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

I. BACKGROUND: A combination of etoposide (VP-16) and cisplatin (CDDP) is the standard treatment for certain colon cancers. 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 non-homologous 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.

Inhibition of DNA-PK potentiates the synergistic effect of NK314 and etoposide combination on human glioblastoma cells.

Etoposide (VP-16) is the topoisomerase 2 (Top2) inhibitor used for treating of glioma patients however at high dose with serious side effects. It induces DNA double-strand breaks (DSBs). These DNA lesions are repaired by non-homologous DNA end joining (NHEJ) mediated by DNA-dependent protein kinase (DNA-PK). One possible approach to decrease the toxicity of etoposide is to reduce the dose while maintaining the anticancer potential. It could be achieved through combined therapy with other anticancer drugs. We have assumed that this objective can be obtained by (1) a parallel topo2 α inhibition and (2) sensitization of cancer cells to DSBs. In this work we investigated the effect of two Top2 inhibitors NK314 and VP-16 in glioma cell lines (MO59 K and MO59 J) sensitized by DNA-PK inhibitor, NU7441. Cytotoxic effect of VP-16, NK314 alone and in combination on human glioblastoma cell lines, was assessed by a colorimetric assay. Genotoxic effect of anticancer drugs in combination with NU7441 was assessed by comet assay. Cell cycle distribution and apoptosis were analysed by flow cytometry. Compared with VP-16 or NK314 alone, the combined treatment significantly inhibited cell proliferation. Combination treatment was associated with a strong accumulation of DSBs, modulated cell cycle phases distribution and apoptotic cell death. NU7441 potentiated these effects and additionally postponed DNA repair. Our findings suggest that NK314 could overcome resistance of MO59 cells to VP-16 and NU7441 could serve as sensitizer to VP-16/NK314 combined treatment. The combined tripartite approach of chemotherapy could reduce the overall toxicity associated with each individual therapy, while concomitantly enhancing the anticancer effect to treat human glioma cells. Thus, the use of a tripartite combinatorial approach could be promising and more efficacious than mono therapy or dual therapy to treat and increase the survival of the glioblastoma patients.

Current interventions to promote safe and appropriate pain management.

PURPOSE: Describe patient-, clinician-, system-, and community-level interventions for pain management developed and employed by 9 healthcare systems across the United States and report on lessons learned from the implementation of these interventions. SUMMARY: The high cost associated with pain coupled with the frequent use of opioid analgesics as primary treatment options has made novel pain management strategies a necessity. Interventions that target multiple levels within healthcare are needed to help combat the opioid epidemic and improve strategies to manage chronic pain. Patient-level interventions implemented ranged from traditional paper-based educational tools to videos, digital applications, and peer networks. Clinician-level interventions focused on providing education, ensuring proper follow-up care, and establishing multidisciplinary teams that included prescribers, pharmacists, nurses, and other healthcare professionals. System- and community-level interventions included metric tracking and analytics, electronic health record tools, lockbox distribution for safe storage, medication return bins for removal of opioids, risk assessment tool utilization, and improved access to reversal agents. CONCLUSION: Strategies to better manage pain can be implemented within health systems at multiple levels and on many fronts; however, these changes are most effective when accepted and widely used by the population for which they are targeted.

Comparison of the effect of three different topoisomerase II inhibitors combined with cisplatin in human glioblastoma cells sensitized with double strand break repair inhibitors.

Topoisomerase II (Topo2) inhibitors in combination with cisplatin represent a common treatment modality used for glioma patients. The main mechanism of their action involves induction of DNA double-strand breaks (DSBs). DSBs are repaired via the homology-dependent DNA repair (HRR) and non-homologous end-joining (NHEJ). Inhibition of the NHEJ or HRR pathway sensitizes cancer cells to the treatment. In this work, we investigated the effect of three Topo2 inhibitors-etoposide, NK314, or HU-331 in combination with cisplatin in the U-87 human glioblastoma cell line. Etoposide as well as NK314 inhibited Topo2 activity by stabilizing Topo2-DNA cleavable complexes whereas HU-331 inhibited the ATPase activity of Topo2 using a noncompetitive mechanism. To increase the effectiveness of the treatment, we combined cisplatin and Topo2 inhibitor treatment with DSB repair inhibitors (DRIs). The cells were sensitized with NHEJ inhibitor, NU7441, or the novel HRR inhibitor, YU238259, prior to drug treatment. All of the investigated Topo2 inhibitors in combination with cisplatin efficiently killed the U-87 cells. The most cytotoxic effect was observed for the cisplatin + HU331 treatment scheme and this effect was significantly increased when a DRI pretreatment was used; however, we did not observed DSBs. Therefore, the molecular mechanism of cytotoxicity caused by the cisplatin + HU331 treatment scheme is yet to be evaluated. We observed a concentration-dependent change in DSB levels and accumulation at the G2/M checkpoint and S-phase in glioma cells incubated with NK314/cisplatin and etoposide/cisplatin. In conclusion, in combination with cisplatin, HU331 is the most potent Topo2 inhibitor of human glioblastoma cells.

Screening for Multiple Autoantibodies in Plasma of Patients with Breast Cancer.

BACKGROUND/AIM: Autoantibodies have potential as circulating biomarkers for early cancer detection. This study aimed to screen for known autoantibodies in human plasma using an Autoantibody Profiling System (APS) and quantify the levels in plasma of donors with/without breast cancer. MATERIALS AND METHODS: Plasma from nine female donors diagnosed with breast cancer (test group) and nine matched donors with no personal history of cancer (reference group) were screened with an APS containing probes for 30 autoantibodies. Autoantibody levels ≥1.5 times the mean concentration of the group were considered elevated, and test/reference ratios ≥1.3 were considered higher in the test group compared to the reference group. RESULTS: Twenty percent of the probes detected elevated levels of autoantibodies against proteins involved in different cancer mechanisms. Amongst these, the levels of autoantibodies against interleukin 29 (IL29), osteoprotegerin (OPG), survivin (SUR), growth hormone (GRH) and resistin (RES) were significantly higher in the cancer group compared to the reference group (p<0.05), whereas the level of autoantibody against cytotoxic T-lymphocyte associated antigen-4 (CTLA4) was not significantly different between the two groups (p=0.38). CONCLUSION: Disease-relevant autoantibodies were detected in the plasma of patients with breast cancer and donors without breast cancer. This means that identifying the type and level of autoantibodies in samples will be important in determining their significance in the disease process. A microtiter plate-based array system could be a fast and inexpensive screening method for identifying and quantifying autoantibodies in human plasma.

A Low-density Antigen Array for Detection of Disease-associated Autoantibodies in Human Plasma.

BACKGROUND/AIM: The ability to easily detect autoantibodies will help in the early diagnosis and treatment of certain diseases. Currently, available methods for autoantibody detection are time-consuming and cumbersome. The present study aimed to evaluate the performance of an easy-to-use antigen array developed for autoantibody detection. MATERIALS AND METHODS: Plasma from 9 female donors diagnosed with ovarian cancer (test group) and 9 matched donors with no history of cancer (reference group) were screened and results were compared. Autoantibody levels ≥1.5-times the background were classified as positive. RESULTS: A total of 29 autoantibodies were detected, out of which the autoantibody against osteoprotegerin was found to be significantly higher in the "test" group (p<0.001) while those against macrophage migration inhibitor factor, interleukin-2 and vascular endothelial growth factor were lower (p<0.05). CONCLUSION: The evaluated antigen array has potential as a simple method for determining the presence/absence of up to 90 disease-associated autoantibodies in a plasma specimen.

Wortmannin potentiates the combined effect of etoposide and cisplatin in human glioma cells.

The combination of etoposide and cisplatin represents a common modality for treating of glioma patients. These drugs directly and indirectly produce the most lethal DNA double-stand breaks (DSB), which are mainly repaired by non-homologous DNA end joining (NHEJ). Drugs that can specifically inhibit the kinase activity of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the major component of NHEJ, are of special interest in cancer research. These small molecule inhibitors can effectively enhance the efficacy of current cancer treatments that generate DNA damage. In this study, we investigated the effect of DNA-PKcs inhibitor, wortmannin, on the cytotoxic mechanism of etoposide and cisplatin in MO59K and MO59J human glioblastoma cell lines. These cell lines are proficient and deficient in DNA-PKcs, respectively. Wortmannin synergistically increased the cytotoxicity of cisplatin and etoposide, when combined, in NHEJ-proficient MO59K cells. Surprisingly, wortmannin sensitizing effect was also observed in DNA-PKcs-deficient MO59J cells. These data suggest that wortmannin sensitization to etoposide and cisplatin in human glioma cells is mediated by inhibition of not only DNA-PKcs activity but other enzymes from PI3-K family, e.g. ATM and ATR. A concentration-dependent increase in etoposide and cisplatin-induced DSB levels was potentiated by inhibitor in both cell lines. Moreover, drug-induced accumulation in the G2/M checkpoint and S-phase was increased by wortmannin. Wortmannin significantly inhibited drug-induced DSB repair in MO59 cells and this effect was more pronounced in MO59J cells. We conclude that the mechanism of wortmannin potentiation of etoposide and cisplatin cytotoxicity involves DSBs induction, DSBs repair inhibition, G2/M checkpoint arrest and inhibition of not only DNA-PKcs activity.

Base damage immediately upstream from double-strand break ends is a more severe impediment to nonhomologous end joining than blocked 3'-termini.

Radiation-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions that are typically repaired by nonhomologous end joining (NHEJ) in human cells. Our previous work indicated that the highly cytotoxic DSBs formed by (125)I decay possess base damage clustered within 8 to 10 bases of the break and 3'-phosphate (P) and 3'-OH ends. This study examined the effect of such structures on NHEJ in in vitro assays employing either (125)I decay-induced DSB linearized plasmid DNA or structurally defined duplex oligonucleotides. Duplex oligonucleotides that possess either a 3'-P or 3'-phosphoglycolate (PG) or a ligatable 3'-OH end with either an AP site or an 8-oxo-dG 1 nucleotide upstream (-1n) from the 3'-terminus have been examined for reparability. Moderate to severe end-joining inhibition was observed for modified DSB ends or 8-oxo-dG upstream from a 3'-OH end. In contrast, abolition of end joining was observed with duplexes possessing an AP site upstream from a ligatable 3'-OH end or for a lesion combination involving 3'-P plus an upstream 8-oxo-dG. In addition, base mismatches at the -1n position were also strong inhibitors of NHEJ in this system, suggesting that destabilization of the DSB terminus as a result of base loss or improper base pairing may play a role in the inhibitory effects of these structures. Furthermore, we provide data indicating that DSB end joining is likely to occur prior to removal or repair of base lesions proximal to the DSB terminus. Our results show that base damage or base loss near a DSB end may be a severe block to NHEJ and that complex combinations of lesions presented in the context of a DSB may be more inhibitory than the individual lesions alone. In contrast, blocked DSB 3'-ends alone are only modestly inhibitory to NHEJ. Finally, DNA ligase activity is implicated as being responsible for these effects.

Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining.

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PK(cs) to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PK(cs) appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PK(cs) protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures.

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo- or radiotherapy by pharmacologically modulating NHEJ cellular responses.

Non-homologous DNA end joining in normal and cancer cells and its dependence on break structures

DNA double-strand breaks (DSBs) are a serious threat to the cell, for if not or miss-repaired, they can lead to chromosomal aberration, mutation and cancer. DSBs in human cells are repaired via non-homologous DNA end joining (NHEJ) and homologous recombination repair pathways. In the former process, the structure of DNA termini plays an important role, as does the genetic constitution of the cells, through being different in normal and pathological cells. In order to investigate the dependence of NHEJ on DSB structure in normal and cancer cells, we used linearized plasmids with various, complementary or non-complementary, single-stranded or blunt DNA termini, as well as whole-cell extract isolated from normal human lymphocytes, chronic myeloid leukemia K562 cells and lung cancer A549 cells. We observed a pronounced variability in the efficacy of NHEJ reaction depending on the type of ends. Plasmids with complementary and blunt termini were more efficiently repaired than the substrate with 3' protruding single-strand ends. The hierarchy of the effectiveness of NHEJ was on average, from the most effective to the least, A549/ normal lymphocytes/ K562. Our results suggest that the genetic constitution of the cells together with the substrate terminal structure may contribute to the efficacy of the NHEJ reaction. This should be taken into account on considering its applicability in cancer chemo-or radiotherapy by pharmacologically modulating NHEJ cellular responses.

In vitro non-homologous DNA end joining assays--the 20th anniversary.

DNA double-strand breaks (DSBs) are the most serious forms of DNA damage in cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to mutations or cell death, and consequently, to cancer predisposition. In human cells non-homologous DNA end joining (NHEJ) is the main repair mechanism of these breaks. Systems for DNA end joining study have been developing during the last 20 years. New assays have some advantages over earlier in vitro DSBs repair assays because they are less time-consuming, allow the use of clinical material and examination of the joining DNA ends produced physiologically in mammalian cells. Proteins involved in NHEJ repair pathway can serve as biomarkers or molecular targets for anticancer drugs. Results of studies on NHEJ in cancer could help to select potent repair inhibitors that may selectively sensitize tumor cells to ionizing radiation (IR) and chemotherapy. Here, we review the principles and practice of in vitro NHEJ assays and provide some insights into the future prospects of this assay in cancer diagnosis and treatment.

Non-homologous DNA end joining in anticancer therapy.

Non-homologous DNA end joining (NHEJ) is the major pathway for the repair of double-strand breaks (DSBs) in human cells. Proteins involved in NHEJ pathway can become molecular targets in the treatment of cancer. Inhibition of this pathway leads to radio- and chemosensitization of cancer cells. This review will focus on the new therapeutic strategies for NHEJ pathway inhibition and their application in anticancer therapy.

Proteomics in human cancer research.

Proteomics is now widely employed in the study of cancer. Many laboratories are applying the rapidly emerging technologies to elucidate the underlying mechanisms associated with cancer development, progression, and severity in addition to developing drugs and identifying patients who will benefit most from molecular targeted compounds. Various proteomic approaches are now available for protein separation and identification, and for characterization of the function and structure of candidate proteins. In spite of significant challenges that still exist, proteomics has rapidly expanded to include the discovery of novel biomarkers for early detection, diagnosis and prognostication (clinical application), and for the identification of novel drug targets (pharmaceutical application). To achieve these goals, several innovative technologies including 2-D-difference gel electrophoresis, SELDI, multidimensional protein identification technology, isotope-coded affinity tag, solid-state and suspension protein array technologies, X-ray crystallography, NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulation have evolved, and are being used in different combinations. This review provides an overview of the field of proteomics and discusses the key proteomic technologies available to researchers. It also describes some of the important challenges and highlights the current pharmaceutical and clinical applications of proteomics in human cancer research.

Non-homologous DNA end joining repair in normal and leukemic cells depends on the substrate ends.

Double-strand breaks (DSBs) are the most serious DNA damage which, if unrepaired or misrepaired, may lead to cell death, genomic instability or cancer transformation. In human cells they can be repaired mainly by non-homologous DNA end joining (NHEJ). The efficacy of NHEJ pathway was examined in normal human lymphocytes and K562 myeloid leukemic cells expressing the BCR/ABL oncogenic tyrosine kinase activity and lacking p53 tumor suppressor protein. In our studies we employed a simple and rapid in vitro DSB end joining assay based on fluorescent detection of repair products. Normal and cancer cells were able to repair DNA damage caused by restriction endonucleases, but the efficiency of the end joining was dependent on the type of cells and the structure of DNA ends. K562 cells displayed decreased NHEJ activity in comparison to normal cells for 5' complementary DNA overhang. For blunt-ended DNA there was no significant difference in end joining activity. Both kinds of cells were found about 10-fold more efficient for joining DNA substrates with compatible 5' overhangs than those with blunt ends. Our recent findings have shown that stimulation of DNA repair could be involved in the drug resistance of BCR/ABL-positive cells in anticancer therapy. For the first time the role of STI571 was investigated, a specific inhibitor of BCR/ABL oncogenic protein approved for leukemia treatment in the NHEJ pathway. Surprisingly, STI571 did not change the response of BCR/ABL-positive K562 cells in terms of NHEJ for both complementary and blunt ends. Our results suggest that the various responses of the cells to DNA damage via NHEJ can be correlated with the differences in the genetic constitution of human normal and cancer cells. However, the role of NHEJ in anticancer drug resistance in BCR/ABL-positive cells is questionable.

DNA uptake and repair enzyme access to transfected DNA is under reported by gene expression.

Gene expression is the typical biological end point of interest following transfection. However, transcription may not accurately assess DNA uptake, or the ability of transfected DNA to be acted on by other enzymatic pathways. We have compared DNA uptake to gene expression and the unrelated enzymatic process of DNA double strand break (DSB) repair. Transfection efficiency (at limiting DNA concentration) was assessed as a function of DNA uptake and gene expression in the DSB repair proficient WI38VA13 and MO59K cell lines and the DSB repair defective cell line MO59J, by comparing eGFP expression from the pHygEGFP expression vector with uptake of rhodamine labeled linear pSP189 plasmid (3:1). Repair proficient cells expressed eGFP most efficiently, but never approached DNA uptake levels (>or=90%). Although transfected DNAs were stable in repair proficient cells and degraded in MO59J cells, most cells did not express eGFP, but in the repair proficient cells linear DNA did undergo DSB repair.

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks.

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Non-homologous DNA end joining.

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.