|Year : 2018 | Volume
| Issue : 2 | Page : 93-101
Inhibitor of nonhomologous end joining can inhibit proliferation of diffuse large B-Cell lymphoma cells and potentiate the effect of ionization radiation
Vidya Gopalakrishnan1, Gudapureddy Radha2, Sathees C Raghavan2, Bibha Choudhary3
1 Institute of Bioinformatics and Applied Biotechnology; Department of Biochemistry, n Institute of Science, Bengaluru; Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Biochemistry, n Institute of Science, Bengaluru, Karnataka, India
3 sInstitute of Bioinformatics and Applied Biotechnology, Bengaluru, Karnataka, India
|Date of Web Publication||22-May-2018|
Dr. Bibha Choudhary
Institute of Bioinformatics and Applied Biotechnology, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Aim: Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of non-Hodgkin's lymphoma that accounts for ~40% of all lymphomas. DLBCL is considered to be clinically heterogeneous with highest mortality rate. Recent advances in gene expression profiling helped in identifying different subtypes of DLBCL, and since then, many therapeutic options have been explored to treat DLBCL patients. Although it is effective, a significant proportion of the patients suffer due to drug resistance. One of the potential causes for this could be elevated DNA repair in the resistant cancer cells. Thus, the present study is aimed at investigating the potential of SCR7, a DNA repair inhibitor in inducing cytotoxicity on a DLBCL cell line, and to study its ability to potentiate effect when used in combination with ionizing radiation. Materials and Methods: DLBCL cell line, Standford University Diffuse Histiocytic Lymphoma 8 (SUDHL8) was treated with various concentrations of SCR7, a DNA repair inhibitor that targets nonhomologous DNA end joining. While cytotoxicity induced by SCR7 was evaluated through trypan blue assay and flow cytometry analysis, 5,5',6,6 tetrachloro-1,1',3,3'-tetraethyl benzimidazol-carbocyanine iodide and annexin V-FITC/propidium iodide [PI] double-staining assays were used to study the mechanism of cell death. Modulation in the level of DNA repair and apoptotic proteins following treatment with SCR7 was examined by immunoblotting. Effect of SCR7 on sensitizing radiotherapy was further investigated in the SUDHL8 cells. Results: SCR7 induced cytotoxicity in the DLBCL cell line in a concentration- and time-dependent manner. Cell cycle analysis and annexin V/PI double-staining assay confirmed apoptosis in cells without interfering with cell cycle progression. Change in mitochondrial membrane potential in conjunction with alterations in the levels of apoptotic proteins suggested activation of both intrinsic and extrinsic pathways of apoptosis. Importantly, administration of SCR7 potentiated the effect of radiation upon combination therapy in DLBCL. Conclusion: Our results suggest that SCR7 could be developed as an alternative chemotherapeutic approach against DLBCL and is also effective along with radiotherapy.
Keywords: Cancer therapeutics, chemotherapy, classical nonhomologous DNA end joining, DNA damage, double-strand breaks, genomic instability, lymphoma
|How to cite this article:|
Gopalakrishnan V, Radha G, Raghavan SC, Choudhary B. Inhibitor of nonhomologous end joining can inhibit proliferation of diffuse large B-Cell lymphoma cells and potentiate the effect of ionization radiation. J Radiat Cancer Res 2018;9:93-101
|How to cite this URL:|
Gopalakrishnan V, Radha G, Raghavan SC, Choudhary B. Inhibitor of nonhomologous end joining can inhibit proliferation of diffuse large B-Cell lymphoma cells and potentiate the effect of ionization radiation. J Radiat Cancer Res [serial online] 2018 [cited 2018 Aug 21];9:93-101. Available from: http://www.journalrcr.org/text.asp?2018/9/2/93/232987
| Introduction|| |
Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma (NHL), is known for its unique heterogeneity and biology. Recently, different subtypes of DLBCL have been reported by gene expression profiling which includes germinal center B-cell (GCB)-like subtype, activated B-cell (ABC)-like subtype, and primary mediastinal B-cell lymphoma subtype.,,, Apart from these, there are double-hit lymphomas where MYC, BCL2, and sometimes BCL6 proteins are overexpressed. The heterogenic nature of DLBCL makes it difficult to find an efficient therapeutic option. Although majority of the DLBCL patients have benefited by standard rituximab (R)-based chemotherapy regimens such as cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy, ~40% of the patients develop relapsed/refractory disease due to limited therapeutic options., Many novel therapeutic agents such as lenalidomide, ibrutinib, bortezomib, and CC-122 have been used as either single agents or in combination with R-CHOP to treat patients with relapsed/refractory DLBCL. However, due to its heterogeneity, DLBCL is an aggressive NHL with high relapse and relatively short survival rate. Thus, there is a need for developing novel and targeted therapeutic agents against DLBCL.
Several traditional chemotherapeutic agents induce DNA breaks as intermediate in cancer cells. However, the ability of cancer cells to repair breaks hampers their therapeutic efficiency. Overexpression of nucleotide-excision repair (NER) proteins is one of the factors associated with CHOP resistance seen in DLBCL patients.,, The interstrand crosslinks induced by cyclophosphamide and doxorubicin are repaired by overexpressed NER proteins. Thus, developing anticancer agents that target DNA repair pathways and proteins will be a promising strategy to overcome the drug resistance. The higher expression level of DNA repair proteins in cancerous cells compared to normal cells would make the inhibitors specific to cancer cells.
Gene expression analysis showed high expression of proteins involved in NER, base-excision repair, homologous recombination (HR), nonhomologous end joining (NHEJ), and mismatch repair, especially in ABC subtype of DLBCL, which is known for its poorer outcome as compared to GCB subtype of DLBCL. We have seen the presence of DNA double-strand break (DSB) repair pathways such as classical NHEJ (c-NHEJ), microhomology-mediated end joining (MMEJ), and HR in a DLBCL cell line (VG/BC unpublished).
SCR7 is a known c-NHEJ inhibitor that inhibits binding of ligase IV to the DNA during DNA DSB repair., SCR7 blocked end joining of broken DNA ends, irrespective of type of termini, when incubated with mouse testicular extract. Recently, another study showed that the presence of SCR7 reduced the pairing efficiency of DNA ends by inhibiting the activity of ligase IV. Although cancer therapeutic potential of SCR7 has been explored in breast cancer, cervical cancer, and other cancers of epithelial origin, it is yet to be tested in DLBCL. Besides, studying the combinational effect of SCR7 along with radiation that induces DNA breaks as an intermediate to eliminate cancer cells during radiotherapy will be of interest. In the present study, we report that SCR7 induces cell death in DLBCL cells in a concentration- and time-dependent manner. Although treatment with SCR7 did not result in cell cycle arrest, it led to alteration in mitochondrial membrane potential (MMP) and activation of apoptosis. Interestingly, we have also observed SCR7-dependent activation of DNA DSB repair proteins. More importantly, treatment with SCR7 potentiated the effect of radiation.
| Materials and Methods|| |
Human DLBCL cell line SUDHL8 was a kind gift from Dr. A. Epstein, USA. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin and incubated at 37°C in a humidified atmosphere containing 5% CO2.
Chemicals and reagents
Chemicals and reagents were purchased from Sigma Aldrich, USA, and SRL, India. Antibodies were purchased from Santa Cruz Biotechnology, Calbiochem, USA, and BD, USA. Synthesis of the SCR7 was described previously.
Samples were irradiated using cobalt-60 gamma irradiator (BI 2000, BRIT, India). The dose rate of the source was 2.06 Gy/min at the time of use.
Trypan blue exclusion assay
Effect of SCR7 on SUDHL8 cell viability was determined by trypan blue exclusion assay as described.,, Cells were seeded at a density of 0.50 × 105 cells/ml and incubated for 24 h at 37°C. Cells were then treated with different concentrations of SCR7 (0, 10, 50, 100, and 250 μM) for 24, 48, 72, and 96 h. After each time point, cells were harvested and counted after staining the cells with trypan blue and the IC50 value was determined. Trypan blue is a vital dye which enters the cells only if the cell membrane is compromised, staining the cells blue. Only viable cells were counted. Dimethyl sulfoxide-treated cells served as vehicle control.
Cell cycle analysis by flow cytometry
Cell cycle analysis was performed in SUDHL8 cells as described.,, Cells were seeded at a density of 0.50 × 105 cells/ml and incubated for 24 h at 37°C. After incubation, cells were treated with 100 and 250 μM SCR7 for 48 h. Cells were harvested after 48 h, washed, fixed, and incubated with RNase A. Cells were then stained with propidium iodide (PI) (50 μg/ml) and incubated at 37°C for 15 min and the readings were acquired in flow cytometer (BD Biosciences FACSCalibur, USA). A minimum of 10,000 cells was acquired per sample and the data were analyzed using WinMDI 2.9 software (Scripps Research Institute, US). Experiments were repeated multiple times and the data are presented along with error bars.
Determination of mitochondrial membrane potential
To test the change in MMP after treatment with SCR7, cells were stained with 5,5', 6, 6 tetrachloro-1,1', 3, 3'-tetraethyl benzimidazol-carbocyanine iodide (JC-1) dye. The assay was carried out as described before.,, JC-1 is a carbocyanine dye that selectively enters mitochondria and changes reversibly its color from red (J-aggregate, emission at 590 nm) to green (monomeric form, emission at 530 nm) upon change in MMP, which occurs during apoptosis. Briefly, cells were treated with SCR7 (0, 100, and 250 μM), harvested after 48 h, and incubated with JC-1 dye. The stained cells were then analyzed using flow cytometry using Cell Quest Pro software (BD Biosciences, US) with an excitation at 480 nm laser and an emission at 530 nm laser. Cells treated with 2,4-dinitrophenol served as positive control. The ratio of cells emitting red to green fluorescence for each concentration was plotted.
Annexin V-FITC/propidium iodide double-staining to detect apoptotic stages
To differentiate early apoptotic, late apoptotic, and necrotic cells, annexin V-FITC/PI staining was done as described earlier.,,, Annexin V-FITC binds to phosphatidylserine which gets translocated from inner side of the plasma membrane to the cell surface during earlier stage of apoptosis. PI stains both late apoptotic and necrotic cells. SUDHL8 cells were treated with SCR7 (0, 100, and 250 μM) for 48 h, stained with annexin V-FITC and PI for 20 min, and examined by flow cytometry (BD Biosciences FACSCalibur, USA) using Cell Quest Pro Software (BD Biosciences, US) at an excitation with 488 nm laser and an emission at 530 nm laser. A minimum of 10,000 cells was acquired per sample and the data were analyzed using Flowing Software (version 2, University of Turku, Finland). Experiments were repeated a minimum of two independent times and the data are presented along with error bars.
Gamma irradiation of cells
Cells were subjected to two doses of gamma radiation (2.06 Gy/min), 0.5 Gy and 1 Gy, after 24 h of SCR7 treatment (100 μM). After irradiation, cells were seeded back and incubated for 24 h in CO2 incubator at 37°C. After incubation, cells were harvested and stained with trypan blue, and the viable cells were counted using a hemocytometer as described above. Cells were treated with SCR7 alone (100 μM) and ionizing radiation alone (0.5 and 1 Gy) to compare their individual effects with that of combined effect.
Cell lysate was prepared after treating cells with SCR7 (0, 100, and 250 μM) for 48 and 72 h, and western blotting was performed as described.,, Briefly, ~30 μg protein sample was loaded in 8%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, USA). Membrane was blocked using 5% skimmed milk powder (1 h, RT) and probed with appropriate primary antibodies (Santa Cruz, USA, Calbiochem, USA, and BD, USA) for different DNA repair proteins (ligase IV [1:500], ligase I [1:500], ligase III [1:2000], XRCC4 [1:500], KU70 [1:750], KU80 [1:750], RAD50 [1:750], RAD51 [1:750], and PARP1 [1:1000]) and apoptotic proteins (BCL2 [1:500], cytochrome c [Cyt c] [1:750], caspase 3, caspase 8, and caspase 9 [1:500]). Following incubation with primary antibody, blots were washed in 1X phosphate-buffered saline and 0.1% Tween 20 (PBST) and incubated with biotinylated secondary antibodies (Santa Cruz; 1:10,000) at room temperature for 2 h. The blots were rinsed in PBST, incubated with 250 ng/ml streptavidin-horseradish peroxidase (Sigma) for 30 min, and then washed. The blots were developed using chemiluminescent solution (Immobilon™ Western; Millipore, USA) and scanned by gel documentation system (LAS 3000; Fuji, Japan). Each western blot was repeated a minimum of two independent times.
Values in statistics are expressed as the mean ± standard error of the mean of two to three independent experiments. Statistical comparison was made by one-way anova followed by Student's t-test using GraphPad Prism 4.0 software(GraphPad Software, Inc.). A P < 0.05 was considered statistically significant.
| Results|| |
SCR7 induces cytotoxicity in a time- and concentration-dependent manner in diffuse large B-cell lymphoma cells
To assess the cytotoxic effect of SCR7, a known NHEJ inhibitor, a cell line derived from DLBCL patient (SUDHL8) was treated with increasing concentrations of SCR7 (0, 10, 50, 100, and 250 μM) and incubated for different time points (24, 48, 72, and 96 h). Cytotoxicity was determined using trypan blue exclusion assay. While cell death observed was minimal after 24 h of SCR7 treatment, even at a concentration of 100 μM, incubation for longer time resulted in the elevated levels of cytotoxicity [Figure 1]. We observed >40% cell death at 100 μM when incubated for 48 h [Figure 1]b. Interestingly, longer incubation of cells with SCR7 (72 h and 96 h) resulted in efficient cell death from a concentration of 50 μM onward in SUDHL8 cells [Figure 1]c and [Figure 1]d. The observed cytotoxicity at lower concentrations of SCR7 at later time points suggests the upregulation of target pathway at later time points after SCR7 treatment. Thus, our results reveal that SCR7 can induce cell death in DLBCL cells in a dose- and time-dependent manner.
|Figure 1: Determination of cytotoxic effect of SCR7 on diffuse large B-cell lymphoma cell line, SUDHL8. (a-d) Cell viability was determined by trypan blue assay on SUDHL8 cell line. SUDHL8 was treated with SCR7 for 24 h (a), 48 h (b), 72 h (c), and 96 h (d). Dimethyl sulfoxide-treated cells served as vehicle control (denoted as “C” in each case). Concentrations of compound used were 10, 50, 100, and 250 μM. Error bar indicates standard deviation based on multiple experiments|
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SCR7 induces cell death without cell cycle arrest
Cell cycle analysis was performed to examine whether the observed growth inhibition was due to cell cycle arrest or apoptosis. To study this, SUDHL8 cells were subjected to flow cytometric analysis following treatment with SCR7 (100 and 250 μM for 48 h). Results showed a dose-dependent increase in the sub G1 population, which is an indication of cell death [Figure 2]a and [Figure 2]b. However, no cell cycle arrest was observed upon treatment with SCR7 in DLBCL cells [Figure 2]a. Thus, these results suggest that SCR7 induces cell death in DLBCL cells without interfering with cell cycle progression.
|Figure 2: Effect of SCR7 on cell cycle progression in SUDHL8 cells. (a) SUDHL8 cells (0.50 × 105) were incubated with SCR7 (100 and 250 μM) for 48 h at 37°C. Cells were then harvested, fixed, and stained with propidium iodide and analyzed by flow cytometry. Histogram resulting from the analysis is shown. (b) Bar diagram showing the percentage of cells in different phases of cell cycle. Error bar indicates standard deviation based on independent experiments. P values were calculated by comparing the mean of control with the mean of SCR7-treated samples. *P < 0.05, **P < 0.005, ***P < 0.0001. Dimethyl sulfoxide-treated cells served as vehicle control|
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SCR7 treatment resulted in elevated mitochondrial membrane potential
Loss of MMP is one of the early indications of cell death through apoptosis. The change in MMP was examined following treatment with SCR7 (0, 100, and 250 μM) on SUDHL8 cells for 48 h. Following treatment, cells were stained with JC-1 dye and subjected to flow cytometry [Figure 3]. Results showed that while control cells exhibited red fluorescence indicating unperturbed MMP, treatment with SCR7 resulted in a shift from red to green fluorescence, indicating loss of MMP and accumulation of monomeric JC-1, in a concentration-dependent manner. The difference in membrane potential was determined by analyzing the ratio of red to green fluorescence following SCR7 treatment [Figure 3]b.
|Figure 3: Analysis of mitochondrial membrane potential following treatment with SCR7 in SUDHL8 cells. (a) SUDHL8 cells (0.50 × 105) were treated with 100 and 250 μM of SCR7 for 48 h. Mitochondrial membrane potential was assayed using 5,5',6,6 tetrachloro-1,1',3,3'-tetraethyl benzimidazol-carbocyanine iodide staining followed by flow cytometry. Histogram showing a spectral shift from red to green upon treatment with SCR7. (b) The ratio of red to green fluorescence is represented as bar diagram with error bars. Dimethyl sulfoxide-treated cells served as vehicle control|
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SCR7 treatment leads to apoptosis of SUDHL8 cells
To study whether the observed cytotoxicity is due to the activation of apoptosis or necrosis, DLBCL cells were stained with annexin V-FITC and PI following treatment with SCR7 (0, 100, and 250 μM, for 48 h) and analyzed using flow cytometry. Results showed that SCR7 induced apoptosis in SUDHL8 cells in a concentration-dependent manner [Figure 4]a and [Figure 4]b. As compared to control cells, 13.45% of the cells and 97% of the cells were in the late apoptotic stage in 100 and 250 μM SCR7-treated samples, respectively [Figure 4]a. In contrast, only 0.9% and 1.3% of the cells were in necrotic phase when treated with 100 and 250 μM SCR7, respectively. Thus, our results suggest that SCR7 treatment induced translocation of phosphatidyl serine to cell surface which was stained with annexin V-FITC indicating activation of apoptosis.
|Figure 4: Annexin V-FITC and propidium iodide staining to evaluate the level of apoptosis in SUDHL8 cells following SCR7 treatment. Cells (0.50 × 105) were treated with 100 and 250 μM of SCR7 and incubated for 48 h. After incubation, cells were harvested and incubated with annexin V-FITC and propidium iodide and analyzed using flow cytometry. (a) In each panel, the lower left quadrant showing cells which are negative for both propidium iodide and annexin V-FITC, upper left quadrant showing only propidium iodide positive cells which are necrotic. The lower right quadrant showing annexin V-positive cells (early apoptotic cells) and the upper right quadrant shows annexin V and propidium iodide-positive cells (late apoptotic cells). (b) The percentage of necrotic, early, and late apoptotic cells is represented as bar diagram with error bars|
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Treatment with SCR7 resulted in enhanced level of double-strand break repair proteins and apoptotic markers
Since SCR7 is a known inhibitor of ligase IV and NHEJ, we were interested in testing the levels of DSB repair proteins following treatment with SCR7. Previous studies revealed that SCR7 treatment resulted in the inhibition of NHEJ, leading to accumulation of DSBs within the cells. Therefore, we were interested in checking the expression levels of different DSB proteins after treating the cells with increasing concentrations of SCR7 (100 and 250 μM) for 48 and 72 h. Results revealed an increase in ligase IV expression in SUDHL8 cells following SCR7 treatment as compared to control cells at both the time points [Figure 5]a and [Figure 5]c. This observation is consistent with increased cytotoxicity observed after 48 h post-SCR7 treatment. Interestingly, we also observed elevated expression of ligase I, ligase III, XRCC4, KU70, KU80, RAD51, RAD50, and PARP1 following treatment with SCR7 after 48 h [Figure 5]a and [Figure 5]c. However, not much change in the expression levels of ligase III and RAD51 was observed after 72 h of SCR7 treatment, unlike other DNA repair proteins studied.
|Figure 5: Impact of SCR7 treatment on modulating expression of DNA repair and apoptotic proteins. (a-d) Expression levels of DNA repair and apoptotic proteins following treatment with SCR7. Cell lysate was prepared after treating SUDHL8 cells with SCR7 (100 and 250 μM) for 48 h (a and b) and 72 h (c and d), respectively. Protein lysate was resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting was performed using appropriate primary and secondary antibodies. Expression pattern was studied for repair proteins after 48 and 72 h of treatment (a and c). Antibodies against ligase IV, ligase III, ligase I, XRCC4, RAD51, KU70, KU80, RAD50, and PARP1 were used for the study. Ponceau-stained polyvinylidene fluoride membrane after protein transfer acted as a control for equal loading of protein. Expression levels of apoptotic proteins following treatment with SCR7 for 48 and 72 h, respectively, were also assessed (b and d). Antibodies against cytochrome c, caspase 3, caspase 8, and caspase 9, and BCL2 were used for the study. Dimethyl sulfoxide-treated cells grown for 48 and 72 h, respectively, served as vehicle control|
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Elevated Cyt c release was observed upon treatment with SCR7 after 48 h, suggesting the involvement of intrinsic pathway of apoptosis. This was further supported by the proteolytic cleavage of caspase 9 and caspase 3, which are also involved in intrinsic pathway of apoptosis [Figure 5]b and [Figure 5]d. Besides, cleavage of caspase 8 was seen at both 48 and 72 h of treatment with SCR7, suggesting the involvement of extrinsic pathway of apoptosis as well [Figure 5]b and d]. Hence, our results suggest that SCR7 treatment can lead to the induction of both intrinsic and extrinsic pathway of apoptosis.
SCR7 can potentiate sensitivity of gamma irradiation in SUDHL8 cells
To check whether SCR7 can potentiate the effect of radiation-induced cytotoxicity, SUDHL8 cells were irradiated with gamma radiation (0.5 and 1 Gy) following exposure to SCR7 (100 μM, 24 h). Cells were harvested after 24 h of incubation following irradiation and evaluated for cytotoxicity. Results showed >70% cell death when combined with SCR7 (100 μM) and 0.5 Gy irradiation in contrast to 20% or 40% exhibited by SCR7 alone or irradiation (0.5 Gy) [Figure 6]. The effect was even better when 1 Gy-irradiated cells were cotreated with SCR7 (80% cells were dead) in contrast to 20% or ~50% shown by SCR7 or irradiation alone (1 Gy) [Figure 6]. Thus, our results show that SCR7 can potentiate the effect of gamma radiation in DLBCL cells.
|Figure 6: Effect of combination treatment of SCR7 and gamma irradiation on SUDHL8 cells. To determine the cytotoxic effect of SCR7 when combined with gamma irradiation on SUDHL8, cells were incubated with SCR7 (100 μM) for 24 h and then irradiated with 0.5 Gy or 1 Gy. Trypan blue exclusion assay was performed after 24 h of irradiation and data are represented as bar diagram. Error bar indicates standard deviation based on independent experiments. P values were calculated by comparing the mean of control with the mean of SCR7 treated samples. *P < 0.05, **P < 0.005, ***P < 0.0001. Dimethyl sulfoxide.-treated cells served as vehicle control. Cells treated with SCR7 or gamma irradiation alone were also examined to determine the individual effects|
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| Discussion|| |
In the current study, we investigated the therapeutic effects of c-NHEJ inhibitor, SCR7, on DLBCL cells, SUDHL8. Cell viability assays showed a concentration- and time-dependent cytotoxicity induced by SCR7, which was further supported by a series of apoptotic assays such as JC-1, cell cycle analysis, and annexin V/PI double-staining assays. Expression of DSB repair and apoptotic proteins was significantly enhanced upon SCR7 treatment. Taken together, results suggest that SCR7 induced both extrinsic and intrinsic pathways of apoptosis, leading to cytotoxicity. Coadministration of SCR7 with gamma radiation led to an enhanced effect, sensitizing the DLBCL cells to lower doses of radiation.
Current treatment modalities of diffuse large B-cell lymphoma
Deregulation of DNA repair pathways is a hallmark of almost all cancers. Recent studies have shown deregulated DNA repair pathways in DLBCL tumor samples that include NER, base-excision repair, HR, NHEJ, and mismatch repair. The proteins involved in these DNA repair pathways are significantly high in expression, particularly, in ABC subtype of DLBCL, leading to its poor therapeutic outcome. Thus, targeting any of these overexpressed DNA repair proteins could be developed as one of the therapeutic strategies. Besides, pharmacological inhibition of DNA damage response through targeting checkpoint kinases is another novel therapeutic strategy that is used in DLBCL therapy. Dai et al. have shown functional interaction between extracellular signal-regulated kinase (ERK) and CHK2, a protein kinase involved in DNA damage checkpoint that responds to DSBs. ERK inhibition along with CHK2 inhibition enhances antitumor activity. Other than DNA repair and DNA damage response, drugs targeting epigenetic pathways, such as demethylating agents or disrupting histone modifiers, are in active preclinical and early clinical development.
R-CHOP chemotherapy is the current standard chemotherapy given to DLBCL patients of all subtypes. CHOP therapy includes the combination of four drugs, (1) cyclophosphamide, an alkylating agent, which damages the DNA by binding to it and forming interstrand crosslinks, (2) hydroxydaunorubicin/doxorubicin, an intercalating agent, (3) oncovin/vincristine, which binds to the protein tubulin and prevents duplication of cell, and (4) prednisone, which is a corticosteroid. CHOP therapy combined with monoclonal antibody rituximab (R) showed dramatic improvement and increased overall survival of the patients., However, in spite of this improvement, ~40% of the patients do not respond to the current therapy and develop refractory disease. Different immune chemotherapy combinations are now developed as alternate to R-CHOP such as R-CHOP14, R-EPOCH, and R-CEOP90 for patients with relapsed/refractory DLBCL. Because of the molecular heterogeneity of DLBCL, many different classes of agents with molecular subtype specificity are currently in the development for DLBCL treatment, which includes proteasome inhibitors, immunomodulatory agents, B-cell receptor signaling pathway inhibitors, and BCL2 inhibitors.,, Although many of these have shown in vitro and in vivo antitumor activity, very few are in early human clinical trials.
The resistance observed in patients after treatment has been further investigated. Studies have shown that high-risk DLBCL patients overexpress genes involved in NER pathway that provides resistance to CHOP therapy. We have also seen the presence of double-strand DNA repair pathways such as c-NHEJ, MMEJ, and HR in SUDHL8 cells. Thus, all these studies demonstrated the dependence of cancer cells on DNA repair proteins for their survival. Besides, activation of DNA repair pathways also plays a critical role in developing drug resistance. This requirement can be exploited to develop anticancer agents targeting DNA repair pathways.
SCR7 as a DNA repair inhibitor
DNA DSBs are considered as one of the most deleterious lesions in the genome which when unrepaired can lead to cell death., This feature can be utilized to treat cancer cells by introducing DSBs and blocking repair, which will lead to the accumulation of DSBs and hence cell death. There are two major DNA repair mechanisms in eukaryotes: c-NHEJ and HR., In cancer cells, DSBs are rapidly repaired by c-NHEJ. Most of the NHEJ proteins are overexpressed in many cancers.,
SCR7 has been recently identified and characterized as a potent NHEJ inhibitor that targets DNA ligase IV. SCR7 blocked end joining of DSBs, irrespective of type of termini, when incubated with mouse testicular extract. In a recent study, another group has shown that c-NHEJ in G1 phase of the cells is inhibited when treated with SCR7. Because of this unique property of SCR7, it has been used in several applications including genome editing by CRISPR/Cas. There are various studies showing SCR7-mediated increased precise genome editing by inhibiting NHEJ and favoring error-free HR repair pathway.,, In a recent study, nanopolymer-encapsulated SCR7 was used which showed improved bioavailability and significant increase in cytotoxicity than parental SCR7., Furthermore, SCR7 can also potentiate the effect of other treatment modalities such as radiotherapy and chemotherapy, which can induce DSBs as intermediates. In our current study, we observed that SCR7 was effective in inducing cytotoxicity after prolonged incubation with SUDHL8 cells. Prolonged incubation results in the accumulation of DSBs which in turn inhibited cell division after 48 h of SCR7 treatment. Annexin/PI double-staining assay showed that the observed growth inhibition is due to apoptosis induced by SCR7 and not necrosis. Our study further demonstrated that SCR7 induced cell death without affecting cell cycle progression and activating both intrinsic and extrinsic pathway of apoptosis. Although, in our study, SCR7 was found to be less efficient as a single agent, since the cells were less sensitive to SCR7 at initial time points, there was a >2-fold increase in cytotoxicity when combined with gamma radiation. Radiation induces DSBs in the cells whose repair is blocked by SCR7, leading to cell death. This combination treatment will reduce the dose at which radiation induces cytotoxicity, which will in turn help in reducing radiation-induced side effects.
Previously, it was shown that SCR7 inhibited end joining of DSBs in a ligase IV-dependent manner both in vitro and ex vivo (SV/SCR, unpublished). Similarly, ligase IV-dependent inhibition of tumor progression was observed in three different tumor models.In vivo studies on mice models did not show any significant side effects in terms of growth of the animal or functioning of kidney and liver. Although a small effect on immune response was observed post-SCR7 treatment, the effect was transient and soon recovered back to normal state. Thus, SCR7 treatment alone did not lead to any significant side effects with respect to proliferation of normal cells. Therefore, combining DNA repair inhibitors, such as SCR7 and radiation, could be exploited as one of the strategies with very limited side effects as it reduces the dose at which the inhibitor induces cytotoxicity and also minimizes radiation-induced side effects. Combination of other chemotherapeutic agents that induce DSBs, along with SCR7, is another potential therapeutic strategy that can be used for treating DLBCL patients.
B. C., S. C. R., and V. G. conceived and coordinated the study and wrote the paper. S. C. R., B. C., and V. G. designed the experiments. V. G. and R. R. performed the experiments. B. C., S. C. R., and V. G. analyzed the data. All authors reviewed the results and approved the final version of the manuscript.
Financial support and sponsorship
This work was supported by grants from DST-FIST-SR/FST/LSI-536/2012 for BC and DBT, BT/PR13722/BRB/10/781/2010 and IISc-DBT partnership programme [DBT/BF/PR/INS/2011-12/IISc] to SCR. VG is supported by Senior Research fellowship from CSIR, India.*
Conflicts of interest
There are no conflicts of interest.
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