase I ( IC50 = 300 μM) and DNA ligase III (where the IC25 parameter similar for DNA ligase IV = 300 μM). This nonspecific action of SCR7 may be due to difficulties during its synthesis. SCR7 is a mixture of three different compounds named SCR7-G, SCR7-R, and SCR7-X, which corresponded to different specificities of DNA ligases [40].
Furthermore, as John et al. demonstrated, SCR7 is characterized by poor bioavailability due to its hydrophobic character, leading to a limited solubility in water (it is soluble in organic solvents like DMSO). Therefore, this compound may possess lower activity than other inhibitors with higher solubility. One of the discussed approach is the use of pluronic copolymer to increase the absorption of this DNA ligase IV inhibitor by the cells. Adding P123 copolymer leads to increasing solubility and bioavailability of SCR7 by the L929 cell line after 24-h incubation and is significantly correlated with viability reduction of these cells [41, 42].

Conclusions

As analyzed in our study inhibitors, NU7441 and SCR7 possessed some activity on human colorectal adenocarcinoma cells. Each inhibitor is characterized by different mechanism of action for DNA double-strand breaks repair inhibition. The first is DNA-PK catalytic subunit inhibitor, while the second one is blocked action of DNA ligase IV. Both inhibitors sensitized the LoVo cell line to VP-16 and CDDP alone and in combination. This can enable a lower effective dose for these chemotherapeutics if they are combined with inhibitors. Furthermore, both inhibitors have a wide range of total concentrations of drugs, singly and in combination, with whom they act synergistically.We conclude that the mechanism of action of both inhibitors, NU7441 and SCR7, for potentiation of VP-16 and CDDP combination is based on the induction of DNA damage (increased level of DSBs), reduction of DSBs repair efficiency, G2/M checkpoint of cell cycle arrest, and apoptosis induction.
Our study has one limitation which point at important elements of further research. Our conclusion is based on the data obtained from experiments performed only on one colorectal cell line. Therefore, the evaluation of our strategy in additional models of CRC represented by different CRC cell lines is required. The choice of appropriate CRC cell lines for such study could be challenging as more than 70 CRC cell lines have been established [43] as consequence of CRC heterogeneity. However, some of them share similar molecular phenotypes allowed to group them into three main populations/groups regarding chromosomal instability (CIN), microsatellite instability (MSI) and CpG island methylator (CIMP) phenotypes. Cell lines representing MSI and CIMP phenotype (accounting for 15% of all CRC) should be excluded from DNA repair studies as they showed 3xR chaos (Replication, Recombination and Repair) and DDR is often disturbed and unpredictable. CIN group could be further divided into five subpopulations based on STR profiling [44]. Considering this classification, we proposed that future studies should be performed on SW48, SW480, CACO-2 and SW1116 cell lines to validate our strategy and allow to move from bench to bedside, however different choice is also available as each subpopulation have more than one representative.

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