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.

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.

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.

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.

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.

Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

The lipid lowering drug lovastatin protects against doxorubicin-induced hepatotoxicity.

Liver is the main detoxifying organ and therefore the target of high concentrations of genotoxic compounds, such as environmental carcinogens and anticancer drugs. Here, we investigated the usefulness of lovastatin, which is nowadays widely used for lipid lowering purpose, as a hepatoprotective drug following the administration of the anthracycline derivative doxorubicin in vivo. To this end, BALB/c mice were exposed to either a single high dose or three consecutive low doses of doxorubicin. Acute and subacute hepatotoxicities were analyzed with or without lovastatin co-treatment. Lovastatin protected the liver against doxorubicin-induced acute pro-inflammatory and pro-fibrotic stress responses as indicated by an attenuated mRNA expression of tumor necrosis factor alpha (TNFα) and connective tissue growth factor (CTGF), respectively. Hepatoprotection by lovastatin was due to a reduced induction of DNA damage following doxorubicin treatment. The statin also mitigated subacute anthracycline-provoked hepatotoxicity as shown on the level of doxorubicin- and epirubicin-stimulated CTGF mRNA expression as well as histopathologically detectable fibrosis and serum concentration of marker enzymes of hepatotoxicity (GPT/GLDH). Kidney damage following doxorubicin exposure was not detectable under our experimental conditions.Moreover, lovastatin showed multiple inhibitory effects on doxorubicin-triggered hepatic expression of genes involved in oxidative stress response, drug transport, DNA repair, cell cycle progression and cell death. Doxorubicin also stimulated the formation of ceramides. Ceramide production, however, was not blocked by lovastatin, indicating that hepatoprotection by lovastatin is independent of the sphingolipid metabolism. Overall, the data show that lovastatin is hepatoprotective following genotoxic stress induced by anthracyclines. Based on the data, we hypothesize that statins might be suitable to lower hepatic injury following anthracycline-based anticancer therapy.

Nuclear RAC1 is a modulator of the doxorubicin-induced DNA damage response.

Rho GTPases like RAC1 are localized on the inner side of the outer cell membrane where they act as molecular switches that can trigger signal transduction pathways in response to various extracellular stimuli. Nuclear functions of RAC1 were identified that are related to mitosis, cell cycle arrest and apoptosis. Previously, we showed that RAC1 plays a role in the doxorubicin (Dox)-induced DNA damage response (DDR). In this context it is still unknown whether cytosolic RAC1 modulates the Dox-induced DDR or if a nuclear fraction of RAC1 is involved. Here, we silenced RAC1 in mouse embryonic fibroblasts (MEF) pharmacologically with EHT1864 or by using siRNA against Rac1. Additionally, we transfected MEF with RAC1 mutants (wild-type, dominant-negative, constitutively active) containing a nuclear localization sequence (NLS). Afterwards, we analysed the Dox-induced DDR by evaluation of fluorescent nuclear γH2AX and 53BP1 foci formation, as well as by detection of activated proteins of the DDR by western blot to elucidate the role of nuclear RAC1 in the DDR. Treatment with EHT1864 as well as Rac1 knock-down reduced the Dox-induced DSB-formation to a similar extent. Enhanced nuclear localization of dominant-negative as well as constitutively active RAC1 mimicked these effects. Expression of the RAC1 mutants altered the Dox-induced amount of pP53 and pKAP1 protein. The observed effects were independent of S1981 ATM phosphorylation. We conclude that RAC1 is required for a substantial activation of the Dox-induced DDR and balanced levels of active/inactive RAC1 inside the nucleus are a prerequisite for this response.

Potential use of HMG-CoA reductase inhibitors (statins) as radioprotective agents.

HMG-CoA reductase inhibitors (statins) are widely used in the therapy of hypercholesterolemia. Apart from their lipid-lowering activity, they have pleiotropic effects that are attributed to the inhibition of regulatory proteins, including Ras-homologous (Rho) GTPases. Here, we discuss the potential usefulness of statins to prevent normal tissue damage provoked by radiotherapy. Statins reduce the mRNA expression of pro-inflammatory and pro-fibrotic cytokines stimulated by ionizing radiation in vitro and alleviate IR-induced inflammation and fibrosis in vivo. The currently available data indicate that statins accelerate the rapid repair of DNA double-strand breaks and, moreover, mitigate the DNA damage response induced by IR. Furthermore, statins increase the mRNA expression of DNA repair factors in vivo. Thus, although the molecular mechanisms involved are still ambiguous, preclinical data concordantly show a promising radioprotective capacity of statins.

Distinct contribution of Rac1 expression in cardiomyocytes to anthracycline-induced cardiac injury.

Cardiotoxicity is the dose limiting adverse effect of anthracycline-based anticancer therapy. Inhibitor studies point to Rac1 as therapeutic target to prevent anthracycline-induced cardiotoxicity. Yet, supporting genetic evidence is still missing and the pathophysiological relevance of different cardiac cell types is unclear. Here, we employed a tamoxifen-inducible cardiomyocyte-specific rac1 knock-out mouse model (Rac1flox/flox/MHC-MerCreMer) to investigate the impact of Rac1 expression in cardiomyocytes on cardiac injury following doxorubicin treatment. Distinctive stress responses resulting from doxorubicin treatment were observed, including upregulation of systemic markers of inflammation (IL-6, IL-1α, MCP-1), cardiac damage (ANP, BNP), DNA damage (i.e. DNA double-strand breaks (DSB)), DNA damage response (DDR) and cell death. Measuring the acute doxorubicin response, the serum level of MCP-1 was elevated, cardiac mRNA expression of Hsp70 was reduced and cardiac DDR was specifically enhanced in Rac1 deficient mice. The frequency of apoptotic heart cells remained unaffected by Rac1. Employing a subactue model, the number of doxorubicin-induced DSB was significantly reduced if Rac1 is absent. Yet, the doxorubicin-triggered increase in serum ANP and BNP levels remained unaffected by Rac1. Overall, knock-out of rac1 in cardiomyocytes confers partial protection against doxorubicin-induced cardiac injury. Hence, the data provide first genetic evidence supporting the view that pharmacological targeting of Rac1 is useful to widen the therapeutic window of anthracycline-based anticancer therapy by alleviating acute/subacute cardiomyocyte damage. Furthermore, considering published data obtained from the use of pharmacological Rac1 inhibitors, the results of our study indicate that Rac1-regulated functions of cardiac cell types others than cardiomyocytes additionally influence the adverse outcomes of anthracycline treatment on the heart.

Statins in anthracycline-induced cardiotoxicity: Rac and Rho, and the heartbreakers.

Cancer patients receiving anthracycline-based chemotherapy are at risk to develop life-threatening chronic cardiotoxicity with the pathophysiological mechanism of action not fully understood. Besides the most common hypothesis that anthracycline-induced congestive heart failure (CHF) is mainly caused by generation of reactive oxygen species, recent data point to a critical role of topoisomerase II beta (TOP2B), which is a primary target of anthracycline poisoning, in the pathophysiology of CHF. As the use of the only clinically approved cardioprotectant dexrazoxane has been limited by the FDA in 2011, there is an urgent need for alternative cardioprotective measures. Statins are anti-inflammatory and anti-oxidative drugs that are clinically well established for the prevention of cardiovascular diseases. They exhibit pleiotropic beneficial properties beyond cholesterol-lowering effects that most likely rest on the indirect inhibition of small Ras homologous (Rho) GTPases. The Rho GTPase Rac1 has been shown to be a major factor in the regulation of the pro-oxidative NADPH oxidase as well as in the regulation of type II topoisomerase. Both are discussed to play an important role in the pathophysiology of anthracycline-induced CHF. Therefore, off-label use of statins or novel Rac1 inhibitors might represent a promising pharmacological approach to gain control over chronic cardiotoxicity by interfering with key mechanisms of anthracycline-induced cardiomyocyte cell death.

Rho inhibition by lovastatin affects apoptosis and DSB repair of primary human lung cells in vitro and lung tissue in vivo following fractionated irradiation.

Thoracic radiotherapy causes damage of normal lung tissue, which limits the cumulative radiation dose and, hence, confines the anticancer efficacy of radiotherapy and impacts the quality of life of tumor patients. Ras-homologous (Rho) small GTPases regulate multiple stress responses and cell death. Therefore, we investigated whether pharmacological targeting of Rho signaling by the HMG-CoA-reductase inhibitor lovastatin influences ionizing radiation (IR)-induced toxicity in primary human lung fibroblasts, lung epithelial and lung microvascular endothelial cells in vitro and subchronic mouse lung tissue damage following hypo-fractionated irradiation (4x4 Gy). The statin improved the repair of radiation-induced DNA double-strand breaks (DSBs) in all cell types and, moreover, protected lung endothelial cells from IR-induced caspase-dependent apoptosis, likely involving p53-regulated mechanisms. Under the in vivo situation, treatment with lovastatin or the Rac1-specific small molecule inhibitor EHT1864 attenuated the IR-induced increase in breathing frequency and reduced the percentage of γH2AX and 53BP1-positive cells. This indicates that inhibition of Rac1 signaling lowers IR-induced residual DNA damage by promoting DNA repair. Moreover, lovastatin and EHT1864 protected lung tissue from IR-triggered apoptosis and mitigated the IR-stimulated increase in regenerative proliferation. Our data document beneficial anti-apoptotic and genoprotective effects of pharmacological targeting of Rho signaling following hypo-fractionated irradiation of lung cells in vitro and in vivo. Rac1-targeting drugs might be particular useful for supportive care in radiation oncology and, moreover, applicable to improve the anticancer efficacy of radiotherapy by widening the therapeutic window of thoracic radiation exposure.

Designer Thiopurine-analogues for Optimised Immunosuppression in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: The clinical use of azathioprine and 6-mercaptopurine is limited by their delayed onset of action and potential side effects such as myelosuppression and hepatotoxicity. As these drugs specifically target the Vav1/Rac1 signalling pathway in T lamina propria lymphocytes via their metabolite 6-thio-GTP, we studied expression and optimised suppression of this pathway in inflammatory bowel diseases [IBD]. METHODS: Rac1 and Vav1 expressions were analysed in mucosal immune cells in IBD patients. Targeted molecular modelling of the 6-thio-GTP molecule was performed to optimise Rac1 blockade; 44 modified designer thiopurine-analogues were tested for apoptosis induction, potential toxicity, and immunosuppression. Activation of the Vav1/Rac1 pathway in lymphocytes was studied in IBD patients and in lamina propria immune cells in the presence or absence of thiopurine-analogues. RESULTS: Several thiopurine-analogues induced significantly higher T cell apoptosis than 6-mercaptopurine. We identified a compound, denoted B-0N, based on its capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. B-0N-treatment resulted in accelerated inhibition of Rac1 activity in primary peripheral blood T cells as well as in intestinal lamina propria immune cells. Compared with 6-thio-GTP and 6-mercaptopurine, B-0N-treatment was associated with decreased myelo- and hepatotoxicity. CONCLUSIONS: The Vav1/Rac1 pathway is activated in mucosal immune cells in IBD. The designer thiopurine-analogue B-0N induces immunosuppression more potently than 6-mercaptopurine.

Rho GTPases: Novel Players in the Regulation of the DNA Damage Response?

The Ras-related C3 botulinum toxin substrate 1 (Rac1) belongs to the family of Ras-homologous small GTPases. It is well characterized as a membrane-bound signal transducing molecule that is involved in the regulation of cell motility and adhesion as well as cell cycle progression, mitosis, cell death and gene expression. Rac1 also adjusts cellular responses to genotoxic stress by regulating the activity of stress kinases, including c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinases as well as related transcription factors. Apart from being found on the inner side of the outer cell membrane and in the cytosol, Rac1 has also been detected inside the nucleus. Different lines of evidence indicate that genotoxin-induced DNA damage is able to activate nuclear Rac1. The exact mechanisms involved and the biological consequences, however, are unclear. The data available so far indicate that Rac1 might integrate DNA damage independent and DNA damage dependent cellular stress responses following genotoxin treatment, thereby coordinating mechanisms of the DNA damage response (DDR) that are related to DNA repair, survival and cell death.