Design, Synthesis, and Investigation of the Pharmacokinetics and Anticancer Activities of Indenoisoquinoline Derivatives That Stabilize the G-Quadruplex in the MYC Promoter and Inhibit Topoisomerase I.

G-quadruplexes are noncanonical four-stranded DNA secondary structures. MYC is a master oncogene and the G-quadruplex formed in the MYC promoter functions as a transcriptional silencer and can be stabilized by small molecules. We have previously revealed a novel mechanism of action for indenoisoquinoline anticancer drugs, dual-downregulation of MYC and inhibition of topoisomerase I. Herein, we report the design and synthesis of novel 7-aza-8,9-methylenedioxyindenoisoquinolines based on desirable substituents and π-π stacking interactions. These compounds stabilize the MYC promoter G-quadruplex, significantly lower MYC levels in cancer cells, and inhibit topoisomerase I. MYC targeting was demonstrated by differential activities in Raji vs CA-46 cells and cytotoxicity in MYC-dependent cell lines. Cytotoxicities in the NCI-60 panel of human cancer cell lines were investigated. Favorable pharmacokinetics were established, and in vivo anticancer activities were demonstrated in xenograft mouse models. Furthermore, favorable brain penetration, brain pharmacokinetics, and anticancer activity in an orthotopic glioblastoma mouse model were demonstrated.

APAP-Induced IκBß/NFκB Signaling Drives Hepatic Il6 Expression and Associated Sinusoidal Dilation.

Acetaminophen (APAP) overdose results in high morbidity and mortality, with limited treatment options. Increased understanding of the cellular signaling pathways activated in response to toxic APAP exposure is needed to provide insight into novel therapeutic strategies. Toxic APAP exposure induces hepatic nuclear factor kappa B (NFκB) activation. NFκB signaling has been identified to mediate the proinflammatory response but also induces a prosurvival and regenerative response. It is currently unknown whether potentiating NFkB activation would be injurious or advantageous after APAP overdose. The NFκB inhibitory protein beta (IκBß) dictates the duration and degree of the NFκB response following exposure to oxidative injuries. Thus, we sought to determine whether IκBß/NFκB signaling contributes to APAP-induced hepatic injury. At late time points (24 h) following toxic APAP exposures, mice expressing only IκBß knock-in mice (AKBI mice) exhibited increased serologic evidence of hepatic injury. This corresponded with increased histologic injury, specifically related to sinusoidal dilatation. When compared with wild type mice, AKBI mice demonstrated sustained hepatic nuclear translocation of the NFκB subunits p65 and p50, and enhanced NFκB target gene expression. This included increased expression of interleukin-6 (Il-6), a known contributor to hepatic sinusoidal dilation. This transcriptional response corresponded with increased plasma protein content of Il-6, as well as increased activation of signal transducer and activator of transcription 3.

The Acute Hepatic NF-κB-Mediated Proinflammatory Response to Endotoxemia Is Attenuated in Intrauterine Growth-Restricted Newborn Mice.

Intrauterine growth restriction (IUGR) is a relevant predictor for higher rates of neonatal sepsis worldwide and is associated with an impaired neonatal immunity and lower immune cell counts. During the perinatal period, the liver is a key immunological organ responsible for the nuclear factor kappa B (NF-κB)-mediated innate immune response to inflammatory stimuli, but whether this role is affected by IUGR is unknown. Herein, we hypothesized that the newborn liver adapts to calorie-restriction IUGR by inducing changes in the NF-κB signaling transcriptome, leading to an attenuated acute proinflammatory response to intraperitoneal lipopolysaccharide (LPS). We first assessed the hepatic gene expression of key NF-κB factors in the IUGR and normally grown (NG) newborn mice. Real-time quantitative PCR (RT-qPCR) analysis revealed an upregulation of both IκB proteins genes (Nfkbia and Nfkbib) and the NF-κB subunit Nfkb1 in IUGR vs. NG. We next measured the LPS-induced hepatic expression of acute proinflammatory genes (Ccl3, Cxcl1, Il1b, Il6, and Tnf) and observed that the IUGR liver produced an attenuated acute proinflammatory cytokine gene response (Il1b and Tnf) to LPS in IUGR vs. unexposed (CTR). Consistent with these results, LPS-exposed hepatic tumor necrosis factor alpha (TNF-α) protein concentrations were lower in IUGR vs. LPS-exposed NG and did not differ from IUGR CTR. Sex differences at the transcriptome level were observed in the IUGR male vs. female. Our results demonstrate that IUGR induces key modifications in the NF-κB transcriptomic machinery in the newborn that compromised the acute proinflammatory cytokine gene and protein response to LPS. Our results bring novel insights in understanding how the IUGR newborn is immunocompromised due to fundamental changes in NF-κB key factors.

Pulmonary implications of acetaminophen exposures independent of hepatic toxicity.

Both preclinical and clinical studies have demonstrated that exposures to acetaminophen (APAP) at levels that cause hepatic injury cause pulmonary injury as well. However, whether exposures that do not result in hepatic injury have acute pulmonary implications is unknown. Thus, we sought to determine how APAP exposures at levels that do not result in significant hepatic injury impact the mature lung. Adult male ICR mice (8-12 wk) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice [280 mg/kg, intraperitoneal (ip)], as well as a lower dose previously reported to not cause hepatic injury (140 mg/kg, ip). We confirm that the lower dose exposures did not result in significant hepatic injury. However, like high dose, lower exposure resulted in increased cellular content of the bronchoalveolar lavage fluid and induced a proinflammatory pulmonary transcriptome. Both the lower and higher dose exposures resulted in measurable changes in lung morphometrics, with the lower dose exposure causing alveolar wall thinning. Using RNAScope, we were able to detect dose-dependent, APAP-induced pulmonary Cyp2e1 expression. Finally, using FLIM we determined that both APAP exposures resulted in acute pulmonary metabolic changes consistent with mitochondrial overload in lower doses and a shift to glycolysis at a high dose. Our findings demonstrate that APAP exposures that do not cause significant hepatic injury result in acute inflammatory, morphometric, and metabolic changes in the mature lung. These previously unreported findings may help explain the potential relationship between APAP exposures and pulmonary-related morbidity.

The developing murine lung is susceptible to acetaminophen toxicity.

Acetaminophen (n-acetyl-p-aminophenol, APAP) use in the neonatal intensive care unit is rapidly increasing. Although APAP-related hepatotoxicity is rarely reported in the neonatal literature, other end-organ toxicity can occur with toxic exposures. APAP-induced lung injury has been reported with toxic exposures in adults, but whether this occurs in the developing lung is unknown. Therefore, we tested whether toxic APAP exposures would injure the developing lung. Neonatal C57BL/6 mice (PN7, early alveolar stage of lung development) were exposed to a dose of APAP known to cause hepatotoxicity in adult mice (280 mg/kg, IP). This exposure induced significant lung injury in the absence of identifiable hepatic toxicity. This injury was associated with increased pulmonary expression of Cyp2e1, the xenobiotic enzyme responsible for the toxic conversion of APAP. Exposure was associated with increased pulmonary expression of antioxidant response genes and decreased pulmonary glutathione peroxidase activity level. Furthermore, we observed an increase in pulmonary expression of proinflammatory cytokines and chemokines. Lastly, we were able to demonstrate that this toxic APAP exposure was associated with a shift in pulmonary metabolism away from glycolysis with increased oxidative phosphorylation, a finding consistent with increased mitochondrial workload, potentially leading to mitochondrial toxicity. This previously unrecognized injury and metabolic implications highlight the need to look beyond the liver and evaluate both the acute and long-term pulmonary implications of APAP exposure in the perinatal period.

Lower threshold to NFκB activity sensitizes murine ß-cells to streptozotocin.

The ß-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we used streptozotocin (STZ) to injure the ß-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8 mM, 0-24h) ±TNFα (100 ng/mL) and ±IκBß siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by the expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBß and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBß expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBß-/- and WT mice were exposed to STZ and monitored for diabetes. The IκBß-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBß levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent ß-cell injury and the development of diabetes.

Maturation of the Acute Hepatic TLR4/NF-κB Mediated Innate Immune Response Is p65 Dependent in Mice.

Compared to adults, neonates are at increased risk of infection. There is a growing recognition that dynamic qualitative and quantitative differences in immunity over development contribute to these observations. The liver plays a key role as an immunologic organ, but whether its contribution to the acute innate immune response changes over lifetime is unknown. We hypothesized that the liver would activate a developmentally-regulated acute innate immune response to intraperitoneal lipopolysaccharide (LPS). We first assessed the hepatic expression and activity of the NF-κB, a key regulator of the innate immune response, at different developmental ages (p0, p3, p7, p35, and adult). Ontogeny of the NF-κB subunits (p65/p50) revealed a reduction in Rela (p65) and Nfkb1 (p105, precursor to p50) gene expression (p0) and p65 subunit protein levels (p0 and p3) vs. older ages. The acute hepatic innate immune response to LPS was associated by the degradation of the NF-κB inhibitory proteins (IκBα and IκBß), and nuclear translocation of the NF-κB subunit p50 in all ages, whereas nuclear translocation of the NF-κB subunit p65 was only observed in the p35 and adult mouse. Consistent with these findings, we detected NF-κB subunit p65 nuclear staining exclusively in the LPS-exposed adult liver compared with p7 mouse. We next interrogated the LPS-induced hepatic expression of pro-inflammatory genes (Tnf, Icam1, Ccl3, and Traf1), and observed a gradually increase in gene expression starting from p0. Confirming our results, hepatic NF-κB subunit p65 nuclear translocation was associated with up-regulation of the Icam1 gene in the adult, and was not detected in the p7 mouse. Thus, an inflammatory challenge induces an NF-κB-mediated hepatic innate immune response activation across all developmental ages, but nuclear translocation of the NF-κB subunit p65 and associated induction of pro-inflammatory genes occurred only after the first month of life. Our results demonstrate that the LPS-induced hepatic innate immune response is developmentally regulated by the NF-κB subunit p65 in the mouse.

In utero inflammatory challenge induces an early activation of the hepatic innate immune response in late gestation fetal sheep.

Chorioamnionitis is associated with inflammatory end-organ damage in the fetus. Tissues in direct contact with amniotic fluid drive a pro-inflammatory response and contribute to this injury. However, due to a lack of direct contact with the amniotic fluid, the liver contribution to this response has not been fully characterized. Given its role as an immunologic organ, we hypothesized that the fetal liver would demonstrate an early innate immune response to an in utero inflammatory challenge. Fetal sheep (131 ± 1 d gestation) demonstrated metabolic acidosis and high cortisol and norepinephrine values within 5 h of exposure to intra-amniotic LPS. Likewise, expression of pro-inflammatory cytokines increased significantly at 1 and 5 h of exposure. This was associated with NF-κB activation, by inhibitory protein IκBα degradation, and nuclear translocation of NF-κB subunits (p65/p50). Corroborating these findings, LPS exposure significantly increased pro-inflammatory innate immune gene expression in fetal sheep hepatic macrophages in vitro. Thus, an in utero inflammatory challenge induces an early hepatic innate immune response with systemic metabolic and stress responses. Within the fetal liver, hepatic macrophages respond robustly to LPS exposure. Our results demonstrate that the fetal hepatic innate immune response must be considered when developing therapeutic approaches to attenuate end-organ injury associated with in utero inflammation.

CpG-ODN-mediated TLR9 innate immune signalling and calcium dyshomeostasis converge on the NFκB inhibitory protein IκBß to drive IL1α and IL1ß expression.

Sterile inflammation contributes to many pathological states associated with mitochondrial injury. Mitochondrial injury disrupts calcium homeostasis and results in the release of CpG-rich mitochondrial DNA. The role of CpG-stimulated TLR9 innate immune signalling and sterile inflammation is well studied; however, how calcium dyshomeostasis affects this signalling is unknown. Therefore, we interrogated the relationship beτween intracellular calcium and CpG-induced TLR9 signalling in murine macrophages. We found that CpG-ODN-induced NFκB-dependent IL1α and IL1ß expression was significantly attenuated by both calcium chelation and calcineurin inhibition, a finding mediated by inhibition of degradation of the NFκB inhibitory protein IκBß. In contrast, calcium ionophore exposure increased CpG-induced IκBß degradation and IL1α and IL1ß expression. These results demonstrate that through its effect on IκBß degradation, increased intracellular Ca2+ drives a pro-inflammatory TLR9-mediated innate immune response. These results have implications for the study of innate immune signalling downstream of mitochondrial stress and injury.

Toxic Acetaminophen Exposure Induces Distal Lung ER Stress, Proinflammatory Signaling, and Emphysematous Changes in the Adult Murine Lung.

Clinical studies have demonstrated a strong association between both acute toxic exposure and the repetitive, chronic exposure to acetaminophen (APAP) with pulmonary dysfunction. However, the mechanisms underlying this association are unknown. Preclinical reports have demonstrated that significant bronchiolar injury occurs with toxic APAP exposure, but very little information exists on how the distal lung is affected. However, cells in the alveolar space, including the pulmonary epithelium and resident macrophages, express the APAP-metabolizing enzyme CYP2E1 and are a potential source of toxic metabolites and subsequent distal lung injury. Thus, we hypothesized that distal lung injury would occur in a murine model of toxic APAP exposure. Following exposure of APAP (280 mg/kg, IP), adult male mice were found to have significant proximal lung histopathology as well as distal lung inflammation and emphysematous changes. Toxic APAP exposure was associated with increased CYP2E1 expression in the distal lung and accumulation of APAP-protein adducts. This injury was associated with distal lung activation of oxidant stress, endoplasmic reticulum stress, and inflammatory stress response pathways. Our findings confirm that following toxic APAP exposure, distal lung CYP2E1 expression is associated with APAP metabolism, tissue injury, and oxidant, inflammatory, and endoplasmic reticulum signaling. This previously unrecognized injury may help improve our understanding of the relationship between APAP and pulmonary-related morbidity.

Developmentally Regulated Innate Immune NFκB Signaling Mediates IL-1α Expression in the Perinatal Murine Lung.

Bronchopulmonary dysplasia (BPD) is the most common morbidity complicating premature birth. Importantly, preclinical models have demonstrated that IL-1 receptor antagonism prevents the lung injury and subsequent abnormal development that typically results following perinatal exposure to inflammatory stresses. This receptor is activated by two pro-inflammatory cytokines, IL-1α and IL-1ß. While many studies have linked IL-1ß to BPD development, IL-1α is relatively under-studied. The objective of our study was to determine whether systemic inflammatory stress induces IL-1α expression in the neonatal lung, and if so, whether this expression is mediated by innate immune NFκB signaling. We found that endotoxemia induced IL-1α expression during the saccular stage of neonatal lung development and was not present in the other neonatal organs or the adult lung. This IL-1α expression was dependent upon sustained pulmonary NFκB activation, which was specific to the neonatal lung. Using in vivo and in vitro approaches, we found that pharmacologic and genetic inhibition of NFκB signaling attenuated IL-1α expression. These findings demonstrate that innate immune regulation of IL-1α expression is developmentally regulated and occurs via an NFκB dependent mechanism. Importantly, the specific role of developmentally regulated pulmonary IL-1α expression remains unknown. Future studies must determine the effect of attenuating innate immune IL-1α expression in the developing lung before adopting broad IL-1 receptor antagonism as an approach to prevent neonatal lung injury.

Sex-differences in LPS-induced neonatal lung injury.

Being of the male sex has been identified as a risk factor for multiple morbidities associated with preterm birth, including bronchopulmonary dysplasia (BPD). Exposure to inflammatory stress is a well-recognized risk factor for developing BPD. Whether there is a sex difference in pulmonary innate immune TLR4 signaling, lung injury and subsequent abnormal lung development is unknown. Neonatal (P0) male and female mice (ICR) were exposed to systemic LPS (5 mg/kg, IP) and innate immune signaling, and the transcriptional response were assessed (1 and 5 hours), along with lung development (P7). Male and female mice demonstrated a similar degree of impaired lung development with decreased radial alveolar counts, increased surface area, increased airspace area and increased mean linear intercept. We found no differences between male and female mice in the baseline pulmonary expression of key components of TLR4-NFκB signaling, or in the LPS-induced pulmonary expression of key mediators of neonatal lung injury. Finally, we found no difference in the kinetics of LPS-induced pulmonary NFκB activation between male and female mice. Together, these data support the conclusion that the innate immune response to early postnatal LPS exposure and resulting pulmonary sequelae is similar in male and female mice.

The hepatic innate immune response is lobe-specific in a murine model endotoxemia.

The liver plays a central role in the innate immune response to endotoxemia. While previous studies have demonstrated lobe-specific transcriptional responses to various insults, whether this is true in response to endotoxemia is unknown. We sought to assess whether there were significant intra- and inter-lobe differences in the murine hepatic innate immune transcriptional response to endotoxemia. Adult male ICR mice were exposed to i.p. LPS (5 mg/kg, 30 min, 60 min, 5 h) and primary ( Tnf, Cxcl1, Nfkbia, Tnfiap3) and secondary ( Il6, Nos2) innate immune response gene expression was assessed in the left medial, right medial, left lateral, and right lateral lobes, and the papillary and caudate processes. The expression of all innate immune response genes increased following i.p. LPS challenge. When tested at the early time points (30 and 60 min), the left medial lobe and caudate process consistently demonstrated the highest induction of gene expression. Most inter-lobe differences were attenuated at later time points (5 h). To improve reproducibility of the study of endotoxemia induced by i.p. LPS challenge, inclusion of appropriate methodological details regarding collection of hepatic tissue should be included when reporting scientific results in published manuscripts.