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1.  Metronomic Chemotherapy: Possible Clinical Application in Advanced Hepatocellular Carcinoma1 
Translational Oncology  2013;6(5):511-519.
Hepatocellular carcinoma (HCC) is a hypervascular highly angiogenic tumor usually associated with liver cirrhosis. Vascular endothelial growth factor plays a critical role in vascular development in HCC. In contrast to the treatment of early-stage HCC, the treatment options for advanced HCC are limited and prognosis is often poor, which contributes to this tumor type being the third leading cause of cancer-related deaths worldwide. Metronomic chemotherapy, which was originally designed to inhibit angiogenesis, involves low-dose chemotherapeutic agents administered in a frequent regular schedule with no prolonged breaks and minimizes severe toxicities. We reviewed the potential effects and impact of metronomic chemotherapy in preclinical studies with HCC models and in patients with advanced HCC, especially when combined with a molecular targeted agent. Metronomic chemotherapy involves multiple mechanisms that include antiangiogenesis and antivasculogenesis, immune stimulation by reducing regulatory T cells and inducing dendritic cell maturation, and possibly some direct tumor cell targeting effects, including the cancer stem cell subpopulation. The total number of preclinical studies with HCC models shows impressive results using metronomic chemotherapy-based protocols, especially in conjunction with molecular targeted agents. Four clinical trials and two case reports evaluating metronomic chemotherapy for HCC indicate it to be a safe and potentially useful treatment for HCC. Several preclinical and clinical HCC studies suggest that metronomic chemotherapy may become an alternative type of chemotherapy for advanced unresectable HCC and postsurgical adjuvant treatment of HCC.
PMCID: PMC3799193  PMID: 24151531
2.  Surface α-1,3-Glucan Facilitates Fungal Stealth Infection by Interfering with Innate Immunity in Plants 
PLoS Pathogens  2012;8(8):e1002882.
Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.
Author Summary
Magnaporthe oryzae, Cochlioborus miyabeanus, and Rhizoctonia solani are the top three fungal pathogens that are responsible for devastating damage to the production of rice, a staple cereal for half of the world's population. These fungal pathogens infect host plants despite the plants' innate immunity, which is activated upon recognition of a conserved cell wall component in fungi, such as chitin. Fungal pathogens seem to have evading mechanism(s) against the host innate immunity; however, the mechanisms are still unclear. In this study, we discovered a novel mechanism that is commonly used by fungal pathogens to prevent host innate immunity. In this mechanism, fungal pathogens mask the cell wall surfaces with α-1,3-glucan, a polysaccharide that plants cannot degrade. In fact, a transgenic rice secreting a bacterial α-1,3-glucanase, which is able to remove α-1,3-glucan on the fungal surfaces, obtained strong resistance to all of those fungal pathogens. We also showed that plants rapidly activated defense responses against fungi (even before the fungal penetration) when α-1,3-glucan on the fungal surfaces were damaged or removed. Our study suggests that fungal surface α-1,3-glucan interferes with host immunity in many fungal pathogens and that α-1,3-glucan is a potential target for controlling various fungal diseases in plants.
PMCID: PMC3426526  PMID: 22927818
3.  Use of perioperative ureteral stent in abdominal aortic aneurysm with retroperitoneal fibrosis - A report of two cases - 
Retroperitoneal fibrosis is associated with fibroblast proliferation due to inflammatory changes in adipose/fibrous tissue. Given that aortic dilation in abdominal aortic aneurysm can cause compression of the ureter, abdominal aortic aneurysm complicated by retroperitoneal fibrosis is likely to result in urinary tract obstruction. Accordingly, close attention to changes in perioperative urine volume is warranted when operating on patients with abdominal aortic aneurysm complicated by retroperitoneal fibrosis. We have recently performed laparotomies on two cases of abdominal aortic aneurysm complicated by retroperitoneal fibrosis. In the first case, surgery was performed without the placement of a ureteral stent. The patient developed postrenal acute renal failure caused by postoperative urinary retention. In the second case, ureteral stent placement in advance enabled perioperative management without complications. The clinical course of these cases suggests that, in laparotomy with concomitant retroperitoneal fibrosis, preoperative ureteral stent placement can prevent postoperative complications in the renal and urinary systems.
PMCID: PMC3408521  PMID: 22870371
Acute kidney injury; Aortic aneurysm; Retroperitoneal fibrosis; Ureteral stent; Urinary retention
4.  Loss of the SxxSS Motif in a Human T-Cell Factor-4 Isoform Confers Hypoxia Resistance to Liver Cancer: An Oncogenic Switch in Wnt Signaling 
PLoS ONE  2012;7(6):e39981.
Aberrantly activated Wnt/β-catenin signaling is important in hepatocellular carcinoma (HCC) development. Downstream gene expressions involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. Here, we show the oncogenic potential of human TCF-4 isoforms based on the expression of a single conserved SxxSS motif.
We investigated the TCF-4J and K isoform pair characterized by the presence (K) or absence (J) of the SxxSS motif. The mRNA expression profiles were examined in 47 pairs of human HCCs and adjacent non-cancerous liver tissues by RT-PCR. Proliferation, sphere assays and immunoblot analysis were performed under normoxia and hypoxia conditions. The ability of HCC cells overexpressing TCF-4J (J cells) and K (K cells) to grow as solid tumors in nude mice was explored.
TCF-4J expression was significantly upregulated in HCC tumors compared to corresponding peritumor and normal liver and was preferentially expressed in poorly differentiated HCCs. In contrast, TCF-4K was downregulated in those same HCC tumors. TCF-4J-overexpressing HCC cells (J cells) revealed a survival advantage under hypoxic conditions, high proliferation rate and formation of aggregates/spheres compared to overexpression of TCF-4K (K cells). The hypoxic J cells had high expression levels of HIF-2α and EGFR as possible mechanisms to promote tumorigenesis. Increased stability of HIF-2α under hypoxia in J cells was associated with a decreased level of von Hippel-Lindau (VHL) protein, a known E3 ligase for HIF-αs. In a xenograft model, the J cells rapidly developed tumors compared to K cells. Tumor tissues derived from J cells exhibited high expression levels of HIF-2α and EGFR compared to the slow developing and small K cell derived tumors.
Our results suggest that the specific TCF-4J isoform, which lacks a regulatory SxxSS motif, has robust tumor-initiating potential under hypoxic conditions.
PMCID: PMC3386968  PMID: 22768190
5.  Identification of T-Cell Factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells 
Experimental cell research  2011;317(7):920-931.
The Wnt/β-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). Downstream signaling events involving the Wnt/β-catenin cascade occur through T-cell factor (TCF) proteins. The human TCF-4 gene is composed of 17 exons with multiple alternative splicing sites. However, the role of different TCF-4 isoforms in the pathogenesis of HCC is unknown. The purpose of this study was to identify and characterize TCF-4 isoforms in HCC. We identified 14 novel TCF-4 isoforms from four HCC cell lines. Functional analysis following transfection and expression in HCC cells revealed distinct effects on the phenotype. The TCF-4J isoform expression produced striking features of malignant transformation characterized by high cell proliferation rate, migration and colony formation even though its transcriptional activity was low. In contrast, the TCF-4K isoform displayed low TCF transcriptional activity; cell proliferation rate and colony formation were reduced as well. Interestingly, TCF-4J and TCF-4K differed by only five amino acids (the SxxSS motif). Thus, these studies suggest that conserved splicing motifs may have a major influence on the transcriptional activity and functional properties of TCF-4 isoforms and alter the characteristics of the malignant phenotype.
PMCID: PMC3066145  PMID: 21256126
TCF-4 isoform; HCC; Wnt
6.  Adiponectin suppresses endoplasmic reticulum stress in nonalcoholic steatohepatitis 
In this study, we examined whether adiponectin suppresses endoplasmic reticulum (ER) stress in nonalcoholic steatohepatitis (NASH) using male transgenic mice expressing nSREBP-1c in adipose tissue, nSREBP-1c/adiponectin double-transgenic mice expressing human adiponectin in the liver, and wild-type male mice as the control. Histological findings similar to those observed in liver specimens from patients with NASH were observed in the livers from the nSREBP-1c transgenic mice at 30 weeks of age. By contrast, the NASH-like liver histology was markedly attenuated in age-matched nSREBP-1c/adiponectin double-transgenic mice. The nSREBP-1c/adiponectin double-transgenic mice showed human adiponectin production in the liver and a restored circulating human adiponectin level. Human adiponectin messenger ribonucleic acid (mRNA) expression in the liver was identified in the nSREBP-1c/adiponectin double-transgenic mice, but adiponectin receptor 1 and 2 mRNA expression in the liver was normal. TNFα mRNA was decreased in the liver of the nSREBP-1c/adiponectin double-transgenic mice compared with the nSREBP-1c transgenic mice. The protein expressions of X-box-binding protein-1, activating transcription factor 4, acetyl-CoA carboxylase, TNFα and NFκB were down-regulated in liver tissues from the nSREBP-1c/adiponectin double-transgenic mice. Mouse adiponectin and activating transcription factor 6 expressions were almost the same in the three groups. Post-load plasma glucose levels were significantly lower in the nSREBP-1c/adiponectin double-transgenic mice compared with the nSREBP-1c transgenic mice. These results indicate that adiponectin expressed in the liver suppresses ER stress and attenuates hepatic steatosis, inflammation and insulin resistance in NASH. Adiponectin may open the way to novel therapies for human NASH.
PMCID: PMC3440812  PMID: 22977616
nonalcoholic steatohepatitis; adiponectin; endoplasmic reticulum stress
7.  Noncanonical Wnt11 inhibits hepatocellular carcinoma cell proliferation and migration 
Molecular cancer research : MCR  2010;8(2):254-265.
The canonical Wnt signaling is frequently activated due to over-expression and/or mutations in components of this pathway in hepatocellular carcinoma (HCC). However, the biological role of noncanonical Wnt-mediated signaling in HCC with respect to the signaling pathways involved and their physiologic function is unknown. Here we report the role of Wnt11, a member of the noncanonical cascade, in hepatic oncogenesis. The expression levels of Wnt11 mRNA and protein were significantly downregulated in human HCC tumors compared to the adjacent uninvolved liver as measured by quantitative real-time RT-PCR and western blot analysis. In human HCC cell lines, over-expression of Wnt11 activated PKC signaling. PKC antagonized the canonical signaling through phosphorylation of β-catenin, and reduced TCF mediated transcriptional activity resulting in a decrease of cell proliferation. Furthermore, ectopic expression of Wnt11 promotes RhoA/Rho kinase (ROCK) activation. We found that activated ROCK inhibited Rac1 to reduce cell motility and migration. These observations suggest a novel role for Wnt11 as a tumor suppressor during hepatocarcinogenesis since loss of expression promotes the malignant phenotype via both canonical and noncanonical Wnt signaling pathways.
PMCID: PMC2824771  PMID: 20103596
Wnt11; HCC; PKC; Rac1; noncanonical Wnt
8.  HvCEBiP, a gene homologous to rice chitin receptor CEBiP, contributes to basal resistance of barley to Magnaporthe oryzae 
BMC Plant Biology  2010;10:288.
Rice CEBiP recognizes chitin oligosaccharides on the fungal cell surface or released into the plant apoplast, leading to the expression of plant disease resistance against fungal infection. However, it has not yet been reported whether CEBiP is actually required for restricting the growth of fungal pathogens. Here we evaluated the involvement of a putative chitin receptor gene in the basal resistance of barley to the ssd1 mutant of Magnaporthe oryzae, which induces multiple host defense responses.
The mossd1 mutant showed attenuated pathogenicity on barley and appressorial penetration was restricted by the formation of callose papillae at attempted entry sites. When conidial suspensions of mossd1 mutant were spotted onto the leaves of HvCEBiP-silenced plants, small brown necrotic flecks or blast lesions were produced but these lesions did not expand beyond the inoculation site. Wild-type M. oryzae also produced slightly more severe symptoms on the leaves of HvCEBiP-silenced plants. Cytological observation revealed that these lesions resulted from appressorium-mediated penetration into plant epidermal cells.
These results suggest that HvCEBiP is involved in basal resistance against appressorium-mediated infection and that basal resistance might be triggered by the recognition of chitin oligosaccharides derived from M. oryzae.
PMCID: PMC3020183  PMID: 21190588
9.  Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line 
BMC Cancer  2006;6:292.
Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line.
Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed.
In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours.
We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy.
PMCID: PMC1770931  PMID: 17177986

Results 1-9 (9)