Inhibition of Ape1's DNA Repair Activity as a Target in Cancer: Identification of Novel Small Molecules that have Translational Potential for Molecularly Targeted Cancer Therapy

Indiana University-Purdue University Indianapolis (IUPUI)The DNA Base Excision Repair (BER) pathway repairs DNA damaged by endogenous and exogenous agents including chemotherapeutic agents. Removal of the damaged base by a DNA glycosylase creates an apurinic / apyrimidinic (AP) site. AP endonuclease1 (Ape1), a critical component in this pathway, hydrolyzes the phosphodiester backbone 5’ to the AP site to facilitate repair. Additionally, Ape1 also functions as a redox factor, known as Ref-1, to reduce and activate key transcription factors such as AP-1 (Fos/Jun), p53, HIF-1α and others. Elevated Ape1 levels in cancers are indicators of poor prognosis and chemotherapeutic resistance, and removal of Ape1 via methodology such as siRNA sensitizes cancer cell lines to chemotherapeutic agents. However, since Ape1 is a multifunctional protein, removing it from cells not only inhibits its DNA repair activity but also impairs its other functions. Our hypothesis is that a small molecule inhibitor of the DNA repair activity of Ape1 will help elucidate the importance (role) of its repair function in cancer progression as wells as tumor drug response and will also give us a pharmacological tool to enhance cancer cells’ sensitivity to chemotherapy. In order to discover an inhibitor of Ape1’s DNA repair function, a fluorescence-based high-throughput screening (HTS) assay was used to screen a library of drug-like compounds. Four distinct compounds (AR01, 02, 03 and 06) that inhibited Ape1’s DNA repair activity were identified. All four compounds inhibited the DNA repair activity of purified Ape1 protein and also inhibited Ape1’s activity in cellular extracts. Based on these and other in vitro studies, AR03 was utilized in cell culture-based assays to test our hypothesis that inhibition of the DNA repair activity of Ape1 would sensitize cancer cells to chemotherapeutic agents. The SF767 glioblastoma cell line was used in our assays as the chemotherapeutic agents used to treat gliobastomas induce lesions repaired by the BER pathway. AR03 is cytotoxic to SF767 glioblastoma cancer cells as a single agent and enhances the cytotoxicity of alkylating agents, which is consistent with Ape1’s inability to process the AP sites generated. I have identified a compound, which inhibits Ape1’s DNA repair activity and may have the potential in improving chemotherapeutic efficacy of selected chemotherapeutic agents as well as to help us understand better the role of Ape1’s repair function as opposed to its other functions in the cell

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryp- tochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B–induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent man- ner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fung

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1

DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

<p>Abstract</p> <p>Background</p> <p>Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process.</p> <p>Methods</p> <p>A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry.</p> <p>Results</p> <p>Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone.</p> <p>Conclusions</p> <p>Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.</p

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

[EN] DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.Deutsche Forschungsgemeinschaf; European funds (EuropeanRegional Development Fund), Spanish Ministerio de Economía y Competitividad; Regional Government (Junta deAndalucía

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells.

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors

Extending Lifespan Using Various Prolongevity Interventions And Their Effects On Enhancing Dna Repair Activity

Aging is not a disease; it causes a decrease in the physiological functions of cells, tissues, and organs. Aging has been considered as one of the biggest risk factors for the development of various diseases such as cancer, type-2 diabetes, obesity, atherosclerotic cardiovascular diseases, and neurodegeneration. Numerous studies have shown that lifespan can be extended in mice by genetic, dietary, and pharmacological interventions. A few prolongevity interventions currently being studied include: the drug rapamycin, that has been found to inhibit mTOR expression and exhibit anticancer properties; reduced caloric intake, a broadly acting dietary intervention for preventing carcinogenesis, and ultimately extending lifespan; and more recently, another promising strategy being studied is crowded litter placement in mice starting from a very young age. We hypothesize that the aforementioned interventions will delay the effects of aging, through enhanced DNA base excision repair activity, which will lead to tumorigenesis prevention

APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24h. In cultures where APE1 expression was reduced by ∼ 80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons

E. coli and colon cancer : Is mutY a culprit?

Peer reviewedPreprin

Small intestinal development in suckling rats after enteral obestatin administration

This study investigated the effect of enteral administration of obestatin on the development of small intestine, as well as oxidative stress markers and trancriptomic profile of gastrointestinal genes. Suckling rats were assigned to 3 groups treated with: C-saline solution; OL-obestatin (125 nmol/kg BW); OH-obestatin (250 nmol/kg BW) administered twice daily, from the 14th to the 21st day of life. Enteral administration of obestatin in both studied doses had no effect neither on the body weight of animals nor the BMI calculated in the day of euthanasia. Compared to the control group (C), treatment with obestatin resulted in significant changes in the histometry of the small intestinal wall as well as intestinal epithelial cell remodeling. The observed changes and their possible implications for intestinal development were dependent on the dosage of peptide. The enteral administration of high dose (OH) of obestatin significantly decreased its expression in the stomach and increased markers of oxidative stress. The gene profile revealed MAPK3 (mitogen-activated protein kinase-3) as the key regulator gene for obestatin action in the gastrointestinal track. In conclusion, we have showed that enteral administration of obestatin influences the gut mucosa remodeling. It is also suggested that the administration of high dose (OH) has inhibitory effect on the intestinal maturation of suckling rats