DNA-damaging Anticancer Drugs Research Articles

Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

Developing novel antimicrobials by combining cancer chemotherapeutics with bacterial DNA repair inhibitors.

Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. To tackle this problem, we have isolated several compounds that inhibit bacterial DNA repair, alone they are non-toxic, however in combination with DNA damaging anti-cancer drugs, they prevent bacterial growth. These compounds were identified through screening of an FDA-approved drug library in the presence of the anti-cancer compound cisplatin. Using a series of triage tests, the screen was reduced to a handful of drugs that were tested for specific activity against bacterial nucleotide excision DNA repair (NER). Five compounds emerged, of which three possess promising antimicrobial properties including cell penetrance, and the ability to block replication in a multi-drug resistant clinically relevant E. coli strain. This study suggests that targeting NER could offer a new therapeutic approach tailor-made for infections in cancer patients, by combining cancer chemotherapy with an adjuvant that targets DNA repair.

Multicellular Complex Tumor Spheroid Response to DNA Repair Inhibitors in Combination with DNA-damaging Drugs.

Clinical efficacy of DNA-damaging anticancer drugs can be influenced by the DNA damage response in tumor cells. The potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways was assessed in multicellular tumor spheroids. Although most combinations demonstrated additive cytotoxicity, synergistic cytotoxicity was observed for several drug combinations.

Enhanced cytotoxicity to lung cancer cells by mitochondrial delivery of camptothecin

Delivering traditional DNA-damaging anticancer drugs into mitochondria to damage mitochondria is a promising chemotherapy strategy. The impermeability of this mitochondrial inner membrane, however, impedes the delivery of drug molecules that could impact other important biological roles of mitochondria. Herein, the prodrug camptothecin (CPT)-triphenylphosphine (TPP) modified with hyaluronic acid (HA) via electrostatic adsorption (HA/CPT-TPP, HCT) was used to mediate the mitochondrial accumulation of CPT. These nanoparticles (NPs) showed enhanced drug accumulation in cancer cells through tumor targeting. HCT entered acidic lysosomes through endosomal transport, HA was degraded by hyaluronidase (HAase) in acidic lysosomes, and the positively charged CPT-TPP was exposed and accumulated fully in the mitochondria. Subsequently, CPT-TPP significantly disrupted the mitochondrial structure and damaged mitochondrial function, leading to increased reactive oxygen species (ROS) levels and energy depletion. Finally, HCT enhanced lung cancer cell apoptosis via the activation of caspase-3 and caspase-9. Furthermore, greatly increased tumor growth inhibition was observed in nude mice bearing A549 xenograft tumors after the administration of HCT via tail injection. This study demonstrated that the mitochondria-targeted delivery of CPT may be a promising antitumor therapeutic strategy.

DDDR-13. MGMT FUNCTION DETERMINES THE DIFFERENTIAL RESPONSE OF ATR AND PARP INHIBITORS WITH DNA DAMAGING AGENTS FOR GBM THERAPY

Abstract The most common cancer therapies including chemo- and radiation therapy are designed to induce cell death by DNA damage. However, DNA damage response (DDR) repair pathways initiated by tumor cells can resist the effect of anticancer drugs thus providing the rationale for combining DNA repair pathway inhibitors with radiation and DNA damaging anticancer drugs. The reliance of different cancer to different DDR mechanism can guide for targeted use of specific DDR inhibitor to maximize tumor cell killing. Here, we show the differential activities of two classes of DDR pathway inhibitors, Ataxia Telangiectasia- and Rad3-related kinase (ATRi) and poly(ADP-ribose) polymerases inhibitors (PARPi) across 20 glioma stem cell (GSC) lines. Using a panel of 3 ATRi, we identified a subgroup of GSCs, showing significant synergism with TMZ combination but resistant to ATRi single agent monotherapy. Interestingly, majority of the synergistic cell lines are associated with MGMT promoter methylation, suggesting that only tumor cells with no MGMT repair will benefit from ATR inhibition. Moreover, we also observe that PARPi + TMZ showed synergism only in MGMT unmethylated GSC cells suggesting that this synergism is a result of the dual function of PARPi in blocking BER repair and blocking MGMT PARylation thus abolishing MGMT mediated repair. Altogether, we believe MGMT methylation status in primary GBM could function as a strong biomarker for patient selection for ATRi or PARPi combination with TMZ in the clinic. We are currently exploring the activity of the triple combination of TMZ + PARPi + ATRi for the small subgroup of GSCs that do not show response to either TMZ + ATRi or PARPi + TMZ combinations.

Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

Halofuginone Sensitizes Lung Cancer Organoids to Cisplatin via Suppressing PI3K/AKT and MAPK Signaling Pathways.

Lung cancer is the leading cause of cancer death worldwide. Cisplatin is the major DNA-damaging anticancer drug that cross-links the DNA in cancer cells, but many patients inevitably develop resistance with treatment. Identification of a cisplatin sensitizer might postpone or even reverse the development of cisplatin resistance. Halofuginone (HF), a natural small molecule isolated from Dichroa febrifuga, has been found to play an antitumor role. In this study, we found that HF inhibited the proliferation, induced G0/G1 phase arrest, and promoted apoptosis in lung cancer cells in a dose-dependent manner. To explore the underlying mechanism of this antitumor effect of halofuginone, we performed RNA sequencing to profile transcriptomes of NSCLC cells treated with or without halofuginone. Gene expression profiling and KEGG analysis indicated that PI3K/AKT and MAPK signaling pathways were top-ranked pathways affected by halofuginone. Moreover, combination of cisplatin and HF revealed that HF could sensitize the cisplatin-resistant patient-derived lung cancer organoids and lung cancer cells to cisplatin treatment. Taken together, this study identified HF as a cisplatin sensitizer and a dual pathway inhibitor, which might provide a new strategy to improve prognosis of patients with cisplatin-resistant lung cancer.

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1.

Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for thetreatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses. In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD+) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis. The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices.

Here, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1-) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1- lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies.

Interplay Between Thiamine and p53/p21 Axes Affects Antiproliferative Action of Cisplatin in Lung Adenocarcinoma Cells by Changing Metabolism of 2-Oxoglutarate/Glutamate.

Thiamine (vitamin B1) is often deficient in oncopatients, particularly those undergoing chemotherapy. However, interaction between the thiamine deficiency and anticancer action of drugs has not been characterized. A major natural thiamine derivative, thiamine diphosphate (ThDP), is a coenzyme of central metabolism, also known to affect transcriptional activity of the master metabolic regulator and genome guardian p53. A direct transcriptional target of p53, p21, regulates cell cycle dynamics and DNA damage response. Our work focuses on dependence of the action of the DNA damaging anticancer drug cisplatin on metabolic regulation through p53/p21 axes and cellular thiamine status in human lung adenocarcinoma cells A549. These cells are used as a model of a hardly curable cancer, known to develop chemoresistance to platinum drugs, such as cisplatin. Compared to wild type (A549WT), a stable line with a 60% knockdown of p21 (A549p21–) is less sensitive to antiproliferative action of cisplatin. In contrast, in the thiamine-deficient medium, cisplatin impairs the viability of A549p21– cells more than that of A549WT cells. Analysis of the associated metabolic changes in the cells indicates that (i) p21 knockdown restricts the production of 2-oxoglutarate via glutamate oxidation, stimulating that within the tricarboxylic acid (TCA) cycle; (ii) cellular cisplatin sensitivity is associated with a 4-fold upregulation of glutamic-oxaloacetic transaminase (GOT2) by cisplatin; (iii) cellular cisplatin resistance is associated with a 2-fold upregulation of p53 by cisplatin. Correlation analysis of the p53 expression and enzymatic activities upon variations in cellular thiamine/ThDP levels indicates that p21 knockdown substitutes positive correlation of the p53 expression with the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) for that with the activity of glutamate dehydrogenase (GDH). The knockdown also changes correlations of the levels of OGDHC, GDH and GOT2 with those of the malate and isocitrate dehydrogenases. Thus, a p53/p21-dependent change in partitioning of the glutamate conversion to 2-oxoglutarate through GOT2 or GDH, linked to NAD(P)-dependent metabolism of 2-oxoglutarate in affiliated pathways, adapts A549 cells to thiamine deficiency or cisplatin treatment. Cellular thiamine deficiency may interfere with antiproliferative action of cisplatin due to their common modulation of the p53/p21-dependent metabolic switch between the glutamate oxidation and transamination.

The tyrosine kinase v-Src modifies cytotoxicities of anticancer drugs targeting cell division.

ABSTRACTv‐Src oncogene causes cell transformation through its strong tyrosine kinase activity. We have revealed that v‐Src‐mediated cell transformation occurs at a low frequency and it is attributed to mitotic abnormalities‐mediated chromosome instability. v‐Src directly phosphorylates Tyr‐15 of cyclin‐dependent kinase 1 (CDK1), thereby causing mitotic slippage and reduction in Eg5 inhibitor cytotoxicity. However, it is not clear whether v‐Src modifies cytotoxicities of the other anticancer drugs targeting cell division. In this study, we found that v‐Src restores cancer cell viability reduced by various microtubule‐targeting agents (MTAs), although v‐Src does not alter cytotoxicity of DNA‐damaging anticancer drugs. v‐Src causes mitotic slippage of MTAs‐treated cells, consequently generating proliferating tetraploid cells. We further demonstrate that v‐Src also restores cell viability reduced by a polo‐like kinase 1 (PLK1) inhibitor. Interestingly, treatment with Aurora kinase inhibitor strongly induces cell death when cells express v‐Src. These results suggest that the v‐Src modifies cytotoxicities of anticancer drugs targeting cell division. Highly activated Src‐induced resistance to MTAs through mitotic slippage might have a risk to enhance the malignancy of cancer cells through the increase in chromosome instability upon chemotherapy using MTAs.

Contribution of Histone Deacetylases in Prognosis and Therapeutic Management of Cholangiocarcinoma.

Cholangiocarcinoma (CCA), a malignant tumor that occurs in the epithelium of the biliary tract, has a very poor prognosis because affected patients are frequently diagnosed at an advanced stage and recurrence after resection is common. Over the last two decades, our understanding of the molecular biology of this malignancy has expanded, and various studies have explored targeted therapy for CCA in order to improve patient survival. The histone acetylation/deacetylation equilibrium is affected in carcinogenesis, leading to altered chromatin structure and therefore changes in gene expression. Understanding the molecular identity of histone deacetylases (HDACs), their cellular interactions and potential role as anticancer agents will help us develop new therapeutic strategies for CCA-affected patients. Furthermore, HDAC inhibitors act on cellular stress response pathways and decrease cancer angiogenesis. Downregulation of pro-angiogenic genes such as vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 (HIF-1), and endothelial nitric oxide synthase (eNOS) inhibit formation of new vessels and can negatively affect the metastatic process. Finally, recent clinical trials prove that administration of both HDAC inhibitors and DNA-targeting chemotherapeutic agents, such as topoisomerase inhibitors, DNA intercalating agents, inhibitors of DNA synthesis, covalently modifying DNA agents, and ionizing radiation, maximizes the anticancer effect by increasing the cytotoxic efficiency of a variety of DNA-damaging anticancer drugs. Therefore, combination therapy of classic chemotherapeutic drugs with HDAC inhibitors can act synergistically for the patients' benefit.

An ATM/TRIM37/NEMO Axis Counteracts Genotoxicity by Activating Nuclear-to-Cytoplasmic NF-κB Signaling.

Blocking genotoxic stress-induced NF-κB activation would substantially enhance the anticancer efficiency of genotoxic chemotherapy. Unlike the well-established classical NF-κB pathway, the genotoxic agents-induced "nuclear-to-cytoplasmic" NF-κB pathway is initiated from the nucleus and transferred to the cytoplasm. However, the mechanism linking nuclear DNA damage signaling to cytoplasmic IKK activation remains unclear. Here, we report that TRIM37, a novel E3 ligase, plays a vital role in genotoxic activation of NF-κB via monoubiquitination of NEMO at K309 in the nucleus, consequently resulting in nuclear export of NEMO and IKK/NF-κB activation. Clinically, TRIM37 levels correlated positively with levels of activated NF-κB and expression of Bcl-xl and XIAP in esophageal cancer specimens, which also associated positively with clinical stage and tumor-node-metastasis classification and associated inversely with overall and relapse-free survival in patients with esophageal cancer. Overexpression of TRIM37 conferred resistance to the DNA-damaging anticancer drug cisplatin in vitro and in vivo through activation of the NF-κB pathway. Genotoxic stress-activated ATM kinase directly interacted with and phosphorylated TRIM37 in the cytoplasm, which induced translocation of TRIM37 into the nucleus, where it formed a complex with NEMO and TRAF6 via a TRAF6-binding motif (TBM). Importantly, blocking the ATM/TRIM37/NEMO axis via cell-penetrating TAT-TBM peptide abrogated genotoxic agent-induced NEMO monoubiquitination and NF-κB activity, resulting in hypersensitivity of cancer cells to genotoxic drugs. Collectively, our results unveil a pivotal role for TRIM37 in genotoxic stress and shed light on mechanisms of inducible chemotherapy resistance in cancer.Significance: In response to genotoxic stress, TRIM37 activates NF-κB signaling via monoubiquitination of NEMO, which subsequently promotes cisplatin chemoresistance and tumor relapse in cancer. Cancer Res; 78(22); 6399-412. ©2018 AACR.

MERIT40-dependent recruitment of tankyrase to damaged DNA and its implication for cell sensitivity to DNA-damaging anticancer drugs.

Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, regulates various intracellular responses, such as telomere maintenance, Wnt/β-catenin signaling and cell cycle progression through its interactions with multiple target proteins. Tankyrase contains a long stretch of 24 ankyrin repeats that are further divided into five subdomains, called ANK repeat clusters (ARCs). Each ARC works as an independent ligand-binding unit, which implicates tankyrase as a platform for multiple protein-protein interactions. Furthermore, tankyrase distributes to various intracellular loci, suggesting potential distinct but yet unidentified physiological functions. To explore the novel functions of tankyrase, we performed liquid chromatography-mass spectrometry analysis and identified the BRE-BRCC36-MERIT40 complex, a regulator of homologous recombination, as tankyrase-binding proteins. Among the complex components, MERIT40 was directly associated with tankyrase via a tankyrase-binding consensus motif, as previously reported. In X-ray-irradiated non-small cell lung cancer cells, tankyrase localized to DNA double-stranded break sites in a MERIT40-dependent manner. MERIT40 knockdown increased the cell sensitivity to X-ray, whereas the wild-type, but not the tankyrase-unbound mutant, MERIT40 rescued the phenotype of the knockdown cells. Tankyrase inhibitors, such as G007-LK and XAV939, increased the cellular sensitivity to X-ray irradiation and anticancer drugs that induce DNA double-stranded breaks. These observations suggest that tankyrase plays a role in the DNA damage repair response and implicates a potential therapeutic utility of tankyrase inhibitors in combination treatments with DNA-damaging anticancer drugs.

Efficacy of Asparaginase Erwinia chrysanthemi With and Without Temozolomide Against Glioma Cells and Intracranial Mouse Medulloblastoma.

Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors. We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC50s) of 0.12->10 IU/ml. Erwinaze at <1 IU/ml reduced temozolomide IC50s by 3.6- to 13-fold (300-1,200 μM to 40-330 μM). Seven-week-old SMO/SMO mice treated with Erwinaze (regardless of temozolomide treatment) had better survival 11 weeks post-therapy, compared to those not treated with Erwinaze (81.25% vs. 46.15, p=0.08). Temozolomide-treated mice developed 10% weight loss, impairing survival. All 16 mice treated with temozolomide (regardless of Erwinaze treatment) succumbed by 40-weeks of age, whereas 5/8 animals treated with Erwinaze alone and 2/6 controls survived (p=0.035). Erwinaze enhances cytotoxicity of temozolomide in vitro, and improves survival in SMO/SMO mice, likely by reducing cerebrospinal fluid glutamine. Temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in vivo.

DNA-Damaging Anticancer Drugs - A Perspective for DNA Repair- Oriented Therapy.

DNA-damaging drugs in cancer present two main problems: therapeutic resistance and side effects and both can associate with DNA repair, which can be targeted in cancer therapy. Bleomycin (BLM) induces complex DNA damages, including strand breaks, base loss and 3'-phosphoglycolate (3'PG) residues repaired by several pathways, but 3'PGs must be processed to the 3'-OH ends, usually by tyrosyl-DNA phosphodiesterase 1 (Tdp1). Therefore, targeting Tdp1 can improve anticancer therapy with BLM. Mitomycin C (MMC) produces a variety of adducts with DNA, including inter-strand cross-links (ICLs) and Xeroderma pigmentosum (XP) proteins, including XPG, XPE and XPF can be crucial for the initial stage of ICL repair, so they can be targeted by inhibitors to increase toxicity of MMC in cancer cells. Although these proteins are essential for nucleotide excision repair (NER), their decreased activity may not be fatal in normal cells as almost all NER substrates can be repaired by other pathways. Four-stranded DNA, resulted mainly from guanine quadruplexes (G-4s), are highly overexpressed at the end of telomeres, where they can inhibit telomerase, hence stabilization G-4s at the telomeres ends can hamper proliferation of cancer cells. Quadruplexes are also found in the promoters of genes important for cancer and are resolved by DNA helicases, which can be targeted in cancer along with stabilization of quadruplexes. As cancer cells often have defects in DNA repair pathway(s), they can be subjected by synthetic lethality, with the most promising results with poly(ADP-ribose) polymerase 1 (PARP-1) and DNA-dependent protein kinase, catalytic subunit (DNA-PKCS).

Effect of Spag5 on proliferation and sensitivity to DNA-damaging chemotherapy.

e15556 Background: Chemotherapy based on DNA-damaging anti-cancer drugs remains the most predominant treatment for advanced gastric cancer. The predictors for their sensitivity are under development. Our data showed that sperm-associated antigen 5 (Spag5) contributed to cancer cell proliferation and decreased DNA-damaging drug sensitivity in vivo and vitro. Methods: Spag5 mRNA expression were compared in The Cancer Genome Atlas (TCGA) and GEO datasets (GSE56807, GSE63089, GSE65801), while protein level were detected in 52 patients diagnosed with gastric cancer in Nanfang Hospital by immunohistochemistry. BGC-823 and MGC-803 were transfected with spag5 and control siRNA and then subjected to MTT and EDU assay to verify proliferation rate. Cell viability and apoptosis were tested in spag5-silenced BGC-823 and MGC-803 treated with oxaliplatin, epirubicin and 5-fluorouracil independently. DNA-damaged were detected by comet assay and yH2ax level with western blot. Treatment of oxaliplatin was given intraperitoneally to nude mice carrying xerographs of Shspag5 or control BGC-823 cells every three days. Results: Spag5 mRNA level was significantly upregulated in cancer tissues compared with paired normal tissues in all of the datasets. Immunohistochemistry shows higher protein level of spag5 in paired cancer tissue. Knockdown of Spag5 BGC-823 and MGC-803 showed a significantly decreased proliferation in both MTT and EDU assay. Spag5 silencing led to increase vulnerability to oxaliplatin, epirubicin and 5-fluorouracil, thus less cell viability and more apoptosis. Spag5 knockdown BGC-823 and MGC-803 subjected to DNA-damaging drugs showed upregulated yH2ax level and increased DNA damage in comet assay. Tumor growth rate were significantly reduced in shspag5 group and treatment of oxaliplatin led to relatively higher inhibition of tumor growth. Conclusions: Spag5 serves as a candidate to predict proliferation and chemosensitivity to DNA-damaging chemotherapy.

Caenorhabditis elegans as a powerful alternative model organism to promote research in genetic toxicology and biomedicine.

In view of increased life expectancy the risk for disturbed integrity of genetic information increases. This inevitably holds the implication for higher incidence of age-related diseases leading to considerable cost increase in health care systems. To develop preventive strategies it is crucial to evaluate external and internal noxae as possible threats to our DNA. Especially the interplay of DNA damage response (DDR) and DNA repair (DR) mechanisms needs further deciphering. Moreover, there is a distinct need for alternative in vivo test systems for basic research and also risk assessment in toxicology. Especially the evaluation of combinational toxicity of environmentally present genotoxins and adverse effects of clinically used DNA damaging anticancer drugs is a major challenge for modern toxicology. This review focuses on the applicability of Caenorhabditis elegans as a model organism to unravel and tackle scientific questions related to the biological consequences of genotoxin exposure and highlights methods for studying DDR and DR. In this regard large-scale in vivo screens of mixtures of chemicals and extensive parallel sequencing are highlighted as unique advantages of C. elegans. In addition, concise information regarding evolutionary conserved molecular mechanisms of the DDR and DR as well as currently available data obtained from the use of prototypical genotoxins and preferential read-outs of genotoxin testing are discussed. The use of established protocols, which are already available in the community, is encouraged to facilitate and further improve the implementation of C. elegans as a powerful genetic model system in genetic toxicology and biomedicine.

Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin

BackgroundTranslationally controlled tumour protein TCTP is an anti-apoptotic protein frequently overexpressed in cancers, where high levels are often associated with poor patient outcome. TCTP may be involved in protecting cancer cells against the cytotoxic action of anti-cancer drugs. Here we study the early increase of TCTP levels in human colorectal cancer (CRC) and the regulation of TCTP expression in HCT116 colon cancer cells, in response to treatment with the anti-cancer drugs 5-FU and oxaliplatin.MethodsUsing immunohistochemistry, we assessed TCTP levels in surgical samples from adenomas and adenocarcinomas of the colon, compared to normal colon tissue. We also studied the regulation of TCTP in HCT116 colon cancer cells in response to 5-FU and oxaliplatin by western blotting. TCTP mRNA levels were assessed by RT-qPCR. We used mTOR kinase inhibitors to demonstrate mTOR-dependent translational regulation of TCTP under these conditions. Employing the Real-Time Cell Analysis (RTCA) System and the MTS assay, we investigated the effect of TCTP-knockdown on the sensitivity of HCT116 cells to the anti-cancer drugs 5-FU and oxaliplatin.Results1. TCTP levels are significantly increased in colon adenomas and adenocarcinomas, compared to normal colon tissue. 2. TCTP protein levels are about 4-fold upregulated in HCT116 colon cancer cells, in response to 5-FU and oxaliplatin treatment, whereas TCTP mRNA levels are down regulated. 3. mTOR kinase inhibitors prevented the up-regulation of TCTP protein, indicating that TCTP is translationally regulated through the mTOR complex 1 signalling pathway under these conditions. 4. Using two cellular assay systems, we demonstrated that TCTP-knockdown sensitises HCT116 cells to the cytotoxicity caused by 5-FU and oxaliplatin.ConclusionsOur results demonstrate that TCTP levels increase significantly in the early stages of CRC development. In colon cancer cells, expression of this protein is largely upregulated during treatment with the DNA-damaging anti-cancer drugs 5-FU and oxaliplatin, as part of the cellular stress response. TCTP may thus contribute to the development of anti-cancer drug resistance. These findings indicate that TCTP might be suitable as a biomarker and that combinatorial treatment using 5-FU/oxaliplatin, together with mTOR kinase inhibitors, could be a route to preventing the development of resistance to these drugs.

Chemoresistant lung cancer stem cells display high DNA repair capability to remove cisplatin-induced DNA damage.

The persistence of lung cancer stem cells (LCSCs) has been proposed to be the main factor responsible for the recurrence of lung cancer as they are highly resistant to conventional chemotherapy. However, the underlying mechanisms are still unclear. We examined the cellular response of a human LCSC line to treatment with cisplatin, a DNA-damaging anticancer drug that is used extensively in the clinic. We compared the response to cisplatin of LCSCs and differentiated LCSCs (dLCSCs) by determining the viability of these cells, and their ability to accumulate cisplatin and to implement genomic and transcription-coupled DNA repair. We also investigated the transcription profiles of genes related to drug transport and DNA repair. LCSCs were found to be more stem-like, and more resistant to cisplatin-induced cytotoxicity than dLCSCs, confirming their drug resistance properties. LCSCs accumulated less cisplatin intracellularly than dLCSCs and showed less DNA damage, potentially due to their ability to down-regulate AQP2 and CTR1. The results of the transcription-coupled repair of cisplatin-DNA cross-links indicated a higher level of repair of DNA damage in LCSCs than in dLCSCs. In addition, LCSCs showed a greater ability to repair cisplatin-DNA interstrand cross-links than dLCSCs; this involved the activation of various DNA repair pathways. Our results further clarify the mechanism of cisplatin resistance in LCSCs in terms of reduced cisplatin uptake and enhanced ability to implement DNA repairs. These findings may aid in the design of the next-generation of platinum-based anticancer drugs.