Inhibitory Effect of Pyra-Metho-Carnil on the Differentiation and Maturation of Macrophages in Normal and Cancer Microenvironments.

BACKGROUND/AIM: In a previous study, we have demonstrated heightened Pyra-Metho-Carnil (PMC) efficacy in nude mice with intact innate immunity that lack T and B cells. This has prompted hypothesizing that PMC may target macrophages that promote cancer growth. In this study, we conducted co-culture experiments with macrophages derived from THP-1 human monocyte cell lines and spheroids representing normal and cancer microenvironments. We then performed RNA sequencing and flow cytometry analysis to elucidate the mechanisms by which PMC affects macrophage differentiation and maturation. MATERIALS AND METHODS: THP-1 cells were differentiated by phorbol 12-myristate 13-acetate (PMA) and matured by PMA and lipopolysaccharide (LPS) either with or without PMC. Co-cultures were performed using stimulated THP-1 cells and HKe3-wild-type KRAS or HKe3-mutant (mt) KRAS spheroids. We then performed RNA-seq analysis of THP-1 cells stimulated by PMA (either with or without PMC) and flow cytometry analysis of mice peripheral blood obtained after PMC administration. RESULTS: THP-1 cells matured by PMA and LPS specifically increased the area of HKe3-mtKRAS cancer spheroids and the addition of PMC to THP-1 cells was found to inhibit cancer spheroid growth. RNA-seq data suggested that PMC treatment of THP-1 cells stimulated with PMA suppressed cell motility regulatory functions via down-regulation of the NF[Formula: see text]B pathway. Furthermore, flow cytometry results showed that PMC treatment suppressed monocyte maturation in B6 mice. CONCLUSION: The high level of in vivo tumor suppression caused by PMC may be due to inhibition of the differentiation and maturation of tumor-associated macrophages via the NF[Formula: see text]B signaling pathway.

Inhibitory Effect of Pyra-Metho-Carnil on Cancer Spheroid Growth Through Decrease in Glycolysis-associated Molecules.

BACKGROUND/AIM: Pyra-Metho-Carnil (PMC) has been identified as a novel candidate compound for treating numerous malignancies; however, its mechanism of action remains unknown. In this study, we conducted RNA-sequencing (RNA-seq) analyses to elucidate the mechanism of PMC against human colorectal cancer cells harboring mutant KRAS (mtKRAS). MATERIALS AND METHODS: RNA-seq analyses of the HKe3-wild-type KRAS and HKe3-mtKRAS spheroids treated with DMSO or PMC for 6 days were performed. RESULTS: RNA-seq data suggested that PMC treatment suppresses the aerobic glycolysis pathway in HKe3-mtKRAS spheroids through the down-regulation of the HIF1 pathway. Indeed, treatment with PMC markedly suppresses the absorption of glucose by spheroids and the secretion of lactate from them. CONCLUSION: PMC suppresses growth of cancer spheroid through down-regulation of cancer-specific glucose metabolism.

Cancer Spheroid Proliferation Is Suppressed by a Novel Low-toxicity Compound, Pyra-Metho-Carnil, in a Context-independent Manner.

BACKGROUND/AIM: In a screen of compounds to selectively suppress the growth of cancer spheroids, which contained mutant (mt) KRAS, NPD10621 was discovered and associated derivatives were investigated. MATERIALS AND METHODS: Spheroid areas from HCT116-derived HKe3 spheroids expressing wild type (wt) KRAS (HKe3-wtKRAS) and mtKRAS (HKe3-mtKRAS) were treated with 12 NPD10621 derivatives and measured in three-dimensional floating (3DF) cultures. Several cancers were treated with NPD1018 (pyra-metho-carnil: PMC) in 3DF cultures. In a nude mouse assay, 50% cell growth inhibition (GI50) values were determined. RESULTS: From these 12 derivatives, PMC was the most effective inhibitor of HKe3-mtKRAS spheroid growth with the least toxicity. Furthermore, PMC-mediated growth suppression was observed in all tested cancer cell lines, independent of tissue context, driver gene mutations, and drug resistance, suggesting that the PMC target(s) was crucial for cancer growth in a context-independent manner. The GI50 value of PMC in nude mice assay was 7.7 mg/kg and nude mice that were administered 40 mg/kg PMC for 7 days did not show any abnormal blood cell count values. CONCLUSION: PMC is a low-toxicity compound that inhibits the growth of different tumor cell types.

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress.

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

DNA damage responses that enhance resilience to replication stress.

During duplication of the genome, eukaryotic cells may experience various exogenous and endogenous replication stresses that impede progression of DNA replication along chromosomes. Chemical alterations in template DNA, imbalances of deoxynucleotide pools, repetitive sequences, tight DNA-protein complexes, and conflict with transcription can negatively affect the replication machineries. If not properly resolved, stalled replication forks can cause chromosome breaks leading to genomic instability and tumor development. Replication stress is enhanced in cancer cells due, for example, to the loss of DNA repair genes or replication-transcription conflict caused by activation of oncogenic pathways. To prevent these serious consequences, cells are equipped with diverse mechanisms that enhance the resilience of replication machineries to replication stresses. This review describes DNA damage responses activated at stressed replication forks and summarizes current knowledge on the pathways that promote faithful chromosome replication and protect chromosome integrity, including ATR-dependent replication checkpoint signaling, DNA cross-link repair, and SLX4-mediated responses to tight DNA-protein complexes that act as barriers. This review also focuses on the relevance of replication stress responses to selective cancer chemotherapies.

SLX4-XPF mediates DNA damage responses to replication stress induced by DNA-protein interactions.

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.

GRWD1 directly interacts with p53 and negatively regulates p53 transcriptional activity.

Glutamate-rich WD40 repeat containing 1 (GRWD1) functions as a histone chaperone to promote loading of the MCM replication helicase at replication origins. GRWD1 is overexpressed in several cancer cell lines, and GRWD1 overexpression confers tumorigenic potential in human cells. However, less is known concerning its oncogenic activity. Our previous analysis showed that GRWD1 negatively regulates the tumour suppressor p53 via the RPL11-MDM2-p53 and RPL23-MDM2-p53 axes. Here, we demonstrate that GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation resulted in increased p21 expression. Conversely, GRWD1 co-expression suppressed several p53-regulated promoters. GRWD1 interacted with the p21 and MDM2 promoters, and these interactions required p53. By using the Human Cancer Genome Atlas database, we found that GRWD1 expression levels are inversely correlated with the expression levels of some p53-target genes. Interestingly, high GRWD1 expression in combination with low expression levels of some p53-target genes was significantly correlated with poor prognosis in skin melanoma patients with wild-type p53. Taken together, our findings suggest a novel oncogenic function of GRWD1 as a transcriptional regulator of p53 and that GRWD1 might be an attractive therapeutic target and prognostic marker in cancer therapy.

Identification of candidate molecular targets of the novel antineoplastic antimitotic NP-10.

We previously reported the identification of a novel antimitotic agent with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-iodobenzohydrazide (code number NP-10). However, the mechanism(s) underlying the cancer cell-selective inhibition of mitotic progression by NP-10 remains unclear. Here, we identified NP-10-interacting proteins by affinity purification from HeLa cell lysates using NP-10-immobilized beads followed by mass spectrometry. The results showed that several mitosis-associated factors specifically bind to active NP-10, but not to an inactive NP-10 derivative. Among them, NUP155 and importin ß may be involved in NP-10-mediated mitotic arrest. Because NP-10 did not show antitumor activity in vivo in a previous study, we synthesized 19 NP-10 derivatives to identify more effective NP-10-related compounds. HMI83-2, an NP-10-related compound with a Cl moiety, inhibited HCT116 cell tumor formation in nude mice without significant loss of body weight, suggesting that HMI83-2 is a promising lead compound for the development of novel antimitotic agents.

Inhibiting the MCM8-9 complex selectively sensitizes cancer cells to cisplatin and olaparib.

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.

GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system.

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1-binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1-interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitin-proteasome system. GRWD1 expression reversed the RPL23-mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis.

TRF2 recruits ORC through TRFH domain dimerization.

Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain.

GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis.

The ribosomal protein L11 (RPL11) binds and inhibits the MDM2 ubiquitin ligase, thereby promoting p53 stability. Thus, RPL11 acts as a tumor suppressor. Here, we show that GRWD1 (glutamate-rich WD40 repeat containing 1) physically and functionally interacts with RPL11. GRWD1 is localized to nucleoli and is released into the nucleoplasm upon nucleolar stress. Silencing of GRWD1 increases p53 induction by nucleolar stress, whereas overexpression of GRWD1 reduces p53 induction. Furthermore, GRWD1 overexpression competitively inhibits the RPL11-MDM2 interaction and alleviates RPL11-mediated suppression of MDM2 ubiquitin ligase activity toward p53. These effects are mediated by the N-terminal region of GRWD1, including the acidic domain. Finally, we show that GRWD1 overexpression in combination with HPV16 E7 and activated KRAS confers anchorage-independent growth and tumorigenic capacity on normal human fibroblasts. Consistent with this, GRWD1 overexpression is associated with poor prognosis in cancer patients. Taken together, our results suggest that GRWD1 is a novel negative regulator of p53 and a potential oncogene.

A novel anti-microtubule agent with carbazole and benzohydrazide structures suppresses tumor cell growth in vivo.

BACKGROUND: The mitotic spindles are among the most successful targets of anti-cancer chemotherapy, and they still hold promise as targets for novel drugs. The anti-mitotic drugs in current clinical use, including taxanes, epothilones, vinca alkaloids, and halichondrins, are all microtubule-targeting agents. Although these drugs are effective for cancer chemotherapy, they have some critical problems; e.g., neurotoxicity caused by damage to neuronal microtubules, as well as innate or acquired drug resistance. To overcome these problems, a great deal of effort has been expended on development of novel anti-mitotics. METHODS: We identified novel microtubule-targeting agents with carbazole and benzohydrazide structures: N'-[(9-ethyl-9H-carbazol-3-yl)methylene]-2-methylbenzohydrazide (code number HND-007) and its related compounds. We investigated their activities against cancer cells using various methods including cell growth assay, immunofluorescence analysis, cell cycle analysis, tubulin polymerization assay, and tumor inhibition assay in nude mice. RESULTS: HND-007 inhibits tubulin polymerization in vitro and blocks microtubule formation and centrosome separation in cancer cells. Consequently, it suppresses the growth of various cancer cell lines, with IC50 values in the range 1.3-4.6µM. In addition, HND-007 can inhibit the growth of taxane-resistant cancer cells that overexpress P-glycoprotein. Finally, HND-007 can inhibit HeLa cell tumor growth in nude mice. CONCLUSIONS AND GENERAL SIGNIFICANCE: Taken together, these findings suggest that HND-007 is a promising lead compound for development of novel anti-mitotic, anti-microtubule chemotherapeutic agents.

Laparoscopic distal pancreatectomy for a pancreatic lymphoepithelial cyst: case report and review of literature.

CONTEXT: Lymphoepithelial cysts of the pancreas are a rare disease of true pancreatic cysts, the cause of which is unknown. The differential diagnosis is broad and includes many benign and malignant cystic lesions of the pancreas and surrounding organs. A combination of imaging modalities and fine needle aspiration might narrow the differential diagnosis. However, the final diagnosis can only be achieved with certainty after resection of the cyst. CASE REPORT: The present case report is a lymphoepithelial cyst of the pancreas that was resected laparoscopically. A 53-year-old man was incidentally found to have a cystic tumor in the tail of the pancreas after undergoing an abdominal ultrasound, which showed a 41x33 mm cystic mass in the pancreatic tail. He had no abdominal symptoms. Laparoscopic distal pancreatectomy and splenectomy were performed. Histologic examination revealed a lymphoepithelial cyst. CONCLUSION: Herein, we discuss the diagnostic difficulties and management decisions that face surgeons treating pancreatic cysts.

[Clinical applications of indocyanine green-fluorescent imaging to liver surgery].

BACKGROUND: Intravenous injection of the indocyanine green (ICG) reagent was performed as a liver function test 1 to 2 weeks prior to surgery. ICG fluorescent imaging was performed using the Photodynamic Eye (PDE) infrared camera (Hamamatsu Photonics k. k., Shizuoka, Japan). METHODS: Intravenous injection of the ICG reagent was performed as a liver function test 1 to 2 weeks before surgery. ICG fluorescent imaging was performed using the PDE infrared camera. RESULTS: ICG fluorescent imaging was useful for liver surgical navigation as follows: three cases of recurrent hepatocellular carcinoma(HCC) after transcatheter arterial chemoembolization or thermal ablation therapy; three cases of colorectal metastatic liver tumors after chemotherapy as conversion therapy; two cases of intrahepatic cholangiocellular carcinoma (ICC); and two cases of surface HCC that were not detected by preoperative computed tomography (CT) scan. In the ICC cases, although dilatation of the bile duct could be detected, the tumor margins were ambiguous by ultrasonography(US). ICG fluorescent imaging clearly described the dilatation of the bile duct including the tumor. CONCLUSIONS: ICG fluorescent imaging was useful for liver surgical navigation including selection of the mode of hepatic resection.

Redundant and differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells.

When human cells enter S-phase, overlapping differential inhibitory mechanisms downregulate the replication licensing factors ORC1, CDC6 and Cdt1. Such regulation prevents re-replication so that deregulation of any individual factor alone would not be expected to induce overt re-replication. However, this has been challenged by the fact that overexpression of Cdt1 or Cdt1+CDC6 causes re-replication in some cancer cell lines. We thought it important to analyze licensing regulations in human non-cancerous cells that are resistant to Cdt1-induced re-replication and examined whether simultaneous deregulation of these licensing factors induces re-replication in two such cell lines, including human fibroblasts immortalized by telomerase. Individual overexpression of either Cdt1, ORC1 or CDC6 induced no detectable re-replication. However, with Cdt1+ORC1 or Cdt1+CDC6, some re-replication was detectable and coexpression of Cdt1+ORC1+CDC6 synergistically acted to give strong re-replication with increased mini-chromosome maintenance (MCM) loading. Coexpression of ORC1+CDC6 was without effect. These results suggest that, although Cdt1 regulation is the key step, differential regulation of multiple licensing factors ensures prevention of re-replication in normal human cells. Our findings also show for the first time the importance of ORC1 regulation for prevention of re-replication.