Camptothecin Treatment Research Articles

Topological DNA damage, telomere attrition and T cell senescence during chronic viral infections

BackgroundT cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions.ResultsWe demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival.ConclusionThese findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

Camptothecin activates SIRT1 to promote lipid catabolism through AMPK/FoxO1/ATGL pathway in C2C12 myogenic cells.

Caloric restriction activates sirtuin 1 (SIRT1) and induces a variety of metabolic effects that are beneficial for preventing age-related disease. The present study screened a commercially available used drug library to develop small molecule activators of SIRT1 as therapeutics for treatment of metabolic disorders. Using an in vitro fluorescence assay, the cancer therapeutic camptothecin increased SIRT1 enzymatic activity by 5.5-fold, indicating it to be a potent SIRT1 activator. Camptothecin also elevated the nicotinamide adenine dinucleotide (NAD)+/NADH ratio and increased SIRT1 protein levels in differentiated C2C12 myogenic cells. Treatment of C2C12 myotubes with camptothecin increased phosphorylation of AMP-dependent kinase (AMPK) and acetyl-coenzyme A carboxylase, caused nuclear translocation and deacetylation of forkhead box O1 (FoxO1), increased transcription and protein expression of adipose triglyceride lipase (ATGL), decreased the amount of intracellular oil droplets, and significantly increased β-oxidation of fatty acids. These in vitro data were confirmed in vivo as camptothecin treatment of C57BL/6J mice reduced fat and plasma triglyceride levels. All of the above camptothecin-induced alterations were attenuated by the SIRT1-specific inhibitor nicotinamide and/or 6-[4-(2-piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a]pyrimidin (compound C). Thus, camptothecin activation of SIRT1 promotes lipid catabolism through AMPK/FoxO1/ATGL signaling.

Camptothecin induces c-Myc- and Sp1-mediated hTERT expression in LNCaP cells: Involvement of reactive oxygen species and PI3K/Akt

Camptothecin (CPT), a quinoline alkaloid isolated from Camptotheca acuminate, targets topoisomerase I, which is continuously expressed in cancer cells. However, the molecular mechanisms responsible for CPT-induced telomerase inhibition remain unclear. Unexpectedly, we found that CPT upregulates hTERT expression and concomitantly increases telomerase activity. However, transfection of hTERT-targeting siRNA had no effect on CPT-induced G2/M phase arrest, suggesting that CPT-induced telomerase activation was not related to G2/M phase arrest. CPT simultaneously increased Nrf2 expression and the level of intracellular reactive oxygen species (ROS), whereas pretreatment with the antioxidants N-acetyl-cysteine (NAC) or glutathione (GSH) strongly attenuated ROS production, which was accompanied by hTERT downregulation. Additionally, transient Nrf2 knockdown enhanced CPT-induced ROS production and hTERT promoter activity. CPT also upregulated hTERT expression and telomerase activity by inducing c-Myc and Sp1 expression and activity. Moreover, c-Myc stimulated ROS production in response to CPT, leading to Sp1 activation, which promoted hTERT expression and telomerase activity. CPT treatment enhanced the phosphorylation of PI3K and Akt, which led to hTERT phosphorylation into the nucleus. These findings demonstrate that CPT positively regulates telomerase activity by upregulating hTERT expression and phosphorylation via the c-Myc/ROS/Sp1 and PI3K/Akt axis.

Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 Are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants.

DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.

RMI1 contributes to DNA repair and to the tolerance to camptothecin.

Maintenance of genome integrity is critical for faithful propagation of genetic information and the prevention of the mutagenesis induced by various DNA damage events. RecQ-mediated genome instability protein 1 (RMI1), together with Bloom syndrome protein and topoisomerase IIIα, form an evolutionarily conserved complex that is critical for the maintenance of genomic stability. Herein, we report that RMI1 depletion increases cell sensitivity to camptothecin treatment, as shown by an elevation of genotoxic stress-induced DNA double-strand breaks, a stronger activation of the DNA damage response, and a greater G2/M cell cycle delay. Our findings support that, upon DNA damage, RMI1 forms nuclear foci at the damaged regions, interacts with RAD51, and facilitates the recruitment of RAD51 to initiate homologous recombination. Our data reveal the importance of RMI1 in response to DNA double-strand breaks and shed light on the molecular mechanisms by which RMI1 contributes to maintain genome stability.-Fang, L., Sun, X., Wang, Y., Du, L., Ji, K., Wang, J., He, N., Liu, Y., Wang, Q., Zhai, H., Hao, J., Xu, C., Liu, Q. RMI1 contributes to DNA repair and to the tolerance to camptothecin.

Camptothecin inhibits the progression of NPC by regulating TGF-β-induced activation of the PI3K/AKT signaling pathway.

Nasopharyngeal carcinoma (NPC) is a type of cancer that is characterized by increased invasiveness, metastatic potential and tumor recurrence. Camptothecin has been demonstrated to exhibit anticancer activity. However, the potential underlying molecular mechanisms mediated by camptothecin in NPC cells remain elusive. In the present study, the efficacy of camptothecin for NPC was investigated in vitro and in vivo. Additionally, the potential signaling pathway mediated by camptothecin in NPC cells was also examined. The results indicated that the viability and aggressiveness of NPC cells were suppressed by camptothecin treatment in a dose-dependent manner. Camptothecin administration downregulated the expression levels of cell-cycle-associated proteins including cyclin 1, cyclin-dependent kinase (CDK)1 and CDK2 in NPC cells. Expression levels of migration-associated proteins including vimentin, fibronectin and epithelial cadherin were regulated by camptothecin treatment in NPC cells. Additionally, camptothecin inhibited the expression of transforming growth factor-β (TGF-β), phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT), whereas TGF-β overexpression abrogated camptothecin-mediated inhibition of PI3K and AKT expression and camptothecin-mediated inhibition of the viability and aggressiveness of NPC cells. Camptothecin significantly inhibited tumor growth and increased survival times in a mouse model of cancer. In conclusion, these results indicate that camptothecin treatment may inhibit the viability of NPC cells and aggressiveness by regulating the TGF-β-induced PI3K/AKT signaling pathways, which in turn may be a potential molecular target for the treatment of NPC.

Roles of Alternative RNA Splicing of the Bif-1 Gene by SRRM4 During the Development of Treatment-induced Neuroendocrine Prostate Cancer

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that becomes more prevalent when hormonal therapy, chemotherapy, or radiation therapy is applied to patients with metastatic prostate adenocarcinoma (AdPC). How AdPC cells survive these anti-cancer therapies and progress into t-NEPC remains unclear. By comparing the whole transcriptomes between AdPC and t-NEPC, we identified Bif-1, an apoptosis-associated gene, which undergoes alternative RNA splicing in t-NEPC. We found that while Bif-1a is the predominant variant of the Bif-1 gene in AdPC, two neural-specific variants, Bif-1b and Bif-1c, are highly expressed in t-NEPC patients, patient derived xenografts, and cell models. The neural-specific RNA splicing factor, SRRM4, promotes Bif-1b and Bif-1c splicing, and the expression of SRRM4 in tumors is strongly associated with Bif-1b/-1c levels. Furthermore, we showed that Bif-1a is pro-apoptotic, while Bif-1b and Bif-1c are anti-apoptotic in PCa cells under camptothecin and UV light irritation treatments. Taken together, our data indicate that SRRM4 regulates alternative RNA splicing of the Bif-1 gene that enables PCa cells resistant to apoptotic stimuli under anti-cancer therapies, and may contribute to AdPC progression into t-NEPC.

DNA damage response signaling does not trigger redistribution of SAMHD1 to nuclear foci

SAMHD1 (Sterile alpha motif and histidine-aspartic acid (HD) domain containing protein 1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) that restricts viral replication in infected cells. This protein is also involved in DNA repair by assisting in DNA end resection by homologous recombination (HR) after DNA double-strand break (DSB) induction with camptothecin (CPT) or etoposide (ETO). We showed that a monoclonal anti-SAMHD1 antibody produced against the full-length protein detected an unspecific 50 kDa protein that colocalized with dot-like structures after CPT treatment in HeLa cells. In contrast, a polyclonal anti-SAMHD1 antibody raised against the N-terminus of this protein specifically detected SAMHD1, as shown in Jurkat, HAP1KO and HEK293T SAMHD1-siRNA cell lysates compared with their respective controls. Our findings showed that SAMHD1 is not localized in dot-like structures under DSB induction in HeLa cells.

Targeting topoisomerases for antileishmanial chemotherapeutics and deciphering the role of Mre11 in topoisomerase induced DNA damage repair in Leishmania donovani

Visceral Leishmaniasis or Kala‐azar, caused by Leishmania donovani, is the second largest cause of human death tally due to parasitic protozoan infection. Researchers across the globe are engaged in identifying and validating novel drug targets specific for Leishmania. Contextually, DNA topoisomerase IB of L. donovani (LdTopIB) has emerged as a promising candidate. Its unique bisubunit structure, completely different from single subunit structure of human topoisomerase I (HTopI), offers an interesting attribute to be exploited to develop novel therapeutics. In a programme to search for novel antileishmanials, our group has been probing into large array of compounds, both from natural and synthetic origin, to identify potential lead molecules. However, when subjected to topoisomerase stressors, the response machineries in trypanosomatid parasites including Leishmania remain largely unknown till date. Parasite death caused by LdTopIB poisons is fundamentally due to the generation of stabilized DNA‐enzyme covalent complex (Top1cc). When replication or transcription machineries collide with this Top1cc, it leads to DNA strand breaks. This molecular threat is detrimental for the parasite. In order to survive, the parasites need to repair this Top1cc and thereby rescue topoisomerase. We found that, among many factors, the Meiotic Recombination Protein 11 (Mre11) got over‐expressed in the parasite when it was subjected to camptothecin treatment. However, reports sugesting direct evidence of Mre11 to resolve Top1cc in vivo remain scarce till date. In the present investigation, we have identified, cloned and sequenced Mre11 gene (LdMre11) from L. donovani for the first time. The LdMre11 protein was expressed and the activity was assayed. We found that the recombinant protein could successfully cleave a phosphotyrosyl substrate in a magnesium dependent way due to its phosphodiesterase activity. The protein also possessed an exonuclease activity. This report is first of its kind to elucidate the possible phosphodiesterase activity of Mre11 protein from a member of trypanosomatid parasite. In order to study the biological significance of Mre11, Mre11 knockout parasites (LdMre11−/−) were next generated by targeted gene deletion method. Mre11 null parasites became hypersensitive to camptothecin. Ectopic expression of wildtype Mre11 in a null background reverted the mutant phenotype. Mre11 null parasites showed perturbed cell cycle progression with a clear arrest in G0/G1 phase in contrast to control cells. Results from immunoband depletion experiments using an antibody raised against the small subunit of LdTopIB clearly indicated that Mre11 null parasites accumulated larger amount of in vivo Top1cc in response to topoisomerase poisoning which was rescued in ectopically overexpressed Mre11 mutants. In addition, we have also found that Mre11 is overexpressed in drug resistant field isolates of L. donovani isolated from endemic zones of India. Interestingly, flow cytometry based experiments showed that Mre11 null parasites were hypersensitive to classical antileishmanial drugs viz. sodium antimony gluconate and miltefosine. Probably this is one of the probable mechanisms in Leishmania to achieve drug resistance. Cumulatively, the study provides the first mechanistic insight into the functioning of Mre11 in Leishmania donovani in response to topoisomerase IB poisoning and probably Mre11 also has a role in drug resistance.Support or Funding InformationDepartment of Science and Technology(DST, Govt. of India) joint Indo‐Brazil project (DST/INT/Brazil/RPO‐01/2009/2)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Melanoma antigen-D2 controls cell cycle progression and modulates the DNA damage response

Overexpression of the ubiquitous type II melanoma antigen-D2 (MAGED2) in numerous types of cancer suggests that this protein contributes to carcinogenesis, a well-documented characteristic of other MAGE proteins. Modification of MAGED2 intracellular localization during cell cycle phases and following treatment with camptothecin (CPT) and phosphorylation by ATM/ATR following ionizing irradiation led us to investigate the molecular functions of MAGED2 in the cellular response to DNA damage. Cell cycle regulators, cell cycle progression, and bromodeoxyuridine (BrdU) incorporation were compared between MAGED2-sufficient and -depleted U2OS cells following exposure to CPT. At 24 h post-CPT removal, MAGED2-depleted cells had lower levels of p21 and p27, and there was an increase in S phase BrdU-positive cells with a concurrent decrease in cells in G2. These cell cycle modifications were p21-independent, but ATR-, SKP2-, and CDC20-dependent. Importantly, while MAGED2 depletion reduced CHK2 phosphorylation after 8 h of CPT treatment, it enhanced and prolonged CHK1 phosphorylation after a 24 h recovery period, indicating sustained ATR activation. MAGED2 depletion had no impact on cell survival under our experimental conditions. In summary, our data indicate that MAGED2 reduced CPT-related replicative stress, suggesting a role for this protein in genomic stability.

Silver Nanoparticles Potentiates Cytotoxicity and Apoptotic Potential of Camptothecin in Human Cervical Cancer Cells

Silver nanoparticles (AgNPs) are widely used metal nanoparticles in health care industries, particularly due to its unique physical, chemical, optical, and biological properties. It is used as an antibacterial, antiviral, antifungal, and anticancer agent. Camptothecin (CPT) and its derivatives function as inhibitors of topoisomerase and as potent anticancer agents against a variety of cancers. Nevertheless, the combined actions of CPT and AgNPs in apoptosis in human cervical cancer cells (HeLa) have not been elucidated. Hence, we investigated the synergistic combinatorial effect of CPT and AgNPs in human cervical cancer cells. We synthesized AgNPs using sinigrin as a reducing and stabilizing agent. The synthesized AgNPs were characterized using various analytical techniques. The anticancer effects of a combined treatment with CPT and AgNPs were evaluated using a series of cellular and biochemical assays. The expression of pro- and antiapoptotic genes was measured using real-time reverse transcription polymerase chain reaction. The findings from this study revealed that the combination of CPT and AgNPs treatment significantly inhibited cell viability and proliferation of HeLa cells. Moreover, the combination effect significantly increases the levels of oxidative stress markers and decreases antioxidative stress markers compared to single treatment. Further, the combined treatment upregulate various proapoptotic gene expression and downregulate antiapoptotic gene expression. Interestingly, the combined treatment modulates various cellular signaling molecules involved in cell survival, cytotoxicity, and apoptosis. Overall, these results suggest that CPT and AgNPs cause cell death by inducing the mitochondrial membrane permeability change and activation of caspase 9, 6, and 3. The synergistic cytotoxicity and apoptosis effect seems to be associated with increased ROS formation and depletion of antioxidant. Certainly, a combination of CPT and AgNPs could provide a beneficial effect in the treatment of cervical cancer compared with monotherapy.

BTG2 is a tumor suppressor gene upregulated by p53 and PTEN in human bladder carcinoma cells.

Although widely deemed as a tumor suppressor gene, the role of B‐cell translocation gene 2 (BTG2) in bladder cancer is still inconclusive. We investigated the role and regulatory mechanism of BTG2 in bladder cancer. BTG2 expression in human bladder tissues was determined by RT‐qPCR and immunoblotting assays. Expressions of BTG2 and PTEN in bladder carcinoma cells were determined by immunoblotting, RT‐qPCR, or reporter assays. The 3H‐thymidine incorporation assay, flow cytometry, and the xenograft animal model were used to determine the cell growth. BTG2 expression was lower in human bladder cancer tissues than normal bladder tissues. Highly differentiated bladder cancer cells, RT4, expressed higher BTG2 than the less‐differentiated bladder cancer cells, HT1376 and T24. Overexpression of BTG2 in T24 cells inhibited cell growth in vitro and in vivo. Camptothecin and doxorubicin treatments in RT‐4 cells or transient overexpression of p53 into p53‐mutant HT1376 cells induced p53 and BTG2 expression. Further reporter assays with site‐mutation of p53 response element from GGGAAAGTCC to GGAGTCC within BTG2 promoter area showed that p53‐induced BTG2 gene expression was dependent on the p53 response element. Ectopic PTEN overexpression in T24 cells blocked the Akt signal pathway which attenuated cell growth via upregualtion of BTG2 gene expression, while reverse effect was found in PTEN‐knockdown RT‐4 cells. PTEN activity inhibitor (VO‐OHpic) treatment decreased BTG2 expression in RT‐4 and PTEN‐overexpressed T24 cells. Our results suggested that BTG2 functioned as a bladder cancer tumor suppressor gene, and was induced by p53 and PTEN. Modulation of BTG2 expression seems a promising way to treat human bladder cancer.

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation.

Abstract 5875: Schlafen 11 (SLFN11) induces lethal S-phase arrest in response to DNA damage: A novel mechanism of how cancer cells are killed by DNA damaging agents

Abstract The putative DNA/RNA helicase, Schlafen 11 (SLFN11), is a recently discovered determinant of sensitivity to DNA damaging agents such as platinums, topoisomerase I and II inhibitors (camptothecins, etoposide, doxorubicin), cisplatin, gemcitabine (1-3) and PARP inhibitors (olaparib, talazoparib) (4). Because SLFN11 expression is suppressed in ~45% cancer cell lines, SLFN11 inactivation is potentially one of the most prevalent mechanisms of resistance to DNA damaging agents. However, the molecular mechanisms of SLFN11 action in the DNA damage response have not been solved. Using isogenic cell lines of SLFN11-knockout and SLFN11-overexpression, we will show that SLFN11 binds to chromatin in response to camptothecin within 2 hours. ChIP-seq analysis reveals that SLFN11 preferentially binds to replication origins under camptothecin treatment. We identify two helicases DHX9 and MCM3, which are involved in replication and origin firing, as binding partners of SLFN11 by IP-MS analysis. Cell cycle analyses revealed that SLFN11 inhibits replication in response to camptothecin, which irreversibly lasts until apoptosis is exerted. Notably, the S-phase arrest by SLFN11 is independent of ATR-mediated S-phase checkpoint. Unscheduled origin firing by ATR inhibition under camptothecin treatment is suppressed in the presence of SLFN11. Hence, SLFN11 enforces irreversible S-phase arrest in damaged cells possibly by inhibiting dormant origin firing and inactivating the MCM helicase in conjunction with DXH9. We conclude that the irreversible S-phase arrest by SLFN11 leads to cell death in response to DNA damaging agents, which is a novel mechanism of how cancer cells are killed by DNA damaging agents. 1. Sousa FG, Matuo R, Tang SW, Rajapakse VN, Luna A, Sander C, et al. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity. DNA Repair (Amst) 2015;28:107-15. 2. Zoppoli G, Regairaz M, Leo E, Reinhold WC, Varma S, Ballestrero A, et al. Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents. Proc Natl Acad Sci U S A 2012;109(37):15030-5. 3. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012;483(7391):603-307. 4. Murai J, Feng Y, Yu GK, Ru Y, Tang SW, Shen Y, et al. Resistance to PARP inhibitors by SLFN11 inactivation can be overcome by ATR inhibition. Oncotarget 2016. Citation Format: Junko Murai, Yves Pommier. Schlafen 11 (SLFN11) induces lethal S-phase arrest in response to DNA damage: A novel mechanism of how cancer cells are killed by DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5875. doi:10.1158/1538-7445.AM2017-5875

Camptothecin Sensitizes Refractory Lung Cancer Cells to Receptor Kinase Inhibitor

BackgroundReceptor tyrosine kinases (RTKs) represent a large class of receptors that are upregulated in many cancers. The Ephrin‐A type‐2 precursor (EphA2) is an important RTK whose overexpression in lung cancer promotes growth, metastasis and acquisition of resistance phenotypes to therapies. Further, EphA2 is a downstream target of the epidermal growth factor receptor (EGFR) which is transcriptionally regulated by the oncogenic SOX2 transcription. This study investigates the effect of combination treatment with camptothecin (CPT) and the RTK inhibitor (RTKI) drug Erlotinib (Erl) in inhibiting survival mechanism in lung cancer cells.MethodsCell viability of H1299 and H1975 was determined using the Resazurin dye assay following single and combination treatments with CPT and Erl for 72 hours. The effect of 48‐hour single and combination CPT (1.0 μM) and Erl (10 μM and 20 μM) treatments on H1299 cell migration, apoptosis, and protein expression were investigated by wound healing assay, acridine orange‐ethidium bromide (AO‐EB) staining, and western blot respectively.ResultsH1299 and H1975 were resistant to Erl treatment (concentrations that killed 50% of cells, IC50 = 291.14±15.84 μM and 92.06±10.06 μM respectively). CPT was relatively more potent in inhibiting H1299 cell proliferation (IC50 = 7.01±0.35 μM). CPT co‐treatment at concentrations of 0.1 μM and 0.5 μM with different concentrations of Erl inhibited H1299 cell proliferation by 2‐(IC50 = 137.12±13.53 μM) and 10‐fold (IC50 = 27.88±2.88 μM) respectively. Combination treatment with CPT and Erl induced apoptotic events and inhibited H1299 cell migration compared to control and single treatment 48 hours after treatment. Additionally, SOX2 and c‐Myc expression as well as EphA2‐mediated cell migration signaling (phospho‐EphA2 Tyr588 and Tyr594 expression) were abrogated.ConclusionCPT is a potent, but highly toxic anticancer agent whose clinical utility is hindered by adverse dose‐dependent hematopoetic and other adverse effects. In this study, however, sub‐toxic doses of CPT was effective in sensitizing highly resistant H1299 cells to Erl through inhibition of cell migration and induction of apoptosis. Combination treatment with low‐dose CPT may be a viable means of sensitizing refractory cancer cells to therapy.Support or Funding InformationDr. Terrick Andey MCPHS UniversityE‐mail address:Terrick.andey@mcphs.edu

Abstract B33: PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy

Abstract Introduction: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor treatment outcomes and prognoses. Treatment usually consists of surgery, radiation and chemotherapy, which often result in severe systemic side effects frequently accompanied by tumor recurrence of highly radioresistant tumor cell populations. PARP-1 is a nuclear enzyme crucial for DNA damage repair (DDR) response. PARP-1 inhibition is a highly studied adjuvant to many anti-cancer treatment strategies as this arrests DNA damage repair thus enhancing the efficacy of conventional chemoradiotherapy at lower doses. However, PARP inhibition also promotes anti-inflammatory responses. Cancer cells suppress the immune cells ability to recognize and eliminate the tumor and promote their release of cancer-supporting growth factors and promote angiogenesis. Microglia the immune cells of the brain, normally protect the brain from pathogens and help in tissue recovery, but when associated with GBM cells, microglia obtain anti-inflammatory state that can promote tumor growth. We are developing a novel bimodal immunotherapeutic/DNA repair suppressive approach to treating GBM through modulation of microglial PARP-1 activity while concurrently sensitizing GBM to DNA damaging therapeutics. We hypothesize that while PARP-1 inhibition can enhance efficacy of chemoradiotherapy it might further suppress microglial ability to promote tumor elimination. Furthermore PARP-1 inhibitors' efficacy in preventing DNA repair is partially linked to their ability to trap PARP-1 at DNA break-site, which might be detrimental for the viability of non-cancerous brain cells. Objectives: 1) to establish the effects of PARP-1 inhibition alone, and in combination with targeted microglial PARP-1 activation, on microglial ability to eliminate glioblastoma cells in vitro (tumor cells vs. healthy glial cells). 2) to identify how microglial PARP-1 modulation (on/off) impacts glioblastoma elimination in vivo (tumor vs. brain). Methods: We will use GBM cells co-cultured with primary microglial cells in the presence of PARP inhibitors and observe their effect on the proliferation and viability of GBM cells. Efficacy of PARP-DNA trapping and corresponding DNA damage repair activity utilizing a variety of PARP inhibitors will be tested by specific biochemical and genotoxicity assays which directly quantify the degree of PARP-trapping in the glial cells and their impact on tumor cell genotoxcity. These studies take advantage of newly-developed and unique high-throughput DNA damage repair assays that we have been optimized to determine potential synergy of PARP-inhibitors with existing chemotherapeutic agents. Results: DNA damage assays (gamma H2AX foci and alkaline comet analysis) show that co-treatment of GBM cells (U87 and U251) with the PARP-inhibitor Olaparib and the chemotherapeutic agent Camptothecin (CPT) significantly increased the level of DNA damage when compared to CPT treatment alone. In the co-culture experiments, microglia enhanced CPT-induced GBM cell death when treated with Olaparib at 50-100 fold lower doses. Real time co-culture experiments also showed that Olaparib does not jeopardize microglial ability to actively phagocytize GBM cells. Conclusion: Preliminary results show that Olaparib is effective in sensitizing GBM cells to chemotherapy while microglia appear to enhance this effect. Further studies linking this combination therapy with microglia-mediated GBM elimination will help to elucidate the mechanism of this bimodal effect. This study emphasizes the importance of: (1) identifying novel effects of PARP inhibition on microglia in association with GBM and (2) the importance of developing microglia-mediated immunotherapeutic intervention to overcome the limitations of current therapies to significantly improve patient outcome. Citation Format: Asha Sinha, Sachin Katyal, Tiina Kauppinen. PARP-dependent co-modulation of DNA repair and microglial activity as a dual-pronged anti-glioblastoma treatment strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B33.

Anticancer Drug Camptothecin Test in 3D Hydrogel Networks with HeLa cells

Development of a biomimetic 3D culture system for drug screening is necessary to fully understand the in vivo environment. Previously, a self-assembling peptide hydrogel has been reported; the hydrogel exhibited physiological properties superior to a 3D cell culture matrix. In this work, further research using H9e hydrogel with HeLa cells was carried out considering H9e hydrogel’s interaction with camptothecin, a hydrophobic drug. According to AFM images, a PGworks solution triggered H9e hydrogel fiber aggregation and forms a 3D matrix suitable for cell culture. Dynamic rheological studies showed that camptothecin was encapsulated within the hydrogel network concurrently with peptide self-assembly without permanently destroying the hydrogel’s architecture and remodeling ability. Fluorescence measurement indicated negligible interaction between the fluorophore part of camptothecin and the hydrogel, especially at concentration 0.25 and 0.5 wt%. Using a dialysis method, we found that H9e hydrogel could not significantly inhibit the diffusion of camptothecin encapsulated inside the hydrogel matrix. In the cell culture experiment, HeLa cells were simultaneously embedded in the H9e hydrogel with the initialization of hydrogelation. Most importantly, cell viability data after camptothecin treatment showed responses that were drug-dose dependent but unaffected by the H9e hydrogel concentration, indicating that the hydrogel did not inhibit the drug.

Automated High-Content Screening of γ-H2AX Expression in HeLa Cells.

Due to their inherent nature, DNA strands can be easily broken by various environmental factors including chemical agents and ionizing radiation. Unrepaired DNA double-stranded breaks (DSBs) may result in genetic instability and have a strong negative impact on the integrity of the genome. It has been found that DSBs are always followed by phosphorylation of histone protein H2AX, a member of the H2A family, and immunocytochemical detection of phosphorylated H2AX (referred to as γ-H2AX) is one of the frequently used techniques for assessing DNA damage. Usually such an assessment is done manually under the microscope which is not practical for analyzing large numbers of cells and prevents researchers from rapid and unbiased testing of novel drug compounds. To solve this problem we attempted to do automated assessment of DSBs by using a High-Content Screening (HCS) platform. As a result of this effort, we developed an easy to run HCS protocol for accurate analysis of DSBs in HeLa cells treated with camptothecin as a model. By varying the time of camptothecin treatment and its concentration we were able to study the dynamics of DSBs and perform a statistical analysis.Results of our study indicate that DSBs can be investigated using a HCS platform that enable the analysis of large numbers of experimental data points in a fast and a highly accurate manner. The protocol presented in this chapter can be easily adapted for screening libraries containing substantial numbers of chemical compounds for their efficiency to induce or/and repair DNA breaks.

Inhibitory Effect of Camptothecin against Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae RS-2.

Camptothecin (CPT) has anticancer, antiviral, and antifungal properties. However, there is a dearth of information about antibacterial activity of CPT. Therefore, in this study, we investigated the inhibitory effect of CPT on Acidovorax avenae subsp. avenae strain RS-2, the pathogen of rice bacterial brown stripe, by measuring cell growth, DNA damage, cell membrane integrity, the expression of secretion systems, and topoisomerase-related genes, as well as the secretion of effector protein Hcp. Results indicated that CPT solutions at 0.05, 0.25, and 0.50 mg/mL inhibited the growth of strain RS-2 in vitro, while the inhibitory efficiency increased with an increase in CPT concentration, pH, and incubation time. Furthermore, CPT treatment affected bacterial growth and replication by causing membrane damage, which was evidenced by transmission electron microscopic observation and live/dead cell staining. In addition, quantitative real-time PCR analysis indicated that CPT treatment caused differential expression of eight secretion system-related genes and one topoisomerase-related gene, while the up-regulated expression of hcp could be justified by the increased secretion of Hcp based on the ELISA test. Overall, this study indicated that CPT has the potential to control the bacterial brown stripe pathogen of rice.