Mechanistic Insight on the Mode of Action of Colletoic Acid.

The natural product colletoic acid (CA) is a selective inhibitor of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which primarily converts cortisone to the active glucocorticoid (GC) cortisol. Here, CA's mode of action and its potential as a chemical tool to study intracellular GC signaling in adipogenesis are disclosed. 11ß-HSD1 biochemical studies of CA indicated that its functional groups at C-1, C-4, and C-9 were important for enzymatic activity; an X-ray crystal structure of 11ß-HSD1 bound to CA at 2.6 Å resolution revealed the nature of those interactions, namely, a close-fitting and favorable interactions between the constrained CA spirocycle and the catalytic triad of 11ß-HSD1. Structure-activity relationship studies culminated in the development of a superior CA analogue with improved target engagement. Furthermore, we demonstrate that CA selectively inhibits preadipocyte differentiation through 11ß-HSD1 inhibition, suppressing other relevant key drivers of adipogenesis (i.e., PPARγ, PGC-1α), presumably by negatively modulating the glucocorticoid signaling pathway. The combined findings provide an in-depth evaluation of the mode of action of CA and its potential as a tool compound to study adipose tissue and its implications in metabolic syndrome.

Studies of Jatrogossone A as a Reactive Oxygen Species Inducer in Cancer Cellular Models.

Natural products continue to provide a platform to study biological systems. A bioguided study of cancer cell models led us to a new member of the jatrophane natural products from Jatropha gossypiifolia, which was independently identified and characterized as jatrogossone A (1). Purification and structure elucidation was performed by column chromatography and high-performance liquid chromatography-mass spectrometry and NMR techniques, and the structure was confirmed via X-ray crystallography. The unique molecular scaffold of jatrogossone A prompted an evaluation of its mode of action. Cytotoxicity assays demonstrated that jatrogossone A displays selective antiproliferative activity against cancer cell models in the low micromolar range with a therapeutic window. Jatrogossone A (1) affects mitochondrial membrane potential (ΔΨm) in a time- and dose-dependent manner. This natural product induces radical oxygen species (ROS) selectively in cancer cellular models, with minimal ROS induction in noncancerous cells. Compound 1 induces ROS in the mitochondria, as determined by colocalization studies, and it induces mitophagy. It promotes also in vitro cell death by causing cell arrest at the G2/M stage, caspase (3/7) activation, and PARP-1 cleavage. The combined findings provide a potential mechanism by which 1 relies on upregulation of mitochondrial ROS to potentiate cytotoxic effects through intracellular signaling.

Cytostatic and Cytotoxic Natural Products against Cancer Cell Models.

The increasing prevalence of drug resistant and/or high-risk cancers indicate further drug discovery research is required to improve patient outcome. This study outlines a simplified approach to identify lead compounds from natural products against several cancer cell lines, and provides the basis to better understand structure activity relationship of the natural product cephalotaxine. Using high-throughput screening, a natural product library containing fractions and pure compounds was interrogated for proliferation inhibition in acute lymphoblastic leukemia cellular models (SUP-B15 and KOPN-8). Initial hits were verified in control and counter screens, and those with EC50 values ranging from nanomolar to low micromolar were further characterized via mass spectrometry, NMR, and cytotoxicity measurements. Most of the active compounds were alkaloid natural products including cephalotaxine and homoharringtonine, which were validated as protein synthesis inhibitors with significant potency against several cancer cell lines. A generated BODIPY-cephalotaxine probe provides insight into the mode of action of cephalotaxine and further rationale for its weaker potency when compared to homoharringtonine. The steroidal natural products (ecdysone and muristerone A) also showed modest biological activity and protein synthesis inhibition. Altogether, these findings demonstrate that natural products continue to provide insight into structure and function of molecules with therapeutic potential against drug resistant cancer cell models.

Development of ergosterol peroxide probes for cellular localisation studies.

Ergosterol peroxide selectively exhibits biological activity against a wide range of diseases; however, its mode of action remains unknown. Here, we present an efficient synthesis of ergosterol peroxide chemical probes for in vitro anticancer evaluation, live cell studies and proteomic profiling. Ergosterol peroxide analogues show promising anti-proliferation activity against triple negative breast cancer cellular models, revealing information on the structure-activity relationship of this natural product in order to develop superior analogues. The combined cellular studies demonstrate that ergosterol peroxide is distributed across the cytosol with significant accumulation in the endoplasmic reticulum (ER). These chemical probes support our efforts towards uncovering the potential target(s) of ergosterol peroxide against triple negative breast cancer cell lines.

Identification of Biologically Active Ganoderma lucidum Compounds and Synthesis of Improved Derivatives That Confer Anti-cancer Activities in vitro.

We previously reported that Ganoderma lucidum extract (GLE) demonstrate significant anti-cancer activity against triple negative inflammatory breast cancer models. Herein, we aimed to elucidate the bioactive compounds of GLE responsible for this anti-cancer activity. We performed NMR, X-ray crystallography and analog derivatization as well as anti-cancer activity studies to elucidate and test the compounds. We report the structures of the seven most abundant GLE compounds and their selective efficacy against triple negative (TNBC) and inflammatory breast cancers (IBC) and other human cancer cell types (solid and blood malignancies) to illustrate their potential as anti-cancer agents. Three of the seven compounds (ergosterol, 5,6-dehydroergosterol and ergosterol peroxide) exhibited significant in vitro anti-cancer activities, while we report for the first time the structure elucidation of 5,6-dehydroergosterol from Ganoderma lucidum. We also show for the first time in TNBC/IBC cells that ergosterol peroxide (EP) displays anti-proliferative effects through G1 phase cell cycle arrest, apoptosis induction via caspase 3/7 activation, and PARP cleavage. EP decreased migratory and invasive effects of cancer cells while inhibiting the expression of total AKT1, AKT2, BCL-XL, Cyclin D1 and c-Myc in the tested IBC cells. Our investigation also indicates that these compounds induce reactive oxygen species, compromising cell fate. Furthermore, we generated a superior derivative, ergosterol peroxide sulfonamide, with improved potency in IBC cells and ample therapeutic index (TI > 10) compared to normal cells. The combined studies indicate that EP from Ganoderma lucidum extract is a promising molecular scaffold for further exploration as an anti-cancer agent.

In-bag enzymatic splenic digestion: a novel alternative to manual morcellation?

BACKGROUND: Contained in-bag spleen morcellation is a conventional extraction technique for safe spleen removal during laparoscopic splenectomy. Existing data for the use of in-bag enzymatic splenic digestion as an alternative to morcellation are lacking. This proof-of-concept study sought to evaluate the effectiveness of single and combinatorial enzyme digestion of murine spleens. MATERIALS AND METHODS: Murine spleens were digested with collagenase alone or with combinations of commercially available enzymes (collagenase, elastase, hyaluronidase, neutral protease) to determine their degradation effect. The primary end point was the percentage of mass reduction at 15 and 30 min. RESULTS: For collagenase alone (n = 15), the mean reduction in mass was 14 ± 10% (range: 2%-31%) at 15 min and 30 ± 25% (range: 7%-100%) at 30 min. Using combinatorial dissolution with collagenase, hyaluronidase, and elastase (n = 8), the mean reduction in mass was 27 ± 16% (range: 6%-42%) at 15 min and 48 ± 27% (range: 3%-100%) at 30 min. Injecting the enzyme solution into whole spleens (n = 9) yielded a mean reduction in mass of 22 ± 13% (range: 9%-42%) at 15 min and 55 ± 31% (range: 9%-100%) at 30 min; mean reduction was 9 ± 13% (range: 0%-39%) at 15 min and 23 ± 13% (range: 3%-53%) with no injection (n = 12). CONCLUSIONS: We provide the first demonstration of successful enzymatic murine spleen digestion as an alternative method for in-bag spleen removal during laparoscopic splenectomy. However, the significant cost and quantities of commercial enzyme required for clinical application dampens the enthusiasm for this novel approach.

Detection of PI3K inhibition in human neuroblastoma using multiplex luminex bead immunoassay: a targeted approach for pathway analysis.

Neuroblastoma (NB) is a common solid tumor in children. Outcomes for advanced stage NB have not improved, at least in part because of multimodality therapy resistance. Better comprehension of novel molecular targets will likely lead to improved therapies with specific cytotoxic agents. For instance, the role of deregulated IGF-1R/AKT/PI3K/mTOR (PI3K) pathway activity has attracted much attention across several tumors, including NB. Thus, modulating this pathway via anti-PI3K drugs has taken center stage in many cancer clinical trials. However, varied clinical effects have hampered the precise application of these agents. Tumor PI3K pathway profiling may reveal a method to enhance the efficacy of these inhibitors. To this end, solid-phase antibody-based array platforms have emerged as a direct, rapid means of profiling intracellular signaling pathways. We tested the efficacy of four PI3K inhibitors against a panel of human NB cell lines using Luminex xMAP bead array technology to establish PI3K phosphoprotein profiles. We demonstrate the utility of the xMAP approach in following intracellular signaling signatures specific for PI3K targeted therapy. Further validation is required before xMAP is used routinely for clinical PI3K pathway evaluation, but this method may eventually be personalized by taking into account each child's basal NB pathway status.

A PCNA-derived cell permeable peptide selectively inhibits neuroblastoma cell growth.

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.

Targeting oxidative stress in embryonal rhabdomyosarcoma.

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Dissecting the PI3K Signaling Axis in Pediatric Solid Tumors: Novel Targets for Clinical Integration.

Children with solid tumors represent a unique population. Recent improvements in pediatric solid tumor survival rates have been confined to low- and moderate-risk cancers, whereas minimal to no notable improvement in survival have been observed in high-risk and advanced-stage childhood tumors. Treatments for patients with advanced disease are rarely curative, and responses to therapy are often followed by relapse, which highlights the large unmet need for novel therapies. Recent advances in cancer treatment have focused on personalized therapy, whereby patients are treated with agents that best target the molecular drivers of their disease. Thus, a better understanding of the pathways that drive cancer or drug resistance is of critical importance. One such example is the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which is activated in many solid cancer patients and represents a target for therapy. PI3K/Akt/mTOR pathway activation has also been observed in tumors resistant to agents targeting upstream receptor tyrosine kinases (RTKs). Agents that target this pathway have the potential to shut down survival pathways, and are being explored both in the setting of pathway-activating mutations and for their ability to restore sensitivity to upstream signaling targeted agents. Here, we examine the role of the PI3K/Akt/mTOR pathway in pediatric solid tumors, review the novel agents being explored to target this pathway, and explore the potential role of the inhibition of this pathway in the clinical development of these agents in children.

Conformational trapping of mismatch recognition complex MSH2/MSH3 on repair-resistant DNA loops.

Insertion and deletion of small heteroduplex loops are common mutations in DNA, but why some loops are prone to mutation and others are efficiently repaired is unknown. Here we report that the mismatch recognition complex, MSH2/MSH3, discriminates between a repair-competent and a repair-resistant loop by sensing the conformational dynamics of their junctions. MSH2/MSH3 binds, bends, and dissociates from repair-competent loops to signal downstream repair. Repair-resistant Cytosine-Adenine-Guanine (CAG) loops adopt a unique DNA junction that traps nucleotide-bound MSH2/MSH3, and inhibits its dissociation from the DNA. We envision that junction dynamics is an active participant and a conformational regulator of repair signaling, and governs whether a loop is removed by MSH2/MSH3 or escapes to become a precursor for mutation.

Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex.

The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.