Andrographolide induces DNA damage in prostate cancer cells.

Prostate cancer (PCa) is the most common diagnosed cancer and is the third cause of cancer mortality in men in the USA. Andrographolide, a diterpenoid lactone isolated from Andrographis paniculata, has shown to possess anticarcinogenic activity in a variety of cancer cells. In this study, we examined the efficacy of Andrographolide in PCa using in vitro and in vivo models. Androgen-independent (PC3) and androgen-dependent (22RV1) cell lines were treated with Andrographolide to determine the effect in cell motility, cell proliferation and apoptosis. Andrographolide decreased PCa cell migration, decreased invasion, and increased cell apoptosis in vitro. Tumor growth was evaluated using an orthotopic xenograft model in which the prostates of SCID mice were injected with 22RV1, and mice were treated three times per week with Andrographolide 10 mg/kg. Andrographolide decreased tumor volume, MMP11 expression and blood vessels formation in vivo. Gene expression analysis identified cellular compromise, cell cycle, and "DNA recombination, replication and repair" as the major molecular and cellular functions altered in tumors treated with Andrographolide. Within DNA repair genes we confirmed increased expression of genes involved in DNA double strand break repair. Consistent with this observation we detected increased γH2AX in Andrographolide treated tumors and in cells in culture. Taken together, these data suggest that Andrographolide inhibits PCa by promoting DNA damage.

Mitochondrial DNA Integrity Is Maintained by APE1 in Carcinogen-Induced Colorectal Cancer.

Changes in mitochondrial DNA (mtDNA) integrity have been reported in many cancers; however, the contribution of mtDNA integrity to tumorigenesis is not well understood. We used a transgenic mouse model that is haploinsufficient for the apurinic/apyrimidinic endonuclease 1 (Apex1+/-) gene, which encodes the base excision repair (BER) enzyme APE1, to determine its role in protecting mtDNA from the effects of azoxymethane (AOM), a carcinogen used to induce colorectal cancer. Repair kinetics of AOM-induced mtDNA damage was evaluated using qPCR after a single AOM dose and a significant induction in mtDNA lesions in colonic crypts from both wild-type (WT) and Apex1+/-animals were observed. However, Apex1+/- mice had slower repair kinetics in addition to decreased mtDNA abundance. Tumors were also induced using multiple AOM doses, and both WT and Apex1+/-animals exhibited significant loss in mtDNA abundance. Surprisingly, no major differences in mtDNA lesions were observed in tumors from WT and Apex1+/- animals, whereas a significant increase in nuclear DNA lesions was detected in tumors from Apex1+/- mice. Finally, tumors from Apex1+/- mice displayed an increased proliferative index and histologic abnormalities. Taken together, these results demonstrate that APE1 is important for preventing changes in mtDNA integrity during AOM-induced colorectal cancer.Implications: AOM, a colorectal cancer carcinogen, generates damage to the mitochondrial genome, and the BER enzyme APE1 is required to maintain its integrity. Mol Cancer Res; 15(7); 831-41. ©2017 AACR.

The beneficial effects of AMP kinase activation against oxidative stress are associated with prevention of PPARα-cyclophilin D interaction in cardiomyocytes.

AMP kinase (AMPK) plays an important role in the regulation of energy metabolism in cardiac cells. Furthermore, activation of AMPK protects the heart from myocardial infarction and heart failure. The present study examines whether or not AMPK affects the peroxisome proliferator-activated receptor-α (PPARα)/mitochondria pathway in response to acute oxidative stress in cultured cardiomyocytes. Cultured H9c2 rat embryonic cardioblasts were exposed to H2O2-induced acute oxidative stress in the presence or absence of metformin, compound C (AMPK inhibitor), GW6471 (PPARα inhibitor), or A-769662 (AMPK activator). Results showed that AMPK activation by metformin reverted oxidative stress-induced inactivation of AMPK and prevented oxidative stress-induced cell death. In addition, metformin attenuated reactive oxygen species generation and depolarization of the inner mitochondrial membrane. The antioxidative effects of metformin were associated with the prevention of mitochondrial DNA damage in cardiomyocytes. Coimmunoprecipitation studies revealed that metformin abolished oxidative stress-induced physical interactions between PPARα and cyclophilin D (CypD), and the abolishment of these interactions was associated with inhibition of permeability transition pore formation. The beneficial effects of metformin were not due to acetylation or phosphorylation of PPARα in response to oxidative stress. In conclusion, this study demonstrates that the protective effects of metformin-induced AMPK activation against oxidative stress converge on mitochondria and are mediated, at least in part, through the dissociation of PPARα-CypD interactions, independent of phosphorylation and acetylation of PPARα and CypD.

Detection of DNA damage in peripheral blood mononuclear cells from pancreatic cancer patients.

DNA repair is a key mechanism in maintaining genomic stability: repair deficiencies increase DNA damage and mutations that lead to several diseases, including cancer. We extracted DNA from peripheral blood mononuclear cells (PBMCs) of 48 pancreatic adenocarcinoma cases and 48 healthy controls to determine relative levels of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) damage by QPCR. All participants were never smokers and between the ages of 60 and 69. Average levels among cases were compared to controls using a rank sum test, and logistic regression adjusted for potential confounding factors (age, sex, and diabetes mellitus). Cases had less DNA damage, with a significant decrease in mtDNA damage (P-value = 0.03) and a borderline significant decrease in nDNA damage (P = 0.08). Across samples, we found mtDNA abundance was higher among non-diabetics compared to diabetics (P = 0.04). Our results suggest that patients with pancreatic adenocarcinoma have less DNA damage in their PBMCs, and that having diabetes, a known pancreatic cancer risk factor, is associated with lower levels of mtDNA abundance.

Mitochondrial DNA damage is associated with reduced mitochondrial bioenergetics in Huntington's disease.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.

Age-related instability in spermatogenic cell nuclear and mitochondrial DNA obtained from Apex1 heterozygous mice.

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.

Requirement of the Saccharomyces cerevisiae APN1 gene for the repair of mitochondrial DNA alkylation damage.

The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1 Delta) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1 Delta strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1 Delta cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1 Delta mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage.

Mitochondrial DNA damage is a hallmark of chemically induced and the R6/2 transgenic model of Huntington's disease.

Many forms of neurodegeneration are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage, however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are primary events in the delayed onset observed in Huntington's disease (HD). We hypothesize that an age-dependent increase in mtDNA damage contributes to mitochondrial dysfunction in HD. Two HD mouse models were studied, the 3-nitropropionic acid (3-NPA) chemically induced model and the HD transgenic mice of the R6/2 strain containing 115-150 CAG repeats in the huntingtin gene. The mitochondrial toxin 3-NPA inhibits complex II of the electron transport system and causes neurodegeneration that resembles HD in the striatum of human and experimental animals. We measured nuclear and mtDNA damage by quantitative PCR (QPCR) in striatum of 5- and 24-month-old untreated and 3-NPA treated C57BL/6 mice. Aging caused an increase in damage in both nuclear and mitochondrial genomes. 3-NPA induced 4-6 more damage in mtDNA than nuclear DNA in 5-month-old mice, and this damage was repaired by 48h in the mtDNA. In 24-month-old mice 3NPA caused equal amounts of nuclear and mitochondrial damage and this damage persistent in both genomes for 48h. QPCR analysis showed a progressive increase in the levels of mtDNA damage in the striatum and cerebral cortex of 7-12-week-old R6/2 mice. Striatum exhibited eight-fold more damage to the mtDNA compared with a nuclear gene. These data suggest that mtDNA damage is an early biomarker for HD-associated neurodegeneration and supports the hypothesis that mtDNA lesions may contribute to the pathogenesis observed in HD.

Opposing effects of ubiquitin conjugation and SUMO modification of PCNA on replicational bypass of DNA lesions in Saccharomyces cerevisiae.

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases zeta (Polzeta) and Poleta and postreplicational repair mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Here we report our studies with a proliferating cell nuclear antigen (PCNA) mutation, pol30-119, which results from a change of the lysine 164 residue to arginine. It has been shown recently that following treatment of yeast cells with DNA-damaging agents, the lysine 164 residue of PCNA becomes monoubiquitinated in a Rad6-Rad18-dependent manner and that subsequently this PCNA residue is polyubiquitinated via a lysine 63-linked ubiquitin chain in an Mms2-Ubc13-, Rad5-dependent manner. PCNA is also modified by SUMO conjugation at the lysine 164 residue. Our genetic studies with the pol30-119 mutation show that in addition to conferring a defect in Polzeta-dependent UV mutagenesis and in Poleta-dependent TLS, this PCNA mutation inhibits postreplicational repair of discontinuities that form in the newly synthesized strand across from UV lesions. In addition, we provide evidence for the activation of the RAD52 recombinational pathway in the pol30-119 mutant and we infer that SUMO conjugation at the lysine 164 residue of PCNA has a role in suppressing the Rad52-dependent postreplicational repair pathway.

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.