Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

The therapeutic efficacy of the anticancer drug cisplatin is limited by acquired drug resistance. Cisplatin forms DNA crosslinks, that, if not removed, lead to replication stress. Due to this, the DNA damage response (DDR) gets activated regulating cell cycle arrest, DNA repair, cell death or survival. This makes DDR components promising targets for the development of new therapeutic approaches aiming to overcome acquired drug resistance. To this end, cisplatin-resistant bladder cancer cells were analyzed regarding their sensitivity to combination treatments with selected pharmacological DDR inhibitors. Synergistic cytolethal effects were achieved after combined treatment with low to moderate doses of the non-genotoxic RAD51-inhibitor (RAD51i) B02 and CHK1-inhibitor (CHK1i) PF477736. This effect was also found in cisplatin resistant tumor cells of other origin as well as with other RAD51i and CHK1i. Combined treatments promoted decelerated replication, S-phase blockage, accumulation of DNA strand breaks, DDR activation and stimulation of apoptotic cell death as compared to mono-treatment, which is independent of the expression of RAD51, CHK1, and PrimPol. Based on these data, we suggest combined inhibition of RAD51 and CHK1 to overcome acquired cisplatin resistance of malignant cells. We propose that the molecular mechanism of this synergistic toxicity relies on a simultaneous inactivation of two key DNA damage tolerance pathways regulating replication fork restart, thereby circumventing the activation of alternative compensatory mechanisms and, in consequence, eventually effectively triggering apoptotic cell death by replication fork collapse.

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.

Targeting Mechanisms of the DNA Damage Response (DDR) and DNA Repair by Natural Compounds to Improve cAT-Triggered Tumor Cell Death.

Recently, we identified secalonic acid F (SA), 5-epi-nakijiquinone Q (NQ) and 5-epi-ilimaquinone (IQ) as natural compounds (NC) affecting mechanisms of the DNA damage response (DDR). Here, we further characterized their effects on DDR, DNA repair and cytotoxicity if used in mono- and co-treatment with conventional anticancer therapeutics (cAT) (cisplatin (Cis), doxorubicin (Doxo)) in vitro. All three NC influence the phosphorylation level of selected DDR-related factors (i.e., pCHK1, pKAP1, pP53, pRPA32) in mono- and/or co-treatment. Both SA and NQ attenuate the Cis- and Doxo-induced G2/M-phase arrest and effectively stimulate caspase-mediated apoptosis. Notably, SA impacts DNA repair as reflected by enhanced steady-state levels of Cis-(1,2-GpG)-DNA adducts and Doxo-induced DNA double-strand breaks (DSB). Moreover, SA decreased the mRNA and protein expression of the homologous recombination (HR)-related DSB repair factors RAD51 and BRCA1. Both SA and NQ promote Cis- and Doxo-induced cytotoxicity in an additive to synergistic manner (CI ≤ 1.0). Summarizing, we conclude that SA promotes cAT-driven caspase-dependent cell death by interfering with DSB repair and DDR-related checkpoint control mechanisms. Hence, SA is considered as the most promising lead compound to evaluate its therapeutic window in forthcoming pre-clinical in vivo studies.

Cisplatin-induced neurotoxicity involves the disruption of serotonergic neurotransmission.

Neurotoxicity is a frequent side effect of cisplatin (CisPt)-based anticancer therapy whose pathophysiology is largely vague. Here, we exploited C. elegans as a 3R-compliant in vivo model to elucidate molecular mechanisms contributing to CisPt-induced neuronal dysfunction. To this end, we monitored the impact of CisPt on various sensory functions as well as pharyngeal neurotransmission by recording electropharyngeograms (EPGs). CisPt neither affected food and odor sensation nor mechano-sensation, which involve dopaminergic and glutaminergic neurotransmission. However, CisPt reduced serotonin-regulated pharyngeal pumping activity independent of changes in the morphology of related neurons. CisPt-mediated alterations in EPGs were fully rescued by addition of serotonin (5-HT) (≤ 2 mM). Moreover, the CisPt-induced pharyngeal injury was prevented by co-incubation with the clinically approved serotonin re-uptake inhibitory drug duloxetine. A protective effect of 5-HT was also observed with respect to CisPt-mediated impairment of another 5-HT-dependent process, the egg laying activity. Importantly, CisPt-induced apoptosis in the gonad and learning disability were not influenced by 5-HT. Using different C. elegans mutants we found that CisPt-mediated (neuro)toxicity is independent of serotonin biosynthesis and re-uptake and likely involves serotonin-receptor subtype 7 (SER-7)-related functions. In conclusion, by measuring EPGs as a surrogate parameter of neuronal dysfunction, we provide first evidence that CisPt-induced neurotoxicity in C. elegans involves 5-HT-dependent neurotransmission and SER-7-mediated signaling mechanisms and can be prevented by the clinically approved antidepressant duloxetine. The data highlight the particular suitability of C. elegans as a 3R-conform in vivo model in molecular (neuro)toxicology and, moreover, for the pre-clinical identification of neuroprotective candidate drugs.

Effects of Manganese on Genomic Integrity in the Multicellular Model Organism Caenorhabditis elegans.

Although manganese (Mn) is an essential trace element, overexposure is associated with Mn-induced toxicity and neurological dysfunction. Even though Mn-induced oxidative stress is discussed extensively, neither the underlying mechanisms of the potential consequences of Mn-induced oxidative stress on DNA damage and DNA repair, nor the possibly resulting toxicity are characterized yet. In this study, we use the model organism Caenorhabditis elegans to investigate the mode of action of Mn toxicity, focusing on genomic integrity by means of DNA damage and DNA damage response. Experiments were conducted to analyze Mn bioavailability, lethality, and induction of DNA damage. Different deletion mutant strains were then used to investigate the role of base excision repair (BER) and dePARylation (DNA damage response) proteins in Mn-induced toxicity. The results indicate a dose- and time-dependent uptake of Mn, resulting in increased lethality. Excessive exposure to Mn decreases genomic integrity and activates BER. Altogether, this study characterizes the consequences of Mn exposure on genomic integrity and therefore broadens the molecular understanding of pathways underlying Mn-induced toxicity. Additionally, studying the basal poly(ADP-ribosylation) (PARylation) of worms lacking poly(ADP-ribose) glycohydrolase (PARG) parg-1 or parg-2 (two orthologue of PARG), indicates that parg-1 accounts for most of the glycohydrolase activity in worms.

Targeting the DNA damage response (DDR) by natural compounds.

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.

In Vitro Assessment of the Genotoxic Hazard of Novel Hydroxamic Acid- and Benzamide-Type Histone Deacetylase Inhibitors (HDACi).

Histone deacetylase inhibitors (HDACi) are already approved for the therapy of leukemias. Since they are also emerging candidate compounds for the treatment of non-malignant diseases, HDACi with a wide therapeutic window and low hazard potential are desirable. Here, we investigated a panel of 12 novel hydroxamic acid- and benzamide-type HDACi employing non-malignant V79 hamster cells as toxicology guideline-conform in vitro model. HDACi causing a ≥10-fold preferential cytotoxicity in malignant neuroblastoma over non-malignant V79 cells were selected for further genotoxic hazard analysis, including vorinostat and entinostat for control. All HDACi selected, (i.e., KSK64, TOK77, DDK137 and MPK77) were clastogenic and evoked DNA strand breaks in non-malignant V79 cells as demonstrated by micronucleus and comet assays, histone H2AX foci formation analyses (γH2AX), DNA damage response (DDR) assays as well as employing DNA double-strand break (DSB) repair-defective VC8 hamster cells. Genetic instability induced by hydroxamic acid-type HDACi seems to be independent of bulky DNA adduct formation as concluded from the analysis of nucleotide excision repair (NER) deficient mutants. Summarizing, KSK64 revealed the highest genotoxic hazard and DDR stimulating potential, while TOK77 and MPK77 showed the lowest DNA damaging capacity. Therefore, these compounds are suggested as the most promising novel candidate HDACi for subsequent pre-clinical in vivo studies.

Hepatic Rac1 GTPase contributes to liver-mediated basal immune homeostasis and LPS-induced endotoxemia.

BACKGROUND: The Ras-homologous GTPase Rac1 plays a key role in the regulation of gene expression, cytoskeleton-associated processes and cell death as well as carcinogenesis and inflammation. Here, we investigated the impact of Rac1 signaling on liver-mediated immune homeostasis. METHODS: We employed a constitutive Alb-Cre-driven rac1 knock-out and a poly I:C-inducible Mx1-Cre-based knock-out model and analyzed cytokine expression profiles in liver and other organs under basal situation and following LPS-induced endotoxemia by flow cytometry, qRT-PCR and immunocytochemistry. RESULTS: Constitutive Alb-Cre-driven rac1 knockout in hepatocytes altered the basal distribution and activation of immune cells in the liver and likewise in kidney and lung. Early systemic alterations in cytokine serum levels following LPS treatment remained unaffected by Rac1. Furthermore, lack of Rac1 in hepatocytes of untreated animals shifted the liver to a chronic inflammatory state, as depicted by an enhanced mRNA expression of marker genes related to activated macrophages. Upon acute LPS-induced endotoxemia, increased IL-10 mRNA expression in the liver of Alb-Cre Rac1-deficient mice provided an anti-inflammatory response. Employing a poly I:C-inducible Mx1-Cre-based rac1 knock-out, which allows a more widespread rac1 deletion in both hepatocytes and non-hepatocytes, we observed substantial differences regarding both basal and LPS-stimulated cytokine expression profiles as compared to the Alb-Cre system. CONCLUSIONS: Rac1-dependent mechanisms in hepatocytes and non-hepatocytes contribute to the maintenance of liver immune homeostasis under basal situation and following LPS-induced endotoxemia. Disturbed Rac1-regulated hepatocyte functions may promote liver damage under pathophysiological situation involving inflammatory stress.

Profiling of a suramin-derived compound library at recombinant human P2Y receptors identifies NF272 as a competitive but non-selective P2Y2 receptor antagonist.

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y2 receptor antagonist with a Ki of 19 µM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 receptors. Up to 250 µM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.

Rac1-mediated cardiac damage causes diastolic dysfunction in a mouse model of subacute doxorubicin-induced cardiotoxicity.

The anticancer efficacy of anthracyclines is limited by congestive heart failure. Clinically established markers of early onset of cardiotoxicity following anthracycline treatment and preventive measures are missing. Although statins are reported to alleviate anthracycline-induced cardiotoxicity in vivo, the molecular mechanisms involved remain elusive. In vitro data point to Rac1 as major target of the cytoprotective statin effects. Here we investigated whether specific inhibition of Rac1 by NSC23766 is as effective as lovastatin in preventing subacute cardiotoxicity following doxorubicin treatment. C57BL/6 mice were treated over 3 weeks with multiple low doses of doxorubicin (6 × 3 mg/kg BW, i.p.) and the level of DNA damage, apoptosis and regenerative proliferation as well as pro-inflammatory, pro-fibrotic and oxidative stress responses were investigated. Moreover, heart function was monitored by echocardiography. Doxorubicin induced subacute cardiotoxicity which was reflected on the level of residual DNA damage, frequency of apoptotic and mitotic cells as well as elevated mRNA expression of markers of heart failure, remodeling and mitochondrial biogenesis. These molecular markers of cardiotoxicity were mitigated to a similar extent by co-treatment with either lovastatin (10 mg/kg BW, p.o.) or NSC23766 (5 mg/kg BW, i.p.) three times a week. Moreover, doxorubicin caused diastolic dysfunction as reflected by increased E-wave acceleration time (EAT), which again was prevented by pharmacological inhibition of Rac1. Inhibition of Rac1 signaling is of major relevance for the cardioprotective effects of lovastatin in the context of anthracycline-induced cardiotoxicity. Moreover, EAT is a useful marker of subacute cardiotoxicity caused by persisting harmful effects of doxorubicin.

Lovastatin protects keratinocytes from DNA damage-related pro-apoptotic stress responses stimulated by anticancer therapeutics.

BACKGROUND: Oral mucositis (OM) is a relevant adverse effect of anticancer therapy involving ionizing radiation (IR) and doxorubicin (Doxo). Because DNA damage of keratinocytes is causative for the pathogenesis of OM, we aim to identify pharmacological measures for geno- and cytoprotection of keratinocytes. METHODS: We investigated the influence of the lipid-lowering drug lovastatin on cell death, proliferation and DNA damage response (DDR) mechanisms of human keratinocytes following treatment with IR and Doxo. RESULTS: Lovastatin protected keratinocytes from the cytotoxic and genotoxic effects of IR and Doxo as shown by a diminished induction of apoptosis as well as a reduced formation and slightly improved repair of DNA damage following Doxo and IR treatment, respectively. Lovastatin selectively blocked the activation of Chk1 and ATR kinases following treatment with IR, Doxo and the ribonucleotide reductase inhibitor hydroxyurea, indicating that the statin antagonizes ATR/Chk1-regulated replicative stress responses. Part of the cytoprotective activity of lovastatin seems to rest on a delayed entry of lovastatin treated cells into S-phase. Yet, because the statin also protected non-proliferating keratinocytes from IR- and Doxo-induced cytotoxicity, cell cycle independent protective mechanisms are involved, too. CONCLUSIONS: Lovastatin attenuates pro-toxic DNA damage-related responses of keratinocytes stimulated by OM-inducing anticancer therapeutics. The data encourage forthcoming in vivo and clinical studies addressing the usefulness of statins in the prevention of OM.

Integrin αvß3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418).

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvß3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit ß3 cDNA, as compared to control cells demonstrate: expression of integrin αvß3; increased expression of integrin αvß5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin ß5 in cells expressing integrin αvß3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvß3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvß3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury.

BACKGROUND: Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. METHODS: We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. RESULTS: CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. CONCLUSIONS: There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt.

Fatal Lymphoproliferative Disease in Two Siblings Lacking Functional FAAP24.

Hereditary defects in several genes have been shown to disturb the normal immune response to EBV and to give rise to severe EBV-induced lymphoproliferation in the recent years. Nevertheless, in many patients, the molecular basis of fatal EBV infection still remains unclear. The Fanconi anemia-associated protein 24 (FAAP24) plays a dual role in DNA repair. By association with FANCM as component of the FA core complex, it recruits the FA core complex to damaged DNA. Additionally, FAAP24 has been shown to evoke ATR-mediated checkpoint responses independently of the FA core complex. By whole exome sequencing, we identified a homozygous missense mutation in the FAAP24 gene (cC635T, pT212M) in two siblings of a consanguineous Turkish family who died from an EBV-associated lymphoproliferative disease after infection with a variant EBV strain, expressing a previously unknown EBNA2 allele.In order to analyze the functionality of the variant FAAP24 allele, we used herpes virus saimiri-transformed patient T cells to test endogenous cellular FAAP24 functions that are known to be important in DNA damage control. We saw an impaired FANCD2 monoubiquitination as well as delayed checkpoint responses, especially affecting CHK1 phosphorylation in patient samples in comparison to healthy controls. The phenotype of this FAAP24 mutation might have been further accelerated by an EBV strain that harbors an EBNA2 allele with enhanced activities compared to the prototype laboratory strain B95.8. This is the first report of an FAAP24 loss of function mutation found in human patients with EBV-associated lymphoproliferation.

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells.

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

Rac1 promotes diethylnitrosamine (DEN)-induced formation of liver tumors.

To elucidate the function of the Ras-homologous GTPase Rac1 in hepatocarcinogenesis induced by diethylnitrosamine (DEN), mice lacking hepatic Rac1 expression were treated with DEN and compared to the wild-type (WT). Rac1 knock-out (KO) mice were found to have a lower tumor yield as compared to Rac1 proficient mice. The small-sized tumors formed in the absence of Rac1 lack an activated Ras/Raf/mitogen-activated protein kinase pathway, as indicated by the absence of p-ERK expression. Apparently, Rac1 is required for Ras-driven oncogenic pathways. Moreover, tumors in Rac1 deficient mice were glutamine synthase (GS) negative. They displayed a high number of p-H3-positive and cyclinB1 expressing cells, pointing to a defect in mitotic progression. To elucidate the influence of Rac1 on mechanisms of tumor initiation, acute DEN-induced hepatic stress responses were monitored. Rac1 deficiency caused fairly complex, partially time-dependent, alterations in both basal and/or DEN-induced messenger RNA (mRNA) and protein levels of susceptibility-related genes. Basal protein expression of DNA repair factors Brca1 and DNA repair protein RAD51 homolog (Rad51) and the cell cycle regulatory factor p27 was enhanced in the absence of Rac1. Following DEN treatment, p21 mRNA and protein expression was stimulated independent of the Rac1 status. Lack of Rac1 increased mechanisms of the DNA damage response (DDR), as shown by elevated protein levels of p-ATR, p-p53 and γH2AX 24h after DEN treatment. The data show that Rac1 is essential for DEN-stimulated hepatocarcinogenesis. We hypothesize that it promotes tumor initiation by counteracting the elimination of initiated cells and, moreover, alleviates the outgrowth of transformed cells. Hence, pharmacological targeting of Rac1 could be suitable for chemoprevention.

Chronic heart damage following doxorubicin treatment is alleviated by lovastatin.

The anticancer efficacy of anthracyclines is limited by cumulative dose-dependent early and delayed cardiotoxicity resulting in congestive heart failure. Mechanisms responsible for anthracycline-induced heart damage are controversially discussed and effective preventive measures are preferable. Here, we analyzed the influence of the lipid lowering drug lovastatin on anthracycline-induced late cardiotoxicity three month after treatment of C57BL/6 mice with five low doses of doxorubicin (5×3mg/kg BW; i.p.). Doxorubicin increased the cardiac mRNA levels of BNP, IL-6 and CTGF, while the expression of ANP remained unchanged. Lovastatin counteracted these persisting cardiac stress responses evoked by the anthracycline. Doxorubicin-induced fibrotic alterations were neither detected by histochemical collagen staining of heart sections nor by analysis of the mRNA expression of collagens. Extensive qRT-PCR-array based analyses revealed a large increase in the mRNA level of heat shock protein Hspa1b in doxorubicin-treated mice, which was mitigated by lovastatin co-treatment. Electron microscopy together with qPCR-based analysis of mitochondrial DNA content indicate that lovastatin attenuates doxorubicin-stimulated hyperproliferation of mitochondria. This was not paralleled by increased expression of oxidative stress responsive genes or senescence-associated proteins. Echocardiographic analyses disclosed that lovastatin protects from the doxorubicin-induced decrease in the left ventricular posterior wall diameter (LVPWD), while constrictions in fractional shortening (FS) and ejection fraction (EF) evoked by doxorubicin were not amended by the statin. Taken together, the data suggest beneficial effects of lovastatin against doxorubicin-induced delayed cardiotoxicity. Clinical studies are preferable to scrutinize the usefulness of statins for the prevention of anthracycline-induced late cardiotoxicity.

DNA damage response (DDR) induced by topoisomerase II poisons requires nuclear function of the small GTPase Rac.

Here, we investigated the influence of Rac family small GTPases on mechanisms of the DNA damage response (DDR) stimulated by topoisomerase II poisons. To this end, we examined the influence of the Rac-specific small molecule inhibitor EHT1864 on Ser139 phosphorylation of histone H2AX, a widely used marker of the DDR triggered by DNA double-strand breaks. EHT1864 attenuated the doxorubicin-stimulated DDR in a subset of cell lines tested, including HepG2 hepatoma cells. EHT1864 reduced the level of DNA strand breaks and increased viability following treatment of HepG2 cells with topo II poisons. Protection by EHT1864 was observed in both p53 wildtype (HepG2) and p53 deficient (Hep3B) human hepatoma cells and, furthermore, remained unaffected upon pharmacological inhibition of p53 in HepG2. Apparently, the impact of Rac on the DDR is independent of p53. Protection from doxorubicin-induced DNA damage by EHT1864 comprises both S and G2 phase cells. The inhibitory effect of EHT1864 on doxorubicin-stimulated DDR was mimicked by pharmacological inhibition of various protein kinases, including JNK, ERK, PI3K, PAK and CK1. EHT1864 and protein kinase inhibitors also attenuated the formation of the topo II-DNA cleavable complex. Moreover, EHT1864 mitigated the constitutive phosphorylation of topoisomerase IIα at positions S1106, S1213 and S1247. Doxorubicin transport, nuclear import/export of topoisomerase II and Hsp90-related mechanisms are likely not of relevance for doxorubicin-stimulated DDR impaired by EHT1864. We suggest that multiple kinase-dependent but p53- and heat shock protein-independent Rac-regulated nuclear mechanisms are required for activation of the DDR following treatment with topo II poisons.

Spongean alkaloids protect rat kidney cells against cisplatin-induced cytotoxicity.

Nephrotoxicity is the major dose-limiting adverse effect of cisplatin (CisPt) and results from CisPt-induced damage of tubular cells. Nephroprotective strategies are preferential to improve supportive care in cancer. We investigated a subset of purified substances originating from various plants or from marine sponges as to their potency to protect rat renal tubular cells (NRK-52E) against the cytotoxic and genotoxic effects of cisplatin. Cotreatment with a substance pool containing five purified substances originating from marine sponges increased the viability of NRK-52E cells following cisplatin treatment. Cytoprotection was accompanied by a reduced level of DNA damage as indicated by a lower amount of S139 phosphorylated histone H2AX (γH2AX) 24 h after treatment. Cytoprotection and genoprotection by the sponge substance pool did not comprise the anthracycline derivative doxorubicin. The spongean alkaloid aaptamine was identified as major bioactive compound that mediates cisplatin resistance. Aeroplysinin-1 was less cytoprotective than aaptamine. Notably, aaptamine preferentially conferred resistance to cisplatin, but not to oxaliplatin. Cytoprotection by aaptamine was also observed in rat glomerular endothelial cells, but not in RT-112 bladder cancer cells. Protection by aaptamine does not rest on a reduced formation of DNA damage caused by cisplatin treatment. Aaptamine and aeroplysinin-1 affected cisplatin-stimulated DDR as reflected on the level of S15-phosphorlyated p53 and S345-phosphorylated checkpoint kinase-1. Summarizing, the spongean alkaloid aaptamine alleviates cisplatin-induced damage in tubular and glomerular rat kidney cells. Therefore, we hypothesize that aaptamine might be useful to widen the therapeutic window of a cisplatin-based therapeutic regimen.

Cisplatin-induced DNA crosslinks trigger neurotoxicity in C. elegans.

The anticancer drug cisplatin (CisPt) injures post-mitotic neuronal cells, leading to neuropathy. Furthermore, CisPt triggers cell death in replicating cells. Here, we aim to unravel the relevance of different types of CisPt-induced DNA lesions for evoking neurotoxicity. To this end, we comparatively analyzed wild-type and loss of function mutants of C. elegans lacking key players of specific DNA repair pathways. Deficiency in ercc-1, which is essential for nucleotide excision repair (NER) and interstrand crosslink (ICL) repair, revealed the most pronounced enhancement in CisPt-induced neurotoxicity with respect to the functionality of post-mitotic chemosensory AWA neurons, without inducing neuronal cell death. Potentiation of CisPt-triggered neurotoxicity in ercc-1 mutants was accompanied by complex alterations in both basal and CisPt-stimulated mRNA expression of genes involved in the regulation of neurotransmission, including cat-4, tph-1, mod-1, glr-1, unc-30 and eat-18. Moreover, xpf-1, csb-1, csb-1;xpc-1 and msh-6 mutants were significantly more sensitive to CisPt-induced neurotoxicity than the wild-type, whereas xpc-1, msh-2, brc-1 and dog-1 mutants did not distinguish from the wild-type. The majority of DNA repair mutants also revealed increased basal germline apoptosis, which was analyzed for control. Yet, only xpc-1, xpc-1;csb-1 and dog-1 mutants showed elevated apoptosis in the germline following CisPt treatment. To conclude, we provide evidence that neurotoxicity, including sensory neurotoxicity, is triggered by CisPt-induced DNA intra- and interstrand crosslinks that are subject of repair by NER and ICL repair. We hypothesize that especially ERCC1/XPF, CSB and MSH6-related DNA repair protects from chemotherapy-induced neuropathy in the context of CisPt-based anticancer therapy.