Global SUMOylation in mouse oocytes maintains oocyte identity and regulates chromatin remodeling and transcriptional silencing at the end of folliculogenesis.

Meiotically competent oocytes in mammals undergo cyclic development during folliculogenesis. Oocytes within ovarian follicles are transcriptionally active, producing and storing transcripts required for oocyte growth, somatic cell communication and early embryogenesis. Transcription ceases as oocytes transition from growth to maturation and does not resume until zygotic genome activation. Although SUMOylation, a post-translational modification, plays multifaceted roles in transcriptional regulation, its involvement during oocyte development remains poorly understood. In this study, we generated an oocyte-specific knockout of Ube2i, encoding the SUMO E2 enzyme UBE2I, using Zp3-cre+ to determine how loss of oocyte SUMOylation during folliculogenesis affects oocyte development. Ube2i Zp3-cre+ female knockout mice were sterile, with oocyte defects in meiotic competence, spindle architecture and chromosome alignment, and a premature arrest in metaphase I. Additionally, fully grown Ube2i Zp3-cre+ oocytes exhibited sustained transcriptional activity but downregulated maternal effect genes and prematurely activated genes and retrotransposons typically associated with zygotic genome activation. These findings demonstrate that UBE2I is required for the acquisition of key hallmarks of oocyte development during folliculogenesis, and highlight UBE2I as a previously unreported orchestrator of transcriptional regulation in mouse oocytes.

Gene expression signatures associated with suppression of TRAMP prostate carcinogenesis by a kavalactone-rich Kava fraction.

Kava (Piper methysticum Forster) extract and its major kavalactones have been shown to block chemically induced lung tumor initiation in mouse models. Here we evaluated the chemopreventive effect of a kavalactone-rich Kava fraction B (KFB), free of flavokavains, on carcinogenesis in a transgenic adenocarcinoma of mouse prostate (TRAMP) model and characterized the prostate gene expression signatures. Male C57BL/6 TRAMP mice were fed AIN93M diet with or without 0.4% KFB from 8 wk of age. Mice were euthanized at 16 or 28 wk. The growth of the dorsolateral prostate (DLP) lobes in KFB-treated TRAMP mice was inhibited by 66% and 58% at the respective endpoint. Anterior and ventral prostate lobes in KFB-treated TRAMP mice were suppressed by 40% and 49% at 28 wk, respectively. KFB consumption decreased cell proliferation biomarker Ki-67 and epithelial lesion severity in TRAMP DLP, without detectable apoptosis enhancement. Real time qRT-PCR detection of mRNA from DLP at 28 wk showed decreased expression of cell cycle regulatory genes congruent with Ki-67 suppression. Microarray profiling of DLP mRNA indicated that "oncogene-like" genes related to angiogenesis and cell proliferation were suppressed by KFB but tumor suppressor, immunity, muscle/neuro, and metabolism-related genes were upregulated by KFB in both TRAMP and WT DLP. TRAMP mice fed KFB diet developed lower incidence of neuroendocrine carcinomas (NECa) (2 out of 14 mice) than those fed the basal diet (8 out of 14 mice, χ2 = 5.6, P < 0.025). KFB may, therefore, inhibit not only TRAMP DLP epithelial lesions involving multiple molecular pathways, but also NECa. © 2016 Wiley Periodicals, Inc.

Characterization of the Fluorescence Properties of 4-Dialkylaminochalcones and Investigation of the Cytotoxic Mechanism of Chalcones.

Understanding the mechanisms responsible for the various biological activities of chalcones, particularly the direct cellular targets, presents an unmet challenge. Here, we prepared a series of fluorescent chalcone derivatives as chemical probes for their mechanistic investigation. Upon systematic physicochemical characterization, we explored their potential to elucidate the mode of action of chalcones' cytotoxicity. The fluorescence of the chalcones was found to be highly sensitive to structural and environmental factors. Structurally, a 4-dialkylamino group on the B ring, suitable electronic properties of the A ring substituents, and the planar conformation of the chalcone's core structure were essential for optimal fluorescence. Environmental factors influencing fluorescence included solvent polarity, pH, and the interactions of the chalcones with proteins and detergents. It was found that 18 chalcones showed a fluorescent brightness greater than 6000 M(-1) cm(-1) in DMSO. However, water dramatically quenched the fluorescence, although it could be partially recovered in the presence of BSA or detergents. As expected, these fluorescent chalcones showed a sharp structure-activity relationship in their cellular cytotoxicity, leading to the identification of structurally similar cytotoxic and non-cytotoxic fluorescent chalcones as chemical probes. Confocal microscopy results revealed the co-localization of the cytotoxic probe C8 and tubulin in cells, supporting tubulin as the direct cellular target responsible for the cytotoxicity of chalcones.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis.

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor ß (TGFß) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFß signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFß family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings reveal dysfunction of BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

Impaired bone morphogenetic protein signaling pathways disrupt decidualization in endometriosis.

It is hypothesized that impaired endometrial decidualization contributes to decreased fertility in individuals with endometriosis. To identify the molecular defects that underpin defective decidualization in endometriosis, we subjected endometrial stromal cells from individuals with or without endometriosis to time course in vitro decidualization with estradiol, progesterone, and 8-bromo-cyclic-AMP (EPC) for 2, 4, 6, or 8 days. Transcriptomic profiling identified differences in key pathways between the two groups, including defective bone morphogenetic protein (BMP)/SMAD4 signaling (ID2, ID3, FST), oxidate stress response (NFE2L2, ALOX15, SLC40A1), and retinoic acid signaling pathways (RARRES, RARB, ALDH1B1). Genome-wide binding analyses identified an altered genomic distribution of SMAD4 and H3K27Ac in the decidualized stromal cells from individuals without endometriosis relative to those with endometriosis, with target genes enriched in pathways related to signaling by transforming growth factor ß (TGFß), neurotrophic tyrosine kinase receptors (NTRK), and nerve growth factor (NGF)-stimulated transcription. We found that direct SMAD1/5/4 target genes control FOXO, PI3K/AKT, and progesterone-mediated signaling in decidualizing cells and that BMP2 supplementation in endometriosis patient-derived assembloids elevated the expression of decidualization markers. In summary, transcriptomic and genome-wide binding analyses of patient-derived endometrial cells and assembloids identified that a functional BMP/SMAD1/5/4 signaling program is crucial for engaging decidualization.

Impaired bone morphogenetic protein (BMP) signaling pathways disrupt decidualization in endometriosis.

Endometriosis is linked to increased infertility and pregnancy complications due to defective endometrial decidualization. We hypothesized that identification of altered signaling pathways during decidualization could identify the underlying cause of infertility and pregnancy complications. Our study reveals that transforming growth factor ß (TGFß) pathways are impaired in the endometrium of individuals with endometriosis, leading to defective decidualization. Through detailed transcriptomic analyses, we discovered abnormalities in TGFß signaling pathways and key regulators, such as SMAD4, in the endometrium of affected individuals. We also observed compromised activity of bone morphogenetic proteins (BMP), a subset of the TGFß family, that control endometrial receptivity. Using 3-dimensional models of endometrial stromal and epithelial assembloids, we showed that exogenous BMP2 improved decidual marker expression in individuals with endometriosis. Our findings unveil a previously unidentified dysfunction in BMP/SMAD signaling in the endometrium of individuals with endometriosis, explaining decidualization defects and subsequent pregnancy complications in these individuals.

SMAD2/3 signaling in the uterine epithelium controls endometrial cell homeostasis and regeneration.

The regenerative potential of the endometrium is attributed to endometrial stem cells; however, the signaling pathways controlling its regenerative potential remain obscure. In this study, genetic mouse models and endometrial organoids are used to demonstrate that SMAD2/3 signaling controls endometrial regeneration and differentiation. Mice with conditional deletion of SMAD2/3 in the uterine epithelium using Lactoferrin-iCre develop endometrial hyperplasia at 12-weeks and metastatic uterine tumors by 9-months of age. Mechanistic studies in endometrial organoids determine that genetic or pharmacological inhibition of SMAD2/3 signaling disrupts organoid morphology, increases the glandular and secretory cell markers, FOXA2 and MUC1, and alters the genome-wide distribution of SMAD4. Transcriptomic profiling of the organoids reveals elevated pathways involved in stem cell regeneration and differentiation such as the bone morphogenetic protein (BMP) and retinoic acid signaling (RA) pathways. Therefore, TGFß family signaling via SMAD2/3 controls signaling networks which are integral for endometrial cell regeneration and differentiation.

Tanshinones: sources, pharmacokinetics and anti-cancer activities.

Tanshinones are a class of abietane diterpene compound isolated from Salvia miltiorrhiza (Danshen or Tanshen in Chinese), a well-known herb in Traditional Chinese Medicine (TCM). Since they were first identified in the 1930s, more than 40 lipophilic tanshinones and structurally related compounds have been isolated from Danshen. In recent decades, numerous studies have been conducted to investigate the isolation, identification, synthesis and pharmacology of tanshinones. In addition to the well-studied cardiovascular activities, tanshinones have been investigated more recently for their anti-cancer activities in vitro and in vivo. In this review, we update the herbal and alternative sources of tanshinones, and the pharmacokinetics of selected tanshinones. We discuss anti-cancer properties and identify critical issues for future research. Whereas previous studies have suggested anti-cancer potential of tanshinones affecting multiple cellular processes and molecular targets in cell culture models, data from in vivo potency assessment experiments in preclinical models vary greatly due to lack of uniformity of solvent vehicles and routes of administration. Chemical modifications and novel formulations had been made to address the poor oral bioavailability of tanshinones. So far, human clinical trials have been far from ideal in their design and execution for the purpose of supporting an anti-cancer indication of tanshinones.

The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis.

The family of Abelson interactor (Abi) proteins is a component of WAVE regulatory complex (WRC) and a downstream target of Abelson (Abl) tyrosine kinase. The fact that Abi proteins also interact with diverse membrane proteins and intracellular signaling molecules places these proteins at a central position in the network that controls cytoskeletal functions and cancer cell metastasis. Here, we identified a motif in Abi proteins that conforms to consensus sequences found in a cohort of receptor and non-receptor tyrosine kinases that bind to Cbl-tyrosine kinase binding domain. The phosphorylation of tyrosine 213 in this motif is essential for Abi degradation. Double knockout of c-Cbl and Cbl B in Bcr-Abl-transformed leukemic cells abolishes Abi1, Abi2, and WAVE2 degradation. Moreover, knockout of Abi1 reduces Src family kinase Lyn activation in Bcr-Abl-positive leukemic cells and promotes EGF-induced EGF receptor downregulation in breast cancer cells. Importantly, Abi1 depletion impeded breast cancer cell invasion in vitro and metastasis in mouse xenografts. Together, these studies uncover a novel mechanism by which the WRC and receptor/non-receptor tyrosine kinases are regulated and identify Abi1 as a potential therapeutic target for metastatic breast cancer.

Immunogenomic intertumor heterogeneity across primary and metastatic sites in a patient with lung adenocarcinoma.

BACKGROUND: Lung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy. METHODS: In order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a 32-year-old female never-smoker with left lung primary late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors. RESULTS: We observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases. Only TP53 mutation was detected in all tumors suggesting an early event while other cancer gene mutations were later events which may have followed subclonal diversification. A set of prevalent T cell clonotypes were completely excluded from left-side thoracic tumors indicating distinct T cell repertoire profiles between left-side and non left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression. CONCLUSIONS: Our study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes.

BACKGROUND: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical study explores the feasibility of producing a NSCLC TIL product with sufficient numbers and enhanced attributes using an improved culture method. METHODS: TIL from resected NSCLC tumors were initially cultured using (1) the traditional method using interleukin (IL)-2 alone in 24-well plates (TIL 1.0) or (2) IL-2 in combination with agonistic antibodies against CD3 and 4-1BB (Urelumab) in a G-Rex flask (TIL 3.0). TIL subsequently underwent a rapid expansion protocol (REP) with anti-CD3. Before and after the REP, expanded TIL were phenotyped and the complementarity-determining region 3 ß variable region of the T-cell receptor (TCR) was sequenced to assess the T-cell repertoire. RESULTS: TIL 3.0 robustly expanded NSCLC TIL while enriching for CD8+ TIL in a shorter manufacturing time when compared with the traditional TIL 1.0 method, achieving a higher success rate and producing 5.3-fold more TIL per successful expansion. The higher proliferative capacity and CD8 content of TIL 3.0 was also observed after the REP. Both steps of expansion did not terminally differentiate/exhaust the TIL but a lesser differentiated population was observed after the first step. TIL initially expanded with the 3.0 method exhibited higher breadth of clonotypes than TIL 1.0 corresponding to a higher repertoire homology with the original tumor, including a higher proportion of the top 10 most prevalent clones from the tumor. TIL 3.0 also retained a higher proportion of putative tumor-specific TCR when compared with TIL 1.0. Numerical expansion of TIL in a REP was found to perturb the clonal hierarchy and lessen the proportion of putative tumor-specific TIL from the TIL 3.0 process. CONCLUSIONS: We report the feasibility of robustly expanding a T-cell repertoire recapitulating the clonal hierarchy of the T cells in the NSCLC tumor, including a large number of putative tumor-specific TIL clones, using the TIL 3.0 methodology. If scaled up and employed as a sole expansion platform, the robustness and speed of TIL 3.0 may facilitate the testing of TIL-ACT approaches in NSCLC.

Interception Targets of Angelica Gigas Nakai Root Extract versus Pyranocoumarins in Prostate Early Lesions and Neuroendocrine Carcinomas in TRAMP Mice.

We reported efficacy of Angelica gigas Nakai (AGN) root ethanol extract and equimolar decursin (D)/decursinol angelate (DA) through daily gavage starting at 8 weeks of age (WOA) to male transgenic adenocarcinoma of mouse prostate (TRAMP) mice such that these modalities suppressed precancerous epithelial lesions in their dorsolateral prostate (DLP) to similar extent, but AGN extract was better than the D/DA mixture at promoting the survival of mice bearing prostate neuroendocrine carcinomas to 28 WOA. Here, we compared by microarray hybridization the mRNA levels in pooled DLP tissues and individual neuroendocrine carcinomas to characterize potential molecular targets of AGN extract and D/DA. Clustering and principal component analyses supported distinct gene expression profiles of TRAMP DLP versus neuroendocrine carcinomas. Pathway Enrichment, Gene Ontology, and Ingenuity Pathway Analyses of differential genes indicated that AGN and D/DA affected chiefly processes of lipid and mitochondrial energy metabolism and oxidation-reduction in TRAMP DLP, while AGN affected neuronal signaling, immune systems and cell cycling in neuroendocrine carcinomas. Protein-Protein Interaction Network analysis predicted and reverse transcription-PCR verified multiple hub genes common in the DLP of AGN- and D/DA-treated TRAMP mice at 28 WOA and select hub genes attributable to the non-D/DA AGN components. The vast majority of hub genes in the AGN-treated neuroendocrine carcinomas differed from those in TRAMP DLP. In summary, the transcriptomic approach illuminated vastly different signaling pathways and networks, cellular processes, and hub genes of two TRAMP prostate malignancy lineages and their associations with the interception efficacy of AGN and D/DA. PREVENTION RELEVANCE: This study explores potential molecular targets associated with in vivo activity of AGN root alcoholic extract and its major pyranocoumarins to intercept precancerous epithelial lesions and early malignancies of the prostate. Without an ethically-acceptable, clearly defined cancer initiation risk reduction strategy available for the prostate, using natural products like AGN to delay formation of malignant tumors could be a plausible approach for prostate cancer prevention.

Deep learning-based prediction of the T cell receptor-antigen binding specificity.

Neoantigens play a key role in the recognition of tumor cells by T cells. However, only a small proportion of neoantigens truly elicit T cell responses, and fewer clues exist as to which neoantigens are recognized by which T cell receptors (TCRs). We built a transfer learning-based model, named pMHC-TCR binding prediction network (pMTnet), to predict TCR-binding specificities of neoantigens, and T cell antigens in general, presented by class I major histocompatibility complexes (pMHCs). pMTnet was comprehensively validated by a series of analyses, and showed advance over previous work by a large margin. By applying pMTnet in human tumor genomics data, we discovered that neoantigens were generally more immunogenic than self-antigens, but HERV-E, a special type of self-antigen that is re-activated in kidney cancer, is more immunogenic than neoantigens. We further discovered that patients with more clonally expanded T cells exhibiting better affinity against truncal, rather than subclonal, neoantigens, had more favorable prognosis and treatment response to immunotherapy, in melanoma and lung cancer but not in kidney cancer. Predicting TCR-neoantigen/antigen pairs is one of the most daunting challenges in modern immunology. However, we achieved an accurate prediction of the pairing only using the TCR sequence (CDR3ß), antigen sequence, and class I MHC allele, and our work revealed unique insights into the interactions of TCRs and pMHCs in human tumors using pMTnet as a discovery tool.

CRISPR/CAS9-mediated knockout of Abi1 inhibits p185Bcr-Abl-induced leukemogenesis and signal transduction to ERK and PI3K/Akt pathways.

BACKGROUND: Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear. METHODS: In this study, we knocked out Abi1 expression in p185Bcr-Abl-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model. RESULTS: We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1α-mediated chemotaxis in p185Bcr-Abl-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185Bcr-Abl-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo. CONCLUSIONS: Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.

Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin.

Bufalin is a major cardiotonic compound in the traditional Chinese medicine, Chansu, prepared from toad skin secretions. Cell culture studies have suggested an anticancer potential involving multiple cellular processes, including differentiation, apoptosis, senescence, and angiogenesis. In prostate cancer cell models, P53-dependent and independent caspase-mediated apoptosis and androgen receptor (AR) antagonism have been described for bufalin at micromolar concentrations. Because a human pharmacokinetic study indicated that single nanomolar bufalin was safely achievable in the peripheral circulation, we evaluated its cellular activity within range with the AR-positive and P53 wild-type human LNCaP prostate cancer cells in vitro Our data show that bufalin induced caspase-mediated apoptosis at 20 nmol/L or higher concentration with concomitant suppression of AR protein and its best-known target, PSA and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Bufalin exposure induced protein abundance of P53 (not mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased senescence-like phenotype (SA-galactosidase). Small RNAi knocking down of P53 attenuated bufalin-induced senescence, whereas knocking down of P21CIP1 exacerbated bufalin-induced caspase-mediated apoptosis. In vivo, daily intraperitoneal injection of bufalin (1.5 mg/kg body weight) for 9 weeks delayed LNCaP subcutaneous xenograft tumor growth in NSG SCID mice with a 67% decrease of final weight without affecting body weight. Tumors from bufalin-treated mice exhibited increased phospho-P53 and SA-galactosidase without detectable caspase-mediated apoptosis or suppression of AR and PSA. Our data suggest potential applications of bufalin in therapy of prostate cancer in patients or chemo-interception of prostate precancerous lesions, engaging a selective activation of P53 senescence. Mol Cancer Ther; 17(11); 2341-52. ©2018 AACR.

Chemopreventive Effects of Korean Angelica versus Its Major Pyranocoumarins on Two Lineages of Transgenic Adenocarcinoma of Mouse Prostate Carcinogenesis.

We showed previously that daily gavage of Angelica gigas Nakai (AGN) root ethanolic extract starting 8 weeks of age inhibited growth of prostate epithelium and neuroendocrine carcinomas (NE-Ca) in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Because decursin (D) and its isomer decursinol angelate (DA) are major pyranocoumarins in AGN extract, we tested the hypothesis that D/DA represented active/prodrug compounds against TRAMP carcinogenesis. Three groups of male C57BL/6 TRAMP mice were gavage treated daily with excipient vehicle, AGN (5 mg per mouse), or equimolar D/DA (3 mg per mouse) from 8 weeks to 16 or 28 weeks of age. Measurement of plasma and NE-Ca D, DA, and their common metabolite decursinol indicated similar retention from AGN versus D/DA dosing. The growth of TRAMP dorsolateral prostate (DLP) in AGN- and D/DA-treated mice was inhibited by 66% and 61% at 16 weeks and by 67% and 72% at 28 weeks, respectively. Survival of mice bearing NE-Ca to 28 weeks was improved by AGN, but not by D/DA. Nevertheless, AGN- and D/DA-treated mice had lower NE-Ca burden. Immunohistochemical and mRNA analyses of DLP showed that AGN and D/DA exerted similar inhibition of TRAMP epithelial lesion progression and key cell-cycle genes. Profiling of NE-Ca mRNA showed a greater scope of modulating angiogenesis, epithelial-mesenchymal transition, invasion-metastasis, and inflammation genes by AGN than D/DA. The data therefore support D/DA as probable active/prodrug compounds against TRAMP epithelial lesions, and they cooperate with non-pyranocoumarin compounds to fully express AGN efficacy against NE-Ca.