Targeting nuclear NAD+ synthesis inhibits DNA repair, impairs metabolic adaptation increases chemosensitivity of U-2OS osteosarcoma cells

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Targeting Nuclear NAD+ Synthesis Inhibits DNA Repair, Impairs Metabolic Adaptation and Increases Chemosensitivity of U-2OS Osteosarcoma Cells

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1-/- cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and the necroptosis inhibitor necrostatin-1. Activation of the DNA damage sensor enzyme poly(ADP-ribose) polymerase 1 (PARP1), a major consumer of NAD+ in the nucleus, was fully blocked by NMNAT1 inactivation, leading to increased DNA damage (phospho-H2AX foci). The PARP inhibitor, olaparib, sensitized wild type but not NMNAT1-/- cells to cisplatin-induced anti-clonogenic effects, suggesting that impaired PARP1 activity is important for chemosensitization. Cisplatin-induced cell death of NMNAT1-/- cells was also characterized by a marked drop in cellular ATP levels and impaired mitochondrial respiratory reserve capacity, highlighting the central role of compromised cellular bioenergetics in chemosensitization byNMNAT1 inactivation. Moreover, NMNAT1 cells also displayed markedly higher sensitivity to cisplatin when grown as spheroids in 3D culture. In summary, our work provides the first evidence that NMNAT1 is a promising therapeutic target for osteosarcoma and possibly other tumors as well

OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2′-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1β, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways

Poly(ADP-Ribose) Polymerase 1 Promotes Inflammation and Fibrosis in a Mouse Model of Chronic Pancreatitis

Chronic pancreatitis (CP) is an inflammatory disease of the pancreas characterized by ductal obstructions, tissue fibrosis, atrophy and exocrine and endocrine pancreatic insufficiency. However, our understanding is very limited concerning the disease’s progression from a single acute inflammation, via recurrent acute pancreatitis (AP) and early CP, to the late stage CP. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor enzyme activated mostly by oxidative DNA damage. As a co-activator of inflammatory transcription factors, PARP1 is a central mediator of the inflammatory response and it has also been implicated in acute pancreatitis. Here, we set out to investigate whether PARP1 contributed to the pathogenesis of CP. We found that the clinically used PARP inhibitor olaparib (OLA) had protective effects in a murine model of CP induced by multiple cerulein injections. OLA reduced pancreas atrophy and expression of the inflammatory mediators TNFα and interleukin-6 (IL-6), both in the pancreas and in the lungs. Moreover, there was significantly less fibrosis (Masson’s trichrome staining) in the pancreatic sections of OLA-treated mice compared to the cerulein-only group. mRNA expression of the fibrosis markers TGFβ, smooth muscle actin (SMA), and collagen-1 were markedly reduced by OLA. CP was also induced in PARP1 knockout (KO) mice and their wild-type (WT) counterparts. Inflammation and fibrosis markers showed lower expression in the KO compared to the WT mice. Moreover, reduced granulocyte infiltration (tissue myeloperoxidase activity) and a lower elevation of serum amylase and lipase activity could also be detected in the KO mice. Furthermore, primary acinar cells isolated from KO mice were also protected from cerulein-induced toxicity compared to WT cells. In summary, our data suggest that PARP inhibitors may be promising candidates for repurposing to treat not only acute but chronic pancreatitis as well

Potential effects of ursodeoxycholic acid on accelerating cutaneous wound healing.

Among the initial responses to skin injury, triggering inflammatory mediators and modifying oxidative status provide the necessary temple for the subsequent output of a new functional barrier, fibroplasia and collagen deposition, modulated by NF-κB and TGF-β1 expressions. Hence, the current study aimed to investigate the effect of local application of ursodeoxycholic acid (UDCA) on cutaneous wound healing induced in Swiss mice. Wound contraction progression was monitored by daily photographing the wounds. Enhanced fibroblast cell migration was observed after incubation with UDCA. Topical application of UDCA (500 μM) cream on excised wounds significantly enhanced wound contraction and improved morphometric scores. In addition, UDCA ameliorated the unbalanced oxidative status of granulated skin tissues. Interestingly, it showed increased expression of TGF-β1 and MMP-2 with decreased expression of NF-κB. On the other hand, UDCA significantly increased collagen fibers deposition and hydroxyproline content and enhanced re-epithelization. UDCA also modified the mitochondrial function throughout the healing process, marked by lower consumption rates of mitochondrial ATP, complex I contents as well as intracellular NAD+ contents accompanied by elevated levels of nicotinamide compared to the untreated controls. In chronic gamma-irradiated (6Gy) model, the illustrated data showed enhanced wound contraction via increased TGF-β1/MMP-2 and collagen deposition incurred by topical application of UDCA without effect on NF-κB level. In sum, the present findings suggest that UDCA may accelerate wound healing by regulating TGF-β1 and MMP-2 and fibroplasia/collagen deposition in either the two wound healing models

L-thyroxine modifies nephrotoxicity by regulating the apoptotic pathway: The possible role of CD38/ADP-ribosyl cyclase-mediated calcium mobilization - Fig 3

<p>(A-D) Kidneys of CIS (15mg/kg) administrated mouse showing (A) vacuolar degeneration (arrow) and necrosis of the renal tubular epithelial linings, hyaline droplets (dashed arrow) and hyaline cast (arrow head). (B) Many apoptotic cells and bodies (arrow) with presence of granular cast (dashed arrow) in the lumen of renal tubules. (C) Wide spread hyaline cast (H) in the lumen of the medullary tubules. (D) Glomerular congestion, hyperplasia of the podocytes and shrinkage of the glomerular tuft (arrow), notice the wide spread of hyaline droplets (upper left) in many tubules. (E-F) Kidney of IRR (6 Gy) mouse showing marked swelling, granular and vacuolar (upper left) degeneration of the renal tubular epithelial linings with many necrotic cells and granular cast (arrow) in the lumen of some tubules. (G) Kidney of CIS and L-TH pretreated mouse showing mild degeneration of the renal tubular epithelium with scattered necrotic cells and small granular cast in the lumen of few tubules (arrow). (H) Kidney of IRR and L-TH pretreated mouse showing good restoration of renal tissue with only mild tubular epithelial degenerative changes. (H&E, X400).</p

Effect of L-TH on serum uric acid (A), creatinine (B) and urea nitrogen (C) levels as well as biochemical analysis of oxidative status; H₂O₂ content (D) and catalase activity (E) in kidney tissues of all experimental groups.

<p>Acute renal injury was induced in male Swiss mice by single <i>i</i>.<i>p</i>. injection of cisplatin (15mg/kg) or gamma irradiation (6 Gy). Animals were pretreated with L-TH (1μg/kg) four hours before induction of nephrotoxicity. L-TH- and vehicle-treated mice were served as normal controls. Values are expressed as mean ± SEM (n = 6–8). ^<i>#</i><i>P</i>< 0.05: significantly different versus vehicle treated control; *<i>P</i>< 0.05, **<i>P</i>< 0.01: significantly different versus untreated control. CIS, cisplatin; H₂O₂, hydrogen peroxide; IRR, gamma irradiated; L-TH, L-thyroxine.</p

L-TH protects against CIS and IRR-induced apoptosis.

<p>Immunohistochemistry for caspase-3 and Bax showing positive expression among the renal tubular epithelium in both models of nephrotoxicity. Very mild to negative expressions of caspase-3 and Bax among the tubular epithelial linings in kidneys of pre-administered L-TH/cisplatin or L-TH/gamma irradiated mice (X 400).</p