Cellular Depletion Research Articles

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed ‘metabolic efficiency’) when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.

DDX5 resolves R-loops at DNA double-strand breaks to promote DNA repair and avoid chromosomal deletions.

R-loops are three-stranded structures consisting of a DNA/RNA hybrid and a displaced DNA strand. The regulatory factors required to process this fundamental genetic structure near double-strand DNA breaks (DSBs) are not well understood. We previously reported that cellular depletion of the ATP-dependent DEAD box RNA helicase DDX5 increases R-loops genome-wide causing genomic instability. In this study, we define a pivotal role for DDX5 in clearing R-loops at or near DSBs enabling proper DNA repair to avoid aberrations such as chromosomal deletions. Remarkably, using the non-homologous end joining reporter gene (EJ5-GFP), we show that DDX5-deficient U2OS cells exhibited asymmetric end deletions on the side of the DSBs where there is overlap with a transcribed gene. Cross-linking and immunoprecipitation showed that DDX5 bound RNA transcripts near DSBs and required its helicase domain and the presence of DDX5 near DSBs was also shown by chromatin immunoprecipitation. DDX5 was excluded from DSBs in a transcription- and ATM activation-dependent manner. Using DNA/RNA immunoprecipitation, we show DDX5-deficient cells had increased R-loops near DSBs. Finally, DDX5 deficiency led to delayed exonuclease 1 and replication protein A recruitment to laser irradiation-induced DNA damage sites, resulting in homologous recombination repair defects. Our findings define a role for DDX5 in facilitating the clearance of RNA transcripts overlapping DSBs to ensure proper DNA repair.

Cardiac tissue citric acid cycle intermediates in exercised very long-chain acyl-CoA dehydrogenase-deficient mice fed triheptanoin or medium-chain triglyceride.

Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes. VLCAD-/- mice exhibited lower succinate in cardiac muscle at exhaustion than WT mice suggesting lower CACi in VLCAD-/- with prolonged exercise. In mice fed either MCT or triheptanoin, succinate and malate were greater in VLCAD-/- mice fed triheptanoin compared to VLCAD-/- animals fed MCT but lower than WT mice fed triheptanoin. Long-chain odd acylcarnitines such as C19 were elevated in VLCAD-/- and WT mice fed triheptanoin suggesting some elongation of the heptanoate, but it is unknown what proportion of heptanoate was oxidized vs elongated. Prolonged exercise was associated with decreased cardiac muscle succinate in VLCAD-/- mice in comparison to WT mice. VLCAD-/- fed triheptanoin had increased succinate compared to VLCAD-/- mice fed MCT but lower than WT mice fed triheptanoin. Cardiac CACi were higher following dietary ingestion of an anaplerotic substrate, triheptanoin, in comparison to MCT.

Reactive Metamizole Metabolites Enhance the Toxicity of Hemin on the ATP Pool in HL60 Cells by Inhibition of Glycolysis.

Metamizole is an analgesic, whose pharmacological and toxicological properties are attributed to N-methyl-aminoantipyrine (MAA), its major metabolite. In the presence of heme iron, MAA forms reactive metabolites, which are toxic for granulocyte precursors. Since decreased cellular ATP is characteristic for MAA-associated toxicity, we studied the effect of MAA with and without hemin on energy metabolism of HL60 cells, a granulocyte precursor cell line. The combination MAA/hemin depleted the cellular ATP stronger than hemin alone, whereas MAA alone was not toxic. This decrease in cellular ATP was observed before plasma membrane integrity impairment. MAA/hemin and hemin did not affect the proton leak but increased the maximal oxygen consumption by HL60 cells. This effect was reversed by addition of the radical scavenger N-acetylcysteine. The mitochondrial copy number was not affected by MAA/hemin or hemin. Hemin increased mitochondrial superoxide generation, which was not accentuated by MAA. MAA decreased cellular ROS accumulation in the presence of hemin. In cells cultured in galactose (favoring mitochondrial ATP generation), MAA/hemin had less effect on the cellular ATP and plasma membrane integrity than in glucose. MAA/hemin impaired glycolysis more than hemin or MAA alone, and N-acetylcysteine blunted this effect of MAA/hemin. MAA/hemin decreased protein expression of pyruvate kinase more than hemin or MAA alone. In conclusion, cellular ATP depletion appears to be an important mechanism of MAA/hemin toxicity on HL60 cells. MAA itself is not toxic on HL60 cells up to 100 µM but boosts the inhibitory effect of hemin on glycolysis through the formation of reactive metabolites.

Herpetosiphon Secondary Metabolites Inhibit Amyloid-β Toxicity in Human Primary Astrocytes.

The accumulation of extracellular plaques containing amyloid-β protein (Aβ) in the brain is one of the main pathological hallmarks of Alzheimer's disease (AD). Aβ peptide can promote the production of highly volatile free radicals and reactive oxygen species (ROS) that can induce oxidative damage to neurons and astrocytes. At present, numerous studies have investigated the neuroprotective and glioprotective effects of natural products derived from plants, animals, and microorganisms. We investigated the glioprotective effect of secondary metabolites obtained from Herpetosiphon sp. HM 1988 against Aβ40-induced toxicity in human primary astrocytes. The protective effect of bacterial secondary metabolites against Aβ40-induced inducible nitric oxide synthase (iNOS) activity was evaluated using the citrulline assay. To confirm the iNOS activity, nitrite production was assessed using the fluorometric Griess diazotization assay. Intracellular NAD+ depletion and lactate dehydrogenase (LDH) release in human primary astrocytes were also examined using well-established spectrophotometric assays. Our results indicate that Aβ40 can induce elevation in iNOS and LDH activities, nitrite production, and cellular energy depletion. Importantly, extract of Herpetosiphon sp. HM 1988 decreased iNOS activity, nitrite production, and LDH release. In addition, metabolites of the strain were able to restore cellular energy deficits through inhibition of NAD+ depletion mediated by Aβ40. These findings suggest that Herpetosiphon metabolites may represent a promising, novel source for the prevention of Aβ toxicity in AD.

Acireductone dioxygenase 1 (ADI1) is regulated by cellular iron by a mechanism involving the iron chaperone, PCBP1, with PCBP2 acting as a potential co-chaperone

The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.

Influence of the introduction of a triphenylphosphine group on the anticancer activity of a copper complex

Aiming at obtaining new copper complexes with good cytotoxicity against cancer cells, triphenylphosphine (TPP) was introduced to obtain insight into the influence of the co-ligands. In this paper, two copper complexes, Cu(2-pbmq)(CH3OH)Br2 (1) and [Cu(2-pbmq)(TPP)Br]2 (2) were designed, synthesized, and characterized by X-ray crystallography, 2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl))quinolone (2-pbmq), to investigate the influence of the TPP group on the anticancer activity of the metal complex. Although the presence of the TPP group diminished the intensity of the interaction properties of the complex with DNA, the in vitro anticancer activity and cellular uptake of the TPP-containing complex were markedly superior to those of its TPP-lacking counterpart. Detailed studies on the more potently cytotoxic complex 2 revealed that it accumulated in nucleus, arrested the cell cycle at the G0-G1 phase, causing mitochondrial dysfunction, involving the potential simultaneous mitochondrial membrane collapse, cellular ATP level depletion, and Ca2+ leakage, eventually inducing cell apoptosis. In summary, the introduction of a TPP group enhances the biological activity and cytotoxicity of the complex.

Cigarette Smoke Induces Intestinal Inflammation via a Th17 Cell-Neutrophil Axis

Abstract Epidemiological evidence finds cigarette smoking is a common risk factor for a number of diseases, not only in the lung but also in other tissues, such as the gastrointestinal tract. While it is well-documented that smoking directly drives lung inflammatory disease, how it promotes disease in peripheral tissues is incompletely understood. In this study, we utilized a mouse model of short-term smoke exposure and found increased Th17 cells and neutrophilia in the lung as well as in the circulation. Following intestinal inflammatory challenge, smoke exposed mice showed increased pathology which corresponds to enhanced intestinal Th17 cells, ILC3 and neutrophils within intestinal tissue. Using cellular depletion and genetic deficiencies, we define a cellular loop by which IL-17A and downstream neutrophils drive cigarette smoke-enhanced intestinal inflammation. Collectively, cigarette smoke induced local lung Th17 responses lead to increased systemic susceptibility to inflammatory insult through enhanced circulating neutrophils. These data demonstrate a cellular pathway by which inflammatory challenge in the lung can sensitize the intestine to enhanced pathological innate and adaptive immune responses

Tumor cell-intrinsic PD-L1 signals promote DNA damage responses that mediate resistance to Chk1 and PARP inhibitors in vivo

Abstract Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (>15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.

Glucose availability but not changes in pancreatic hormones sensitizes hepatic AMPK activity during nutritional transition in rodents

The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to nutritional variations to maintain a proper energy balance in cells. We show here that suckling-weaning and fasting-refeeding transitions in rodents are associated with changes in AMPK activation and the cellular energy state in the liver. These nutritional transitions were characterized by a metabolic switch from lipid to glucose utilization, orchestrated by modifications in glucose levels and the glucagon/insulin ratio in the bloodstream. We therefore investigated the respective roles of glucose and pancreatic hormones on AMPK activation in mouse primary hepatocytes. We found that glucose starvation transiently activates AMPK, whereas changes in glucagon and insulin levels had no impact on AMPK. Challenge of hepatocytes with metformin-induced metabolic stress strengthened both AMPK activation and cellular energy depletion under limited-glucose conditions, whereas neither glucagon nor insulin altered AMPK activation. Although both insulin and glucagon induced AMPKα phosphorylation at its Ser485/491 residue, they did not affect its activity. Finally, the decrease in cellular ATP levels in response to an energy stress was additionally exacerbated under fasting conditions and by AMPK deficiency in hepatocytes, revealing metabolic inflexibility and emphasizing the importance of AMPK for maintaining hepatic energy charge. Our results suggest that nutritional changes (i.e. glucose availability), rather than the related hormonal changes (i.e. the glucagon/insulin ratio), sensitize AMPK activation to the energetic stress induced by the dietary transition during fasting. This effect is critical for preserving the cellular energy state in the liver.

Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called “legal highs”. The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

STAT1 upregulates glutaminase and modulates amino acids and glutathione metabolism

We previously reported the upregulation of cellular Glu and glutathione levels in human ABCB5- and murine Abcb5-transfected cells. Here, we demonstrate the upregulation of STAT1 and glutaminase (GLS) in ABCB5/Abcb5-transfected cells. Among a total of four ABCB5/Abcb5 high-expressing clones with docetaxel resistance, three of the clones expressed STAT1 and GLS highly and showed resistance to docetaxel and buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. Neither STAT1 nor GLS upregulation was observed in the remaining ABCB5 high-expressing clone, as well as in another two ABCB5 low-expressing clones; these three clones did not show BSO resistance. The ABCB5/STAT1 high-expressing clones showed higher cellular levels of Ala, Glu, and Asp and lower cellular levels of Phe, Trp, Leu, Ile, Gly, Met, Tyr, Val, and His compared to the ABCB5/STAT1 low-expressing clones. The former clones also showed a higher resistance to Glu. The STAT1-transfected clones expressed high levels of GLS and the corresponding mRNA, suggesting the transactivation of GLS by STAT1. These clones showed resistance to Glu and BSO, similar to the ABCB5/STAT1 high-expressing clones. The cellular glutathione levels of the STAT1-transfected clones were significantly higher than that of the control. The STAT1-transfected clones also showed greater resistance to the effect of BSO on the cellular glutathione depletion compared to the control. These results demonstrate that STAT1 upregulates GLS and modulates amino acids and glutathione metabolism. Although we were unable to directly prove STAT1 upregulation by ABCB5, our results suggest that ABCB5 expression, directly or indirectly, leads to the overexpression of STAT1.

Hepatoprotective role of berberine against paraquat-induced liver toxicity in rat.

Paraquat (PQ) is a herbicide agent commonly used in agricultural applications. Hepatotoxicity is among clinical complications associated with PQ intoxication. Oxidative stress and its subsequent events are major mechanisms identified in PQ-induced liver toxicity. Berberine (BBR) is a natural antioxidant widely investigated for its hepatoprotective effects. The present study designed to evaluate the potential cytoprotective properties of BBR against PQ-induced cytotoxicity in primary cultured rat hepatocytes and in vivo test of liver function enzymes. Cellular and biochemical parameters including lactate dehydrogenase (LDH), cell viability, ROS formation, glutathione (GSH) content, and mitochondrial membrane potential in the PQ-treated hepatocytes were measured, and the mentioned markers were evaluated in the presence of BBR. BBR treatment caused significant decrease in PQ-induced cell death, ROS formation, and LDH release. On the other hand, it was found that BBR inhibits cellular glutathione depletion in PQ-treated hepatocytes. Also, BBR treatment significantly diminished PQ-induced the liver function enzyme elevation. These data mention the potential hepatoprotective effect of BBR with therapeutic capability against PQ-induced liver damage.

The Xc- inhibitor sulfasalazine improves the anti-cancer effect of pharmacological vitamin C in prostate cancer cells via a glutathione-dependent mechanism.

Traditional treatment regimens for advanced prostate cancer, especially castration-resistant prostate cancer, result in low survival times with severe side effects. Therefore, new treatment options are required. Vitamin C (VC) has been identified as a promising anti-cancer agent of which the effects depend on the accumulation of H2O2 that is produced through autoxidation. Sulfasalazine (SAS), a cystine transporter (Xc-) inhibitor, is known to suppress cellular glutathione (GSH) biosynthesis. Here, we hypothesized that targeting the Xc- transporter via SAS may improve the anti-cancer activity of VC through regulating GSH biosynthesis, which in turn may result in the accumulation of reactive oxygen species (ROS). The anti-cancer effect of VC and/or SAS on prostate cancer cells was assessed using WST-8, colony formation and annexin V-FITC/PI FACS assays. Changes in cellular ROS and GSH levels were determined to verify our hypothesis. Finally, BALB/c nude mice bearing prostate cancer xenografts were used to assess the anti-cancer effects of single or combined VC and SAS therapies. We found that SAS could potentiate the short- and long-term cytotoxicity of VC in prostate cancer cells. We also found that the synergistic effect of SAS and VC led to significant cellular GSH depletion, resulting in increased ROS accumulation. This synergistic effect could be reversed by the antioxidant N-acetyl-L-cysteine (NAC). The synergistic effect of SAS and VC was also noted in prostate cancer xenografts and correlated with immunohistochemistry results. Our results strongly indicate that SAS, a relatively non-toxic drug that targets cystine transporters, in combination with VC may be superior to their single applications in the treatment of prostate cancer.

Biomimetic Magnetic Nanostructures: A Theranostic Platform Targeting Lipid Metabolism and Immune Response in Lymphoma.

B-cell lymphoma cells depend upon cholesterol to maintain pro-proliferation and pro-survival signaling via the B-cell receptor. Targeted cholesterol depletion of lymphoma cells is an attractive therapeutic strategy. We report here high-density lipoprotein mimicking magnetic nanostructures (HDL-MNSs) that can bind to the high-affinity HDL receptor, scavenger receptor type B1 (SR-B1), and interfere with cholesterol flux mechanisms in SR-B1 receptor positive lymphoma cells, causing cellular cholesterol depletion. In addition, the MNS core can be utilized for its ability to generate heat under an external radio frequency field. The thermal activation of MNS can lead to both innate and adaptive antitumor immune responses by inducing the expression of heat shock proteins that lead to activation of antigen presenting cells and finally lymphocyte trafficking. In the present study, we demonstrate SR-B1 receptor mediated binding and cellular uptake of HDL-MNS and prevention of phagolysosome formation by transmission electron microscopy, fluorescence microscopy, and ICP-MS analysis. The combinational therapeutics of cholesterol depletion and thermal activation significantly improves therapeutic efficacy in SR-B1 expressing lymphoma cells. HDL-MNS reduces the T2 relaxation time under magnetic resonance imaging (MRI) more effectively compared with a commercially available contrast agent, and the specificity of HDL-MNS toward the SR-B1 receptor leads to differential contrast between SR-B1 positive and negative cells suggesting its utility in diagnostic imaging. Overall, we have demonstrated that HDL-MNSs have cell specific targeting efficiency, can modulate cholesterol efflux, can induce thermal activation mediated antitumor immune response, and possess high contrast under MRI, making it a promising theranostic platform in lymphoma.

Design, Synthesis, and Anticancer Effect Studies of Iridium(III) Polypyridyl Complexes against SGC-7901 Cells.

Three iridium(III) complexes ([Ir(Hppy)2(L)](PF6) (Hppy = 2-phenylpyridine, L = 5-nitrophenanthroline, NP), 1; 5-nitro-6-amino-phenanthroline (NAP), 2; and 5,6-diamino-phenanthroline (DAP) 3 were synthesized and characterized. The cytotoxicities of Ir(III) complexes 1–3 against cancer cell lines SGC-7901, A549, HeLa, Eca-109, HepG2, BEL-7402, and normal NIH 3T3 cells were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) method. The results showed that the three iridium(III) complexes had moderate in vitro anti-tumor activity toward SGC-7901 cells with IC50 values of 3.6 ± 0.1 µM for 1, 14.1 ± 0.5 µM for 2, and 11.1 ± 1.3 µM for 3. Further studies showed that 1–3 induce cell apoptosis/death through DNA damage, cell cycle arrest at the S or G0/G1 phase, ROS elevation, increased levels of Ca2+, high mitochondrial membrane depolarization, and cellular ATP depletion. Transwell and Colony-Forming assays revealed that complexes 1–3 can also effectively inhibit the metastasis and proliferation of tumor cells. These results demonstrate that 1–3 induce apoptosis in SGC-7901 cells through ROS-mediated mitochondrial damage and DNA damage pathways, as well as by inhibiting cell invasion, thereby exerting anti-tumor cell proliferation activity in vitro.

Interleukin-1β inhibits estrogen receptor-α, progesterone receptors A and B and biomarkers of human endometrial stromal cell differentiation: implications for endometriosis

Human blastocyst nidation in the uterus and successful pregnancy require coordinated endometrial expression of estrogen receptor (ER)-α, progesterone receptors (PR)-A and -B and the gap junction protein, connexin (Cx)43. Our prior work established that inflammation associated with conditions of reduced fecundity, particularly endometriosis, can perturb eutopic decidual function. In the current studies, we have modeled endometrial decidualization in primary human endometrial stromal cell cultures derived from normal controls (NESC) and from the eutopic endometria of women with endometriosis (EESC) to test the hypothesis that a proinflammatory cytokine, interleukin (IL)-1β, can disrupt stromal cell differentiation. The cells were grown under a standard protocol with hormones (10nM 17β-estradiol, 100nM progesterone and 0.5mM dibutyryl cAMP) for up to 7days in the absence or presence of IL-1β. Time-course experiments showed that IL-1β compromised decidual function in both NESC and EESC, which was accompanied by rapid phosphorylation of ER-α, PR and Cx43 and their cellular depletion. Inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway by a selective pharmacological blocker (PD98059) or siRNA interference, or the addition of hormones themselves, blocked the phosphorylation of ERK mediators; increased the production of steroid receptors, Cx43, prolactin, insulin-like growth factor binding protein-1 (IGFBP)-1 and vascular endothelial growth factor (VEGF) and accelerated the differentiation. The results indicate that inhibition of IL-1β can enhance decidualization in NESC and EESC in vitro. Strategies to interfere with this pathway might be implemented as an in vivo approach to enhance fertility in women with endometriosis and, potentially, other inflammatory pathologies.

Salivary glands require Aurora Kinase B\xa0for regeneration after transient innate immune-mediated injury

Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren’s syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

Abstract 1902: The comparative superiority of a bispecific antibody targeting alpha v and alpha 5 integrins is uniquely characterized by induced degradation of integrins

Abstract Background: Bispecific antibody blockade of alpha v and alpha 5 integrins (BsAbαvα5) resulted in superior anti-tumor activity in prostate cancer cells over monospecific αv (MsAbαv) and α5 blockade (MsAbα5), singly or in combination. We sought to define a molecular basis for this observation. Methods: We examined the comparative impact of monospecific and bispecific antibody strategies on membrane-specific and cellular expression of integrins as well as their transcription, translation and degradation fate in integrin av/a5+ prostate cancer cells (VCAP, C4-2b, DU-145 and PC-3). These dynamic changes were assessed in the context of functional studies including tumor-stromal interactions and downstream signaling. Results: With MsAbαv or MsAbα5 therapy, alone or in combination, upregulation of target integrin and/or the alternative integrin was observed which suggested both autoregulation as well as a cross-regulation among the target integrins. However, with BsAbαvα5 therapy, either a block in upregulation of target integrin/s or a marked decrease in cellular integrin expression was noted. Unexpectedly, these dynamic changes in cellular integrin expression with the BsAbαvα5 were retained up to 72 hours after antibody exposure with recovery of integrin expression in viable cells noted by 7 days. In cells recovered at 48-72 hours, a marked decrease in clonogenicity, adhesion and migration was observed. Flow cytometry indicated persistent membrane loss of integrin expression at 48-72 hours confirming that the impact of the BsAbαvα5 is not related to altered subcellular integrin trafficking alone. QRT-PCR studies did not identify upregulation of integrin transcription to account for upregulated integrin expression. Blockade of protein translation with cycloheximide failed to abrogate the differential impact of MsAbs or BsAbαvα5 on integrin expression. However, lysosomal inhibition with chloroquine blocked the downregulation of integrin expression with BsAbαvα5 therapy. Variable effects on downstream signaling in the Akt and MAPK pathway observed across cell types contrasted with consistent functional impairment of tumor-stromal interactions. Conclusions: BsAbαvα5 therapy uniquely induces lysosomal degradation of target integrins compared to MsAbs, alone or in combination. Cellular depletion of integrin expression serves as a pharmacodynamic marker of BsAbαvα5activity. By contrast, upregulated cellular expression secondary to MsAb therapy is likely secondary to a block in both integrin degradation and membrane recycling within the endosomal/lysosomal pathway. These observations provide additional insight into the mechanistic impact of the BsAbαvα5 as well as a pharmacodynamic method for monitoring in vivo activity. Citation Format: Raghav Joshi, Wenying Ren, Paul Mathew. The comparative superiority of a bispecific antibody targeting alpha v and alpha 5 integrins is uniquely characterized by induced degradation of integrins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1902.

Depletion of the cellular cholesterol content reduces the dynamics of desmosomal cadherins and interferes with desmosomal strength.

Desmosomal cadherins, desmocollins, and desmogleins are cholesterol-dependent entities responsible for the stable adhesion of desmosomes in epithelial cells. Here, we investigated the influence of cellular cholesterol depletion on the dynamic properties of the desmosomal cadherin desmocollin, particularly the lateral mobility and distribution of desmocollin 2 (Dsc2-YFP) in the plasma membrane, and how these properties influence the adhesion strength of desmosomes. Depletion of cellular cholesterol decreased the lateral mobility of Dsc2-YFP and caused dispersion of Dsc2-YFP in the plasma membrane of epithelial MDCK cells. As a consequence of the altered Dsc2-YFP dynamics, the adhesive strength of desmosomes was weakened. Moreover, our study is the first to show and quantify the co-association of desmosomes with cholesterol/sphingomyelin-enriched membrane domains at the ultrastructural level. Taken together, our data emphasize a critical role for the cellular cholesterol content in regulating the lateral mobility and distribution of Dsc2 and show that cholesterol depletion reduces the strength of desmosomal adhesions.