Cisplatin-Mediated Upregulation of APE2 Binding to MYH9 Provokes Mitochondrial Fragmentation and Acute Kidney Injury.

Cisplatin chemotherapy is standard care for many cancers but is toxic to the kidneys. How this toxicity occurs is uncertain. In this study, we identified apurinic/apyrimidinic endonuclease 2 (APE2) as a critical molecule upregulated in the proximal tubule cells (PTC) following cisplatin-induced nuclear DNA and mitochondrial DNA damage in cisplatin-treated C57B6J mice. The APE2 transgenic mouse phenotype recapitulated the pathophysiological features of C-AKI (acute kidney injury, AKI) in the absence of cisplatin treatment. APE2 pulldown-MS analysis revealed that APE2 binds myosin heavy-Chain 9 (MYH9) protein in mitochondria after cisplatin treatment. Human MYH9-related disorder is caused by mutations in MYH9 that eventually lead to nephritis, macrothrombocytopenia, and deafness, a constellation of symptoms similar to the toxicity profile of cisplatin. Moreover, cisplatin-induced C-AKI was attenuated in APE2-knockout mice. Taken together, these findings suggest that cisplatin promotes AKI development by upregulating APE2, which leads to subsequent MYH9 dysfunction in PTC mitochondria due to an unrelated role of APE2 in DNA damage repair. This postulated mechanism and the availability of an engineered transgenic mouse model based on the mechanism of C-AKI provides an opportunity to identify novel targets for prophylactic treatment of this serious disease. SIGNIFICANCE: These results reveal and highlight an unexpected role of APE2 via its interaction with MYH9 and suggest that APE2 has the potential to prevent acute kidney injury in patients with cisplatin-treated cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/713/F1.large.jpg.

Characterization of Single Amino Acid Variations in an EDTA-Tolerating Non-specific Nuclease from the Ice-Nucleating Bacterium Pseudomonas syringae.

Non-specific nuclease (NSN) can be applied in industrial downstream processing to remove nucleic acids from crude protein extracts or in cell-sorting systems to degrade nucleic acids derived from lysed cells. PsNuc from the ice-nucleating bacterium Pseudomonas syringae has the ability to decompose double- and single-stranded DNA in linear or circular form and RNA. It is not affected by the presence of metal-ion chelators such as EDTA and tolerates several protease inhibitors and reducing agents. A multiple sequence alignment of PsNuc with closely related enzymes (97-99% identity on the protein level) within the family Pseudomonaceae revealed the presence of only six amino acid residues that are variable in putative NSN from different members of the genus Pseudomonas. Single amino acid variants were produced in recombinant form in Escherichia coli, purified, and characterized. They showed similar activity compared to PsNuc, but a single variant even displayed an improved performance with an activity of > 20,000 U/mg at 35 °C, while amino acid residues S148 and V161 were found to be essential for enzymatic functionality. These results suggest that homologous nucleases from Pseudomonaceae display high activity levels in a metal-ion-independent manner and are therefore of interest for applications in biotechnology.

Curcumin activates DNA repair pathway in bone marrow to improve carboplatin-induced myelosuppression.

Carboplatin, a second-generation platinum agent, has been used as a cancer therapy for decades and exhibits strong anti-tumor activity. However, the wide application of carboplatin is largely limited due to its side effects, especially myelosuppression. Here, we combined carboplatin with curcumin, a natural product that improves tumor-induced anemia, for the treatment of fibrosarcoma to improve the side effects of carboplatin. We first examined the synergistic and attenuated effects of the two agents in a T241-bearing mouse model. The combination therapy caused no obvious synergistic effect, but curcumin significantly improved the survival rate of carboplatin-treated mice. Histologic analysis of the kidney and bone marrow revealed that curcumin improved carboplatin-induced myelosuppression but did not affect the kidney. To determine the mechanism involved, we introduced a probe derived from curcumin to identify its targets in bone marrow cells and the results provided us a clue that curcumin might affect the DNA repair pathway. Western blot analysis revealed that curcumin up-regulated BRCA1, BRCA2 and ERCC1 expression in bone marrow. In conclusion, curcumin attenuates carboplatin-induced myelosuppression by activating the DNA repair pathway in bone marrow cells.

Small molecule-triggered Cas9 protein with improved genome-editing specificity.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

Cross-protection of influenza A virus infection by a DNA aptamer targeting the PA endonuclease domain.

Amino acid residues in the N-terminal of the PA subunit (PAN) of the influenza A virus polymerase play critical roles in endonuclease activity, protein stability, and viral RNA (vRNA) promoter binding. In addition, PAN is highly conserved among different subtypes of influenza virus, which suggests PAN to be a desired target in the development of anti-influenza agents. We selected DNA aptamers targeting the intact PA protein or the PAN domain of an H5N1 virus strain using systematic evolution of ligands by exponential enrichment (SELEX). The binding affinities of selected aptamers were measured, followed by an evaluation of in vitro endonuclease inhibitory activity. Next, the antiviral effects of enriched aptamers against influenza A virus infections were examined. A total of three aptamers targeting PA and six aptamers targeting PAN were selected. Our data demonstrated that all three PA-selected aptamers neither inhibited endonuclease activity nor exhibited antiviral efficacy, whereas four of the six PAN-selected aptamers inhibited both endonuclease activity and H5N1 virus infection. Among the four effective aptamers, one exhibited cross-protection against infections of H1N1, H5N1, H7N7, and H7N9 influenza viruses, with a 50% inhibitory concentration (IC50) of around 10 nM. Notably, this aptamer was identified at the 5th round but disappeared after the 10th round of selection, suggesting that the identification and evaluation of aptamers at early rounds of selection may be highly helpful for screening effective aptamers. Overall, our study provides novel insights for screening and developing effective aptamers for use as anti-influenza drugs.

Influence of Morinda citrifolia (Noni) on Expression of DNA Repair Genes in Cervical Cancer Cells.

BACKGROUND: Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer risk. OBJECTIVE: The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination on DNA repair genes in the SiHa cervical cancer cell line. MATERIALS AND METHODS: SiHa cells were cultured and treated with 10% Noni, 10 µg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted, RNA extracted, and processed for investigating DNA repair genes by real time PCR. RESULTS: The expression of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold, 1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). CONCLUSIONS: Noni enhanced the expression of DNA repair genes by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate the DNA repair protein expression.

Expression of hENTl and ERCC1 genes in tumor tissues non-small cell lung cancer.

OBJECTIVE: To investigate the expression of hENTl and ERCC1 genes in tumor tissues non-small cell lung cancer (NSCLC). METHODS: Fresh non-small lung cancer specimens were transplanted into nude mice. Twenty mice were randomized into two groups: experimental group receiving gemcitabine plus cisplatin and control group receiving 0.9% physiological saline. The expressions of hENTl and ERCC1 mRNA in tumor tissue were detected by real-time fluorescent quantitative PCR. The volume of tumor, the weight of nude mice and tumor volume were respectively measured and calculated 2-3 times per week. Tissue samples were collected from NSCLC mice treated with gemcitabine plus carboplatin. RESULTS: The histological examination showed that many tumor cells were well preserved in nude mice. The rate of transplanted tumor cells was 86.7%. The concomitant treatment study showed that the rate of TV, RTV, T/C in GEM + DDP group was the lowest. LBP + DOC, DDP + DOC obviously influenced the body weight. Compared with NS group, DDP group, GEM group, the survival period and the level of hENTl of DDP+GEM group increased obviously, the level of ERCC1 decreased significantly (P<0.05). CONCLUSIONS: The expression of hENT1 and ERCC1 genes in tumor tissues were closely correlated with the response to chemotherapy and prognosis of patients with NSCLC treated with gemcitabine plus cisplatin.

Effects of emodin extracted from Chinese herbs on proliferation of non-small cell lung cancer and underlying mechanisms.

To aim of this was to observe emodin-mediated cytotoxicity and its influence on Rad51 and ERCC1 expressionin non-small cell lung cancer (NSCLC). NSCLC cells were cultured in vitro with emodin at various concentrations (0, 25, 50, 75 and 100 µmol/L) for 48 h and the proliferation inhibition rate was determined by the MTT method. Then, NSCLC were treated with emodin (SK-MES-1 40 µmol/L, A549 70 µmol/L) or 20 µmol/L U0126 (an ERK inhibitor) for 48 h, or with various concentrations of emodin for 48 h and the protein and mRNA expressions of ERCC1 and Rad51 were determined by RT-PCR and Western blot assay, respectively. Emodin exerted a suppressive effect on the proliferation of NSCLC in a concentration dependent manner. Protein and mRNA expression of ERCC1 and Rad51 was also significantly decreased with the dose. Vacuolar degeneration was observed in A549 and SK-MES-1 cell lines after emodin treatment by transmission electron microscopy. Emodin may thus inhibited cell proliferation in NSCLC cells by downregulation ERCC1 and Rad51.

Sunitinib synergizes the antitumor effect of cisplatin via modulation of ERCC1 expression in models of gastric cancer.

We evaluated the effects of sunitinib monotherapy and in combination with cisplatin in human gastric cancer cell lines. Sunitinib showed antiproliferative effect in gastric cancer cells line with high PDGFRA expression. Knockdown of PDGFRA showed that sunitinib sensitivity was correlated with the basal expression of PDGFRA. Synergistic growth inhibitory activity in combination with cisplatin was identified. We further explored how sunitinib potentiated the activity of cisplatin. We found that sunitinib treatment resulted in the down-regulation of ERCC1 expression via the modulation of PDGFRA expression in gastric cancer cells. The effect was verified via SNU484 xenograft model. Our data support the rationale of clinical trial using sunitinib in combination of cisplatin in gastric cancer.

[Examination of ERCC1 expression in lung cancer patients treated with platinum-based chemotherapy]. / ERCC1-expresszió vizsgálata platinabázisú kezelésben részesülõ tüdõrákos betegekben.

Platinum-based chemotherapies are widely used in the treatment of lung cancer. However, little is known about their effect in the expression of certain tissue biomarkers. We have studied the ERCC1 (excision repair cross-complementation group 1) expression in tissue samples of patients treated with neoadjuvant chemotherapy. Fifty lung cancer tissue blocks of 25 patients (15 males, 10 females) were studied. They included 25 bronchoscopic biopsies (14 squamous cell carcinomas and 11 adenocarcinomas) together with their corresponding surgical biopsies after neoadjuvant chemotherapy. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues to study the expression of ERCC1. Staining scores (0-300) were calculated by multiplying the percentage of positive tumor cells (0-100) by the staining intensity (0-3). All but one bronchosopic squamous cell carcinoma tissues (13/14) expressed ERCC1. Four of these cases became negative after neoadjuvant therapy, and in 8 cases the level of expression decreased. In the adenocarcinoma group all but one bronchosopic tissues (10/11) expressed ERCC1. Six of these cases became negative after neoadjuvant therapy, and in 4 cases the level of expression decreased. Comparison of staining scores before and after chemotherapy revealed more pronounced decrease in adenocarcinomas and in female patients. There was no newly expressed ERCC1-positive case in the surgical biopsy group. The results of the present study suggest that platinum-based chemotherapy affects the expression of tissue biomarker (ERCC1) which may have predictive value, and probably induces a selection of tumor cells with more aggressive phenotype. This knowledge might be of importance when designing treatment protocols for non-small cell lung cancer patients.

The sequence-dependent cytotoxic effect of trastuzumab in combination with 5-Fluorouracil or cisplatin on gastric cancer cell lines.

The effect of trastuzumab on patients with HER-2/neu (HER2)-positive gastric cancer has been confirmed in a phase III clinical trial (ToGA study). However, the optimized sequence and synergic mechanism of trastuzumab and chemotherapy are not clear. Our study investigated the effects and mechanisms of trastuzumab in combination with 5-Fluorouracil (5-Fu) or cisplatin (DDP) on gastric cancer cell lines. Flow cytometry was used to determine HER2 expression and cell cycle. MTT assay was performed to evaluate cytotoxicity. Western blotting and RT-PCR were used to analyze signaling transduction and mRNA expression. Sequential 5-Fu followed by trastuzumab and trastuzumab plus DDP followed by trastuzumab produced the best inhibitory effects. Inhibition of HER2-PI3K-AKT signal transduction, downregulation of nucleotide excision repair cross-complementation 1 (ERCC1), and interference with cell cycle distribution may elucidate the synergism between trastuzumab and chemotherapy. These results provide some evidence for designing a rational regime when trastuzumab is being considered to be used in patients with gastric cancer.

Platinum-based chemotherapy in lung cancer affects the expression of certain biomarkers including ERCC1.

Chemotherapies are widely used in the treatment of lung cancer. However, little is known about their effect in the expression of different tissue markers. Seventeen lung cancer tissue blocks obtained by bronchoscopic biopsies together with their corresponding surgical biopsies after neoadjuvant chemotherapy were studied. They included 9 adenocarcinomas (ADC) and 8 squamous cell carcinomas (SCC). Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues to study the expression of Ki-67, p53, Bcl-2, Bax, Fas-ligand and ERCC1 (excision repair cross-complementation group 1). Out of 17 NSCLC 6 expressed proapoptotic markers and 4 expressed antiapoptotic markers, while in 7 cases the apoptotic markers did not show detectable changes after neoadjuvant chemotherapy. Six of 17 bronchoscopic NSCLC cases expressed increased level of Ki-67 after neoadjuvant treatment. Eight bronchoscopic NSCLC tissues (6 SCC, 2 ADC) expressed ERCC1. All but one ADC became ERCC1 negative after neoadjuvant therapy. There was no newly expressed ERCC1 positive case in the surgical biopsy group. Platinum-based neoadjuvant chemotherapy had no effect on the apoptotic activity of 17 patients' tumor specimen, however, 6 of 17 bronchoscopic NSCLC cases expressed increased level of Ki-67 after neoadjuvant treatment, in 3 cases the level of Ki-67 became decreased, while 8 cases had no detectable change of proliferation activity. The results of the present study suggest that platinum-based chemotherapy probably induces a selection of tumor cells with more aggressive phenotype, and also affects the expression of tissue marker (ERCC1) that could have predictive value.

Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: a potential treatment strategy.

Resistance of ovarian mucinous adenocarcinoma to standard chemotherapy with paclitaxel and carboplatin is associated with poor prognosis, and an effective treatment is needed. The present study aimed to identify an effective chemotherapy for ovarian mucinous adenocarcinoma. Five human ovarian mucinous adenocarcinoma cell lines (MN-1, OMC-1, RMUG-L, RMUG-S, TU-OM-1) were used in this study. Sensitivity of the cells to the anticancer agents was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and we assessed drug sensitivity by calculating the assay area under the curve for each agent. Protein expression was confirmed by Western blot analysis. We also examined the efficacy of combination chemotherapy on survival in a xenograft model of nude mice. The IC(50) to anticancer agents ranged widely. The assay area under the curve indicated that two of five cell lines (MN-1, TU-OM-1) were sensitive to oxaliplatin, 5-fluorouracil and etoposide, and only one (TU-OM-1) was sensitive to 7-ethyl-10-hydroxycamptothecin, which is an active metabolite of camptothecin. All cell lines were resistant to cisplatin and paclitaxel. The combination of oxaliplatin and 5-fluorouracil resulted in additive or synergistic effects on all cell lines. The combination of oxaliplatin and 5-fluorouracil significantly prolonged survival in a ovarian mucinous adenocarcinoma xenograft model of nude mice. Protein expression levels of the excision repair cross-complementation group 1 were lower in oxaliplatin sensitive cell lines. Exposure to 5-fluorouracil down-regulated cross-complementation group 1 expression in ovarian mucinous adenocarcinoma cells. We conclude that combination chemotherapy consisting of oxaliplatin and 5-fluorouracil was an effective treatment for ovarian mucinous adenocarcinoma and may be a pivotal candidate for a novel treatment strategy.

Comparative toxicity of arsenite metabolites in wild type CHO cells and in cells deficient in excision repair cross-complementing 1 and 2.

Arsenic (As) has been shown to alter one or more DNA repair processes. Excision repair cross-complementing 1 and 2 (ERCC1 and ERCC2) have shown to be associated with arsenic-induced toxicity and carcinogenicity. In this study, we investigated cytotoxic effects of various As metabolites in relation to two nucleotide excision repair genes: ERCC1 and ERCC2. Various arsenate (pentavalent) and arsenite (trivalent) metabolites were tested in ERCC1, ERCC2 deficient and wild type cells. Our results showed that in the selected concentration range pentavalent As metabolites; iAs(V), MMA(V) and DMA(V) were not cytotoxic, unlike the trivalent As metabolites; iAs(III), MMA(III) and DMA(III). The measured LC(50) demonstrated a significant difference (p<0.01) for iAs(III) between the three cell lines, while MMA(III) and DMA(III) are more cytotoxic to all three cell lines. UV5 (ERCC2 deficient) cells also showed a lower resistance to iAs(III) in comparison to AA8 (wild type) and UV20 (ERCC2 deficient) cells. This might be explained through the generation of hydrogen peroxide (H(2)O(2)), which is generated by increase of intracellular Ca(2+) level. Generation of H(2)O(2) in UV5 cells after incubation with iAs(III) is significantly higher than AA8 and UV20 cells (p<0.01). In conclusion, absence of ERCC2 leads to a increased generation of H(2)O(2) by iAs(III) in UV5 cells, which is in contrast to AA8 and UV20 cells.

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression.

BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.

Peroxynitrite induces apoptosis and decline in intracellular free Mg with concomitant elevation in [Ca2+]I in rat aortic smooth muscle cells: possible roles of extracellular and intracellular magnesium ions in peroxynitrite-induced cell death.

The present study demonstrates that exogenous ONOO(-) can result in rapid declines in intracellular free magnesium ions ([Mg(2+)](i)) concomitant with rapid rises in intracellular free calcium ions ([Ca(2+)](i)) and, subsequently, trigger apoptosis but not necrosis in rat aortic SMCs; high [Mg(2+)] significantly attenuates ONOO(-)-induced apoptosis. ONOO(-)-induced apoptosis in vascular SMCs appears to involve activation of Ca(2+)-Mg(2+)-dependent endonucleases and caspase-3. Mg deficiency itself could not induce apoptosis in these SMCs, but it could significantly enhance ONOO(-)-induced apoptosis.

A novel naturally occurring tripyrrole with potential nuclease and anti-tumour properties.

The DNA targeting and membrane damaging activities of a novel tripyrrole 1 obtained as a red pigment from the Micrococcus sp. were investigated. It was found that compound 1 binds with DNA efficiently and facilitates copper-mediated DNA cleavage as well as peroxidation of membrane lipids by a process that does not require any external reducing agent. Compound 1 also showed impressive cytotoxicity to both mouse and human tumour cell lines. The membrane damaging ability of compound 1 might be vital in its nuclease and cytotoxicity properties. Interestingly, compared to the various DNA cleaving agents, compound 1 showed a preferential binding with the G-C rich domain.

Ce(4+) induced down-regulation of ERK-like MAPK and activation of nucleases during the apoptosis of cultured Taxus cuspidata cells.

Ce(4+) (Ce(NH(4))(2)(NO(3))(6)) at 1mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions (O(2)(-)) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 microM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce(4+)-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca(2+)/Mg(2+). Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O(2)(-) generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O(2)(-), activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce(4+)-induced cultured T. cuspidata cells.

Using translational research to tailor the use of chemotherapy in the treatment of NSCLC.

The treatment of advanced non-small-cell lung cancer (NSCLC) has improved over the past two decades with the availability of new agents such as gemcitabine, the taxanes and vinorelbine. Despite these advances, survival prospects remain disappointingly low for most patients. Further improvements in response rate and survival requires the development of new agents with novel mechanisms of action, such as molecularly targeted therapies, which are currently being tested in clinical trials. The conventional treatment approach to NSCLC is beginning to shift towards the application of specific strategies and techniques, such as pharmacogenomics, to tailor treatment to individual patients. Many efforts are in progress to identify the clinical predictors of outcome. The discovery of prognostic molecular markers, such as the putative suppressor gene (RRM1), represents a novel and potential parameter to help guide clinical treatment decisions.