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Diabetic wounds exhibit chronic inflammation and delayed tissue proliferation or remodeling, mainly owing to prolonged proinflammatory (M1) macrophage activity and defects in transition to prohealing/proremodeling (M2a/M2c; CD206

Krüppel-like factor 10 (KLF10) is a phospho-regulated transcriptional factor involved in many biological processes including lipogenesis; however, the transcriptional regulation on lipogenesis by KLF10 remains largely unclear. Lipogenesis is important in the development of nonalcoholic fatty liver disease (NAFLD) which was known regulated mainly by AMP-activated protein kinase (AMPK) and sterol regulatory element-binding protein (SREBP-1C). Interesting, our previous study using phosphorylated site prediction suggested a regulation of AMPK on KLF10. Therefore, we aimed to study the protein–protein interactions of AMPK on the regulation of KLF10, and to delineate the mechanisms of phosphorylated KLF10 in the regulation of NAFLD through SREBP-1C. We performed in vitro and in vivo assays that identified AMPK phosphorylates KLF10 at Thr189 and subsequently modulates the steady state level of KLF10. Meanwhile, a chromatin immunoprecipitation–chip assay revealed the novel target genes and signaling cascades of corresponding to phosphorylated KLF10. SREBP-1C was identified as a target gene suppressed by phosphorylated KLF10 through promoter binding. We further performed high-fat-diet-induced NAFLD models using hepatic-specific KLF10 knockout mice and wild-type mice and revealed that KLF10 knockout markedly led to more severe NAFLD than that in wild-type mice. Taken together, our findings revealed for the first time that AMPK activates and stabilizes the KLF10 protein via phosphorylation at Thr189, thereby repressing the expression of SREBP-1C and subsequent lipogenesis pathways along with metabolic disorders. We suggested that the targeted manipulation of liver metabolism, particularly through increased KLF10 expression, is a potential alternative solution for treating NAFLD.

Krüppel-like factor 10 (KLF10) is a tumor suppressor in multiple cancers. In a murine model of spontaneous pancreatic adenocarcinoma (PDAC), additional KLF10 depletion accelerated distant metastasis. However, Klf10 knockout mice, which suffer from metabolic disorders, do not develop malignancy. The mechanisms of KLF10 in PDAC progression deserve further exploration. KLF10-depleted and KLF10-overexpressing PDAC cells were established to measure epithelial-mesenchymal transition (EMT), glycolysis, and migration ability. A murine model was established to evaluate the benefit of genetic or pharmacological manipulation in KLF10-depleted PDAC cells (PDACshKLF10). Correlations of KLF10 deficiency with rapid metastasis, elevated EMT, and glycolysis were demonstrated in resected PDAC tissues, in vitro assays, and murine models. We identified sirtuin 6 (SIRT6) as an essential mediator of KLF10 that modulates EMT and glucose homeostasis. Overexpressing SIRT6 reversed the migratory and glycolytic phenotypes of PDACshKLF10 cells. Linoleic acid, a polyunsaturated essential fatty acid, upregulated SIRT6 and prolonged the survival of mice injected with PDACshKLF10. Modulating HIF1α and NFκB revealed that EMT and glycolysis in PDAC cells were coordinately regulated upstream by KLF10/SIRT6 signaling. Our study demonstrated a novel KLF10/SIRT6 pathway that modulated EMT and glycolysis coordinately via NFκB and HIF1α. Activation of KLF10/SIRT6 signaling ameliorated the distant progression of PDAC. Clinical Trial Registration: ClinicalTrials.gov. identifier: NCT01666184., Pancreatic cancer: Possible treatment based on tumor suppressor genes Boosting the activity of a tumor suppressor pathway in pancreatic cancer may reduce the risk of metastasis. Reduced expression of a tumor suppressor gene called Krüppel-like factor 10 (KLF10) is found in breast, lung and pancreatic cancers. KLF10 deficiency in pancreatic cancer is linked to accelerated cancer progression and metastasis, but the underlying mechanisms are unclear. In experiments on mouse models and human cell cultures, Hui-Ju Ch’ang at the National Institute of Cancer Research in Zhunan, Taiwan, and co-workers found that KLF10 binds to and upregulates another tumor suppressor gene, SIRT6. This second gene mediates KLF10 activity, including its role in regulating glucose metabolism and a process that transforms epithelial cells into cells with invasive, migratory characteristics. Overexpressing SIRT6 in KLF10-deficient cells reversed glycolysis, migratory cell behavior and reduced metastasis, suggesting a potential treatment option.

Designing multi‐drug regimens often involves target‐ and synergy prediction‐based drug selection, and subsequent dose escalation to achieve the maximum tolerated dose (MTD) of each drug. This approach may improve efficacy, but not the optimal efficacy and often substantially increases toxicity. Drug interactions depend on many pathways in the omics networks and further complicate the design process. The virtually infinite drug–dose parameter space cannot be reconciled using conventional approaches, which are largely based on prediction. This barrier at least partially accounts for the low response rates that are observed with conventional mono‐ and combinatorial chemotherapy. A combination of nonstandard therapies for colorectal cancer (AGCH: adriamycin, gemcitabine, cisplatin, and herceptin) at ¼ MTD is used to treat the rats, the tumor response rates varied in a wide range. Some of the tumor response rates are close to that of control group. This work harnesses an artificial intelligence (AI) platform that is mechanism free and can dynamically optimize combinatorial therapy in rats. The individually optimized AGCH regimen reveal starkly different drug–dose parameters, which can converge each rat toward the same and low tumor response rate. Importantly, this AI‐based drug–dose optimization technology is an actionable platform, which can achieve N‐of‐1 therapy.

Lung cancer is the leading cause of cancer deaths worldwide. The difficulties of early detection and treatment resistance adversely affect prognosis. Major challenges for new drug development include cost, safety, and efficacy. As a result, repurposing existing drugs for new indications offers a more favorable risk–return trade-off than other available drug development strategies. Numerous mouse models for lung cancer have been established, which can primarily be divided into genetically engineered or xenograft mouse models. However, genetically modulated models cannot fully represent the systemic complexity of lung cancer, and xenograft models do not behave identically to human lung tumors in drug repurposing. This chapter reviews the current animal models in lung cancer drug repurposing and assesses the challenges and future directions of this field.

In the present study, we attempted to develop a lecithin-stabilized micellar drug delivery system (LsbMDDs) for loading docetaxel (DTX) to enhance its therapeutic efficacy and minimize systemic toxicity. A novel DTX-loaded LsbMDDs was optimally prepared by a thin-film hydration method and then hydrated with a lecithin nanosuspension while being subjected to ultrasonication. Physical characteristics of the optimized DTX-loaded LsbMDDs formulations were examined and found to have a mean size of 90%, and drug loading of >6% with stability at room temperature and at 4 °C being longer than 2 and 7 days, respectively. The in vitro release of DTX from the DTX-loaded LsbMDDs was slower than that from the generic product of DTX (Tynen®). A cell viability assay demonstrated that the LsbMDDs showed better cytotoxicity than Tynen® against CT26 cancer cells. The in vivo antitumor efficacy of the DTX-loaded LsbMDDs was observed to be better than that of Tynen® in a CT26 tumor-bearing mice model. A high-dose regimen of the DTX-loaded LsbMDDs formulation showed greater inhibition of DU145 tumor growth than did Tynen®, but with less to similar systemic toxicity. An in vivo study also showed that a greater amount of drug was able to accumulate in the tumor site with the DTX-loaded LsbMDDs, and its maximal tolerable doses for single and repeated injections were 2–2.5-fold higher than those of Tynen®. In conclusion, the LsbMDDs could be a promising high drug-loaded nanocarrier for delivering hydrophobic chemotherapeutic agents that can enhance the efficacy of chemotherapy and reduce systemic toxicity.

KLF10 is a transforming growth factor (TGF)-β/Smad downstream regulated gene. KLF10 binds to the promoter of target genes and mimics the effects of TGF-β as a transcriptional factor. In our laboratory, we noted that Klf10 deficiency in mice is associated with significant inflammation of the lungs. However, the precise mechanism of this association remains unknown. We previously identified NPRA as a target gene potentially regulated by KLF10 through direct binding; NPRA knockout have known that prevented lung inflammation in a mouse model of allergic asthma. Here, we further explored the regulatory association between KLF10 and NPRA on the basis of the aforementioned findings. Our results demonstrated that KLF10 acts as a transcriptional repressor of NPRA and that KLF10 binding reduces NPRA expression in vitro. Compared with wild-type mice, Klf10-deficient mice were more sensitive to lipopolysaccharide or ovalbumin challenge and showed more severe inflammatory histological changes in the lungs. Moreover, Klf10-deficient mice showed pulmonary neutrophil accumulation. These findings collectively reveal the precise site where KLF10 signaling affects pulmonary inflammation by attenuating NPRA expression. They also verify the importance of KLF10 and atrial natriuretic peptide/NPRA in exerting influences on chronic pulmonary disease pathogenesis.

Inflammation is the first step that leads to inflammatory cell migration, cytokine release, and myofibroblast formation. Myofibroblasts can deposit excess amounts of extracellular matrix. Cyclooxygenase (COX) inhibitor exhibits strong anti-inflammatory response; however, this is usually achieved with undesirable side effects. In this study, we demonstrated the effects of the fluorine-modified rutaecarpine (RUT), fluoro-2-methoxyrutaecarpine (F-RUT), in inflammatory damage in the lungs. Based on the results, F-RUT retained anti-inflammatory activity both in vitro and in vivo in lungs. Compared to the parent compound, F-RUT showed better COX-2 suppression as a COX-2-selective inhibitor with lower cytotoxicity, and enhanced molecular reactivity and biological activity. F-RUT was also observed to reduce reactive oxygen species (ROS) generation and inflammatory infiltrating neutrophils in lipopolysaccharide (LPS)-stimulated zebrafish and ovalbumin (OVA)/alum-challenged KLF-10-knockout mouse lungs, respectively. Furthermore, F-RUT ameliorated the respiratory function in OVA/alum-challenged BALB/c mice by maintaining the thickness of the blood-air barrier in mouse lungs. Overall, these data suggest that F-RUT may function as an effective therapeutic agent for inflammation-induced lung dysfunction, and a better selection for pharmaceutical purposes than conventionally used anti-inflammatory agents.

Background Chemotherapy is used mainly to treat and control the progression of gynecological cancer. Bone marrow suppression, one of the adverse side effects of chemotherapy, may decrease immune function, increasing the risk of serious, fatal infections. Purpose The aims of this study were to evaluate the effectiveness of noninvasive acupressure in preventing and diminishing chemotherapy-induced myelosuppression in patients with gynecologic cancer and to determine whether this effect is associated with the regulation of the expressions of granulocyte-macrophage colony-stimulating factor and stem cell factor (SCF). Methods In total, 28 women with gynecological cancer were randomly assigned either to the experimental group (n = 10) or to the control group (n = 18). The experimental group received acupressure of 5-minute duration to the Hegu (LI4), Quchi (LI11), Xuehai (SP10), Sanyinjiao (SP6), Taixi (K3), Zusanli (ST36), Taichong (LR3), and Baihui (GV20) points, respectively, three times per day for 6 weeks. The control group did not receive the acupressure intervention. The blood count, including white blood cells, platelets, and hemoglobin, and serum levels for SCF and granulocyte-macrophage colony-stimulating factor were assessed before (pretest) and 6 weeks after (posttest) the participants' first course of chemotherapy. Results At posttest, blood hemoglobin had significantly decreased from (mean ± SD) 11.6 ± 2.2 to 10.8 ±1.6 mg/dl (p = .03) in the control group. However, no significant pretest-posttest difference in hemoglobin concentration (11.4 ± 1.0 vs. 10.9 ± 1.1 mg/dl) was detected in the experimental group. Levels of SCF increased significantly between pretest and posttest in both the control group (from 1196.10 ± 293.17 to 1325.05 ± 253.77 ng/ml; p = .01) and the acupressure group (from 1046.78 ± 469.52 to 1387.06 ± 310.00 ng/ml; p = .007). In addition, a borderline difference (p = .05) in mean pretest-posttest SCF increase was found between the acupressure group (340.28 ± 255.46 ng/ml) and the control group (128.94 ± 250.64 ng/ml). Finally, a significant time-dependent interactive effect was found between acupressure and the increased blood level of SCF at posttest (β = 211.34, p = .02). Conclusions/implications for practice The findings support that acupressure on specific acupoints increases blood SCF levels significantly, which may help protect chemotherapy patients from experiencing reduced hemoglobin levels and may relieve chemotherapy-induced myelosuppression in patients with gynecologic cancer. This noninvasive approach is suggested for practical implementation in patients undergoing a course of chemotherapy.

Proton pump inhibitors (PPIs) are used for the long-term treatment of gastroesophageal disorders and the non-prescription medicines for acid reflux. However, there is growing concerns about PPI misuse, overuse and abuse. This study aimed to develop an animal model to examine the effects of long-term use of PPI in vivo. Twenty one Wistar rats were given omeprazole orally or intravenously for 30 days, and caerulein as a positive control. After euthanization, the serum and stool were collected to perform MS-based quantitative analysis of metabolites. We carried out 16S-based profiling of fecal microbiota, assessed the expression of bile acid metabolism regulators and examined the immunopathological characteristics of bile ducts. After long-term PPI exposure, the fecal microbial profile was altered and showed similarity to those observed in high-fat diet studies. The concentrations of several metabolites were also changed in various specimens. Surprisingly, morphological changes were observed in the bile duct, including ductal epithelial proliferation, micropapillary growth of biliary epithelium, focal bile duct stricture formation and bile duct obstruction. These are characteristics of precancerous lesions of bile duct. FXR and RXRα expressions were significantly reduced, which were similar to that observed in cholangiocarcinoma in TCGA and Oncomine databases. We established a novel animal model to examine the effects of long-term use of omeprazole. The gut microbes and metabolic change are consequences of long-term PPI exposure. And the results showed the environment in vivo tends to a high-fat diet. More importantly, we observed biliary epithelial hyperplasia, which is an indicator of a high-fat diet.

Introduction: Pancreatic adenocarcinoma (PDAC) is well known for its deregulated TGFβ signaling. TGFβ signaling exerts a contextual effect that suppresses malignant growth early in epithelial tumorigenesis but promotes metastasis at later stages. The Krüppel-like factor 10 (Klf10) is an early response gene that mediates the antiproliferative effects of TGFβ but opposes the prometastatic effects of TGFβ by limiting its ability to induce epithelial-to-mesenchymal transition (EMT). The distinction between the antiproliferative versus prometastatic functions of TGFβ makes Klf10 a potential target for the development of therapy in cancers with aberrant TGFβ pathway such as PDAC. Klf10 knock-out mice do not have abnormal pancreas development or carcinogenesis. However, ablating Klf10 in murine PDAC models increases pancreatic tumorigenesis and distant metastasis. Genome-wide comparison of mRNA profiles from wild-type and Klf10 knock-out mice revealed significant enrichment for transcripts involved in glucose metabolism. We propose that Klf10 modulates PDAC progression via enhanced glycolysis, which leads to EMT and cancer stemness phenotypes. Materials and Methods: Genetically manipulated Klf10 of human PDAC cell lines was established to measure EMT markers, glycolysis activity and stemness related proteins, cell migration, and murine liver metastasis after intrasplenic injection. Chip-Chip assay was performed to find sirtuin 6 (Sirt6), governing both cancer metabolism and progression, to be one of the candidate molecules regulated by Klf10. The interaction between Klf10 and Sirt6 was evaluated by ChIP-PCR and luciferase reporter assay. PDAC cell lines with or without Klf10 mRNA silencing were overexpressed with Sirt6 to reverse malignant phenotypes induced by Klf10 deficiency. Linoleic acid, a specific Sirt6 activator, was used in vitro and in vivo to ameliorate the aberrant glucose metabolism, EMT phenotype, and distant metastasis of PDAC with loss of Klf10 expression. Results: In vitro and in vivo studies demonstrated Klf10 deficiency in pancreatic cancer enhanced metastasis, EMT, stemness, and glycolysis. Klf10 bond to the promoter and transcriptionally regulated Sirt6. Sirt6 expression paralleled the level of Klf10 in PDAC. Modulating Sirt6 genetically or pharmacologically, by linoleic acid, reversed the EMT, glycolysis, and metastatic phenotypes of PDAC with KLF10 deficiency. Using PDK1, a glycolysis inhibitor, and BSI#38, an EMT inhibitor, we demonstrated the interaction of cancer metabolism and EMT phenotypes in PDAC. Discussion: Loss of Klf10 contributes to pancreatic cancer progression and enhances glycolysis, EMT, and cancer stemness by transcriptionally regulating Sirt6. Overexpressing Sirt6 genetically or pharmacologically by linoleic acid may ameliorate PDAC progression. Targeting Klf10/Sirt6 signaling is a potential strategy to modulate glycolysis and EMT simultaneously in PDAC for preventing malignant progression. Citation Format: Hui-Ju Ch'ang, Yi-Chih Tsai, Su-Liang Chen, Vincent H.S. Chang. Linoleic acid elevates sirtuin 6 expression and modulates glycolysis and epithelial-mesenchymal transition of pancreatic cancer with loss of Krüpple like factor 10 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C11.

Lung cancer is one of the major cause of cancer-related deaths worldwide. The poor prognosis and resistance to both radiation and chemotherapy urged the development of potential targets for lung cancer treatment. In this study, using a network-based cellular signature bioinformatics approach, we repurposed a clinically approved mTOR inhibitor for renal cell carcinomans, temsirolimus, as the potential therapeutic candidate for lung adenocarcinoma. The PI3K-AKT-mTOR pathway is known as one of the most frequently dysregulated pathway in cancers, including non-small-cell lung cancer. By using a well-documented lung adenocarcinoma mouse model of human pathophysiology, we examined the effect of temsirolimus on the growth of lung adenocarcinoma in vitro and in vivo. In addition, temsirolimus combined with reduced doses of cisplatin and gemcitabine significantly inhibited the lung tumor growth in the lung adenocarcinoma mouse model compared with the temsirolimus alone or the conventional cisplatin-gemcitabine combination. Functional imaging techniques and microscopic analyses were used to reveal the response mechanisms. Extensive immunohistochemical analyses were used to demonstrate the apparent effects of combined treatments on tumor architecture, vasculature, apoptosis, and the mTOR-pathway. The present findings urge the further exploration of temsirolimus in combination with chemotherapy for treating lung adenocarcinoma.

Background: Proton pump inhibitors (PPIs) are used for the long-term treatment of gastroesophageal disorders and the non-prescription medicines for acid reflux. However, there is growing concerns about PPI misuse, overuse and abuse. This study aimed to develop an animal model to examine the effects of long-term use of PPI in vivo. Methods: Twenty-one Wistar rats were given omeprazole orally for 30 days. After euthanization, the serum and stool were collected to perform MS-based quantitative analysis of metabolites. We carried out 16S rRNA gene profiling of fecal microbiota, assessed the expression of bile acid metabolism regulators and examined the immunopathological characteristics of bile ducts. Findings: After long-term PPI exposure, the fecal microbial and metabolites profile was altered and showed similarity to those observed in high-fat diet studies. Surprisingly, morphological changes were observed in the bile duct, including ductal epithelial proliferation, micropapillary growth of biliary epithelium, focal bile duct stricture formation and bile duct obstruction. These are characteristics of precancerous lesions of bile duct. FXR and RXRα expressions were significantly reduced, which were similar to that observed in cholangiocarcinoma in TCGA and Oncomine databases. Interpretation: We established a novel animal model to examine the effects of long-term use of omeprazole. The gut microbes, metabolic change and biliary epithelial hyperplasia are consequences of long-term PPI exposure. And the results showed the environment in vivo tends to a high-fat diet. Funding Statement: This work was financially supported in part by a grant from Taipei Medical University, Taiwan (TMU105-AE1-B41), a grant from Ministry of Science and Technology, Taiwan (MOST106-2320-B-038-005) and the “TMU Research Center of Cancer Translational Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animals were maintained and handled in an accredited facility in accordance with IACUC (Institutional Animal Care and Use Committee) guidelines and institutional ethics review board approval.

// Sheng-Huei Yang 1 , Hung-Yun Lin 1, 2 , Vincent H.S. Chang 3 , Chien-Chung Chen 4 , Yun-Ru Liu 5 , Jinghan Wang 6 , Keqiang Zhang 7 , Xiaoqing Jiang 6 , Yun Yen 1 1 PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 2 Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan 3 Institute of Translational Medicine, Taipei Medical University, Taipei, Taiwan 4 Graduate Institute of Biomedical Materials and Engineering, Taipei Medical University, Taipei, Taiwan 5 Office of Human Research, Taipei Medical University, Taipei, Taiwan 6 The First Department of Biliary Surgery, Eastern Hepatobiliary Surgical Hospital, The Second Military Medical University, Shanghai, China 7 Department of Molecular Pharmacology, City of Hope National Medical Center and Beckman Research Center, Duarte, California, USA Correspondence to: Yun Yen, e-mail: yyen@tmu.edu.tw Keywords: cholangiocarcinomas, combination therapy, lovastatin, gefitinib Received: January 29, 2015 Accepted: June 12, 2015 Published: June 23, 2015 ABSTRACT Gefitinib resistance has been shown to complicate cancer therapy. Lovastatin is a proteasome inhibitor that enhances gefitinib-induced antiproliferation in non-small cell lung cancer. The objective of this study is to investigate the mechanism of lovastatin-induced antiproliferation in gefitinib-resistant human cholangiocarcinoma. Two gefitinib-resistant cholangiocarcinoma cell lines, SSP-25 and HuH-28, were used in this study to determine how to compensate gefitinib resistance. The combined effect of these two drugs was examined using the MTT assay, qPCR, immunoblotting, flow cytometry, and in vivo xenograft. Results indicated that lovastatin enhanced TNF-α-induced cell death in vitro . In addition, the combination of lovastatin with gefitinib enhanced accumulation of TNF-α. Furthermore, the treatment induced a synergistic cytotoxic effect and antiproliferation through apoptosis in SSP-25 cells and cell cycle arrest in HuH-28 cells. Reproductive results were also observed in in vivo xenografts. These observations suggest that the combination of gefitinib and lovastatin might have additive antiproliferative effects against gefitinib-resistant cholangiocarcinoma cells. Based on these observations, we concluded that the combination of gefitinib and lovastatin could be used to overcome gefitinib resistance in cholangiocarcinoma cells.

Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGF-β1-mediated growth inhibition of human carcinoma cells. It is known that KLF10 mimics the anti-proliferative and apoptotic effects that TGF-β1 has on epithelial cell growth and the growth of various tumor cells; based on these findings it is considered as a tumor suppressor. KLF10 protein expression is tightly associated with cell cycle-dependent events. However, the regulatory mechanism and its biological meaning have not been identified. In this study, we have demonstrated that KLF10 is a substrate of CDK2/cyclin E and can be phosphorylated. We also have shown that KLF10 efficiently binds to CDK2, while binding much less to CDK4, and displaying no binding to Cdk6. Using mass spectrometry, site direct mutagenesis, in vitro kinase assays and depletion assays, we have established that CDK2 phosphorylates Ser206, which subsequently affects the steady state level of KLF10 in cells. Our studies have also proved that CDK2 up-regulates the protein level of KLF10 through reducing its association with SIAH1, a KLF10 E3-ubiqutin ligase involved in proteasomal degradation. Taken all together, these findings indicate that CDK2-dependent phosphorylation regulates KLF10 stability and that this affects the role of KLF10 in cell.

// Jinghan Wang 1,2 , Keqiang Zhang 2 , Jinhui Wang 3 , Xiwei Wu 3 , Xiyong Liu 2 , Bin Li 1 , Yan Zhu 4 , Yong Yu 1 , Qingbao Cheng 1 , Zhenli Hu 4 , Chao Guo 3 , Shuya Hu 2 , Bing Mu 3 , Chun-Hao Tsai 5 , Jie Li 2 , Lynne Smith 2 , Lu Yang 3 , Qi Liu 2 , Peiguo Chu 6 , Vincent Chang 7 , Baihe Zhang 1 , Mengchao Wu 1 , Xiaoqing Jiang 1 and Yun Yen 2,8 1 The First Department of Biliary Surgery, Eastern Hepatobiliary Surgical Hospital, The Second Military Medical University, Shanghai, China 2 Department of Molecular Pharmacology, City of Hope National Medical Center, Duarte, California, USA 3 The Integrative Genomics Core lab of Department of Molecular Medicine, City of Hope National Medical Center, Duarte, California, USA 4 Changhai Hospital, The Second Military Medical University, Shanghai, China 5 Department of Orthopedic Surgery, School of Medicine, China Medical University, Taichung, Taiwan 6 Department of Pathology, City of Hope National Medical Center; Duarte, California, USA 7 Program for Translation Medicine, Taipei Medical University, Taipei, Taiwan 8 PhD Program of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Correspondence to: Yun Yen, email: // Xiaoqing Jiang, email: // Keqiang Zhang, email: // Keywords : intrahepatic cholangiocarcinoma (ICC), liver kinase B1 (LKB1), global transcriptional profiling, Wnt/β-catenin, recurrence and metastasis Received : February 28, 2015 Accepted : May 13, 2015 Published : May 27, 2015 Abstract Intrahepatic cholangiocarcinoma (ICC) is a rare and highly aggressive malignancy. In this study, we identified the presence of gene deletion and missense mutation leading to inactivation or underexpression of liver kinase B1 (LKB1) tumor suppressor and excluded the involvement of LKB1 gene hypermethylation in ICC tissues. Immunohistochemical analysis showed that LKB1 was underexpressed in a portion of 326 ICC tissues compared to their adjacent normal tissues. By statistical analysis underexpression of LKB1 in ICC tissues significantly correlated with poor survival and malignant disease characteristics in ICC patients. Moreover, we showed that knockdown of LKB1 significantly enhanced growth, migration, and invasion of three LKB1-competent ICC cell lines. Global transcriptional profiling analysis identified multiple malignancy-promoting genes, such as HIF-1α , CD24 , Talin1 , Vinculin , Wnt5 , and signaling pathways including Hedgehog, Wnt/β-catenin, and cell adhesion as novel targets of LKB1 underexpression in ICC cells. Furthermore, knockdown of LKB1 gene expression dramatically enhanced Wnt/β-catenin signaling in ICC cells, while an inverse correlation between LKB1 and nuclear β-catenin was observed in ICC tissues. Our findings suggest a novel mechanism for ICC carcinogenesis in which LKB1 underexpression enhances multiple signaling pathways including Wnt/β-catenin to promote disease progression.

Various forms of cortical stimulation are capable of modulating motor cortical excitability through plasticity-like mechanisms and thus might have therapeutic potential for neurological diseases. To better understand the neural mechanism underlying the cortical neuromodulation effects and to enable translational research in rodent disease models, we developed a focused brain stimulation method using cortical electrical stimulation (CES) on the motor cortex in anesthetized rats. A specific stimulation scheme using theta burst stimulation (TBS) was then adopted to observe the facilitatory and inhibitory effects in motor cortical excitability. Adult male Sprague–Dawley rats were used for all experiments. Under urethane anesthesia, two epidural stainless steel screw electrodes were unilaterally implanted over the primary motor cortex targeting the forelimb area. Brachioradialis motor evoked potentials (MEPs) were obtained by single-pulse CES. Acute MEP changes were measured before and after intermittent and continuous TBS (iTBS and cTBS). For sham intervention, electrodes were implanted, but no TBS was delivered. To examine TBS-elicited plasticity responses, MEP amplitude was measured at baseline and for 30 min after iTBS or cTBS. The MEPs were significantly enhanced immediately after iTBS (p = 0.001) and remained enhanced for 30 min (p 0.05). The developed TBS scheme uses the focused CES method to produce consistent, rapid, and controllable electrophysiological changes in the motor cortex. In particular, the cortical plasticity can be modulated in rat models via the CES–TBS protocols. These findings may have translational relevance for establishing new therapeutic CES applications in neurological disorders.

Traditional drug delivery systems, using invasive, transdermal, and oral routes, are limited by various factors, such as the digestive system environment, skin protection, and sensory nerve stimulation. To improve the drug delivery system, we fabricated a polysaccharide-based, dissolvable microneedle-based array, which combines the advantages of both invasive and transdermal delivery systems, and promises to be an innovative solution for minimally invasive drug delivery. In this study, we designed a reusable aluminum mold that greatly improved the efficiency and convenience of microneedle fabrication. Physical characterization of the polysaccharides, individual or mixed at different ratios, was performed to identify a suitable molecule to fabricate the dissolvable microneedle. We used a vacuum deposition-based micro-molding method at low temperature to fabricate the model. Using a series of checkpoints from material into product, a systematic feedback mechanism was built into the "all-in-one" fabrication step, which helped to improve production yields. The physical properties of the fabricated microneedle were assessed. The cytotoxicity analysis and animal testing of the microneedle demonstrated the safety and compatibility of the microneedle, and the successful penetration and effective release of a model protein.

BPC 157, a pentadecapeptide with extensive healing effects, has recently been suggested to contribute to angiogenesis. However, the underlying mechanism is not yet clear. The present study aimed to explore the potential therapeutic effect and pro-angiogenic mechanism of BPC 157. As demonstrated by the chick chorioallantoic membrane (CAM) assay and endothelial tube formation assay, BPC 157 could increase the vessel density both in vivo and in vitro, respectively. BPC 157 could also accelerate the recovery of blood flow in the ischemic muscle of the rat hind limb as detected by laser Doppler scanning, indicating the promotion of angiogenesis. Histological analysis of the hind limb muscle confirmed the increased number of vessels and the enhanced vascular expression of vascular endothelial growth factor receptor 2 (VEGFR2) in rat with BPC 157 treatment. In vitro study using human vascular endothelial cells further confirmed the increased mRNA and protein expressions of VEGFR2 but not VEGF-A by BPC 157. In addition, BPC 157 could promote VEGFR2 internalization in vascular endothelial cells which was blocked in the presence of dynasore, an inhibitor of endocytosis. BPC 157 time dependently activated the VEGFR2-Akt-eNOS signaling pathway which could also be suppressed by dynasore. The increase of endothelial tube formation induced by BPC 157 was also inhibited by dynasore. This study demonstrates the pro-angiogenic effects of BPC 157 that is associated with the increased expression, internalization of VEGFR2, and the activation of VEGFR2-Akt-eNOS signaling pathway. BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation.BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation.

Deregulation of transforming growth factor (TGF)-β function is a common feature of pancreatic cancer, rendering these cancers unresponsive to TGF-β–stimulated growth inhibition. Recent findings have supported a primary role for Kruppel-like factor 10 (KLF10) as an important transcription factor involved in mediating TGF-β1 signaling. The aim of this study was to evaluate the correlation between KLF10 expression and the clinical and pathologic features of pancreatic cancer. Tissue specimens from patients with pancreatic adenocarcinoma were retrospectively collected for immunohistochemical analysis. To demonstrate that Klf10 expression was primarily regulated by methylation status, the Klf10 promoter was examined by methylation-specific PCR using a pancreatic cancer cell line (Panc-1). DNA methyltransferase (DNMT) inhibitor and small-interfering RNA depletion of DNMT genes were used to reverse KLF10 expression in the Panc-1 cells. In parallel, DNMT1 expression was evaluated in the pancreatic cancer tissue specimens. In 95 pancreatic cancer tissue specimens, KLF10 expression was inversely correlated with pancreatic cancer stage ( P = 0.01). Multivariable analysis revealed that, in addition to the presence of distant metastasis at diagnosis ( P = 0.001 and 0.001, respectively), KLF10 was another independent prognostic factor related to progression-free and overall survival ( P = 0.018 and 0.037, respectively). The loss of KLF10 expression in advanced pancreatic cancer is correlated with altered methylation status, which seems to be regulated by DNMT1. Our results suggest that KLF10 is a potential clinical predictor for progression of pancreatic cancer.

Developing lung cancer in mouse models that display similarities of both phenotype and genotype will undoubtedly provide further and better insights into lung tumor biology. Moreover, a high degree of pathophysiological similarity between lung tumors from mouse models and their human counterparts will make it possible to use these mouse models for preclinical tests. Ovine pulmonary adenocarcinomas (OPAs) present the same symptoms as adenocarcinomas in humans and are caused by a betaretrovirus. OPAs have served as an exquisite model of carcinogenesis for human lung adenocarcinomas. In this study, we characterized the histopathology and transcriptome profiles of a jaagsiekte sheep retrovirus (JSRV)-envelope protein (Env) transgenic mouse model with spontaneous lung tumors, and associations of the transcriptome profiles with tumor invasion/metastasis, especially the phenomenon of the epithelial-mesenchymal transition (EMT). Genetic information obtained from an expression array was analyzed using an ingenuity pathways analysis (IPA) and human disease database (MalaCards). By careful examination, several novel EMT-related genes were identified from tumor cells using RT-qPCR, and these genes also scored high in MalaCards. We concluded that the JSRV-Env mouse model could serve as a spontaneous lung adenocarcinoma model with a metastatic phenotype, which will benefit the study of early-onset and progression of lung adenocarcinoma. In addition, it can also be a valuable tool for biomarkers and drug screening, which will be helpful in developing intervention therapies.

TGF-β plays a significant role in regulating pancreas islet function and maintaining their mass. KLF10, a TGF-β downstream gene, belongs to a group of Kruppel-like transcription factors that bind to the promoters of target genes and produce effects that mimic TGF-β as a tumor suppressor. Using ChIP-chip screening, SEI-1 was identified as a target gene that may be regulated by KLF10. We conducted a series of assays to verify the presence of unknown regulation events between SEI-1 and KLF10. These showed that KLF10 transcriptionally activates the SEI-1 promoter and, furthermore, induces SEI-1 protein expression in pancreatic carcinoma cells. SEI-1 is one of the key factors involved in cell cycle control through the regulation of other transcription factors such as the p21(Cip1) gene. Interestingly, it has been shown previously that p21(Cip1) is indirectly activated by KLF10. Our results first demonstrated that KLF10 acts as a transcriptional activator on SEI-1, which can then result in increased p21(Cip1) expression. Furthermore, KLF10-deficiency in mice is associated with a decrease in the pancreatic islet mass, which is similar to the effects found in SEI-1 deficient mice. The KLF10-defect was also associated with the nuclear accumulation of the p21(Cip1) in islet cells. Based on our molecular and histological findings, we conclude that KLF10 plays an important role in pancreatic β-cells and this supports a functional link between KLF10 and various cell cycle regulators, most notably in the context of the pancreas.

Background Cancer is the result of a multistep process of genomic alterations, including mutations in key regulatory proteins that result in loss of balanced gene expression and subsequent malignant transformation. Throughout the various stages of colorectal carcinoma (CRC), complex genetic alterations occur, of which over-expression of growth factors, such as vascular endothelial growth factor, fibroblast growth factor and platelet-derive growth factor and their corresponding receptor tyrosine kinases, have been shown to correlate with invasiveness, tumor angiogenesis, metastasis, recurrence, and poor prognosis of colorectal cancer. To evaluate the therapeutic effect, we combined Dovitinib, an orally bioavailable, potent inhibitor of class III-V receptor tyrosine kinases with chemotherapeutic drug, oxaliplatin in preclinical models of colon cancer. Methods Human colon cancer cells with different RAS-RAF mutation status (HCT-116, HT-29, SW-480, CaCO2 and LS174T) were treated with a combination of Dovitinib and Oxaliplatin at low dosage followed by assays to investigate the effect of the combination on cell proliferation, cell migration, cell apoptosis and signaling pathways involved in molecular mechanism of drug(s). The antitumor effects of either of the drugs were compared to the combination using human colon carcinoma cell line HT-29 xenograft model. Treated vs untreated tumor sections were also compared for proliferation and angiogenesis markers by immunohistochemistry. Results The combination of dovitinib and oxaliplatin showed higher in vitro cytotoxicity in colon cell lines irrespective of their RAS-RAF status as compared to either of the drugs alone. Simultaneous inhibition of MAP kinase and AKT pathways and induction of apoptosis via activation of caspases 9/caspases 3 contributed to the synergistic effect of this combination therapy. In the xenograft model, the combination showed a significantly higher antitumor activity. Immunohistochemistry of post treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. Conclusions This study demonstrates the synergistic antitumor activity of combination of dovitinib and oxaliplatin against colon cancer with different RAS-RAF status. The combination also showed its antitumor efficacy in a multidrug resistant phenotype xenograft model. This provides a basis for further investigation for its potential in clinical setting for colorectal cancer.

KLF10 is now classified as a member of the Krüppel-like transcription factor family and acts as a tumor suppressor. Although KLF10 is originally named as TGF-β-inducible early gene-1 and mimicking the anti-proliferative effect of TGF-β in various carcinoma cells, the transcriptional upregulatory function of KLF10 has been described for a variety of cytokines and in many diseases. Through in vivo and in vitro phosphorylation assays, we identified that KLF10 is a phosphorylated protein in cells. Using yeast-two hybrid screening and site direct mutagenesis, we also identified PIN1 as a novel KLF10 associated protein. PIN1 is a peptidyl-prolyl isomerase enzyme belonging to the parvulin family, which specifically recognizes phosphorylated Ser/Thr-Pro containing substrates. Through protein–protein interaction assays, we showed that the Pro-directed Ser/Thr-Pro motif at Thr-93 in the KLF10 N-terminal region is essential for the interaction between KLF10 and PIN1. More importantly, PIN1 interacts with KLF10 in a phosphorylation-dependent manner and this interaction promotes KLF10 protein degradation in cells. Therefore, KLF10 shows shorter protein stability compared with mutant KLF10 that lacks PIN1 binding ability after cycloheximide treatments. The reversely correlated expression profile between KLF10 and PIN1 as observed in cell lines was also shown in clinic pancreatic cancer specimen. Using in vitro kinase assays and depletion assays, we were able to show that RAF-1 phosphorylates the Thr-93 of KLF10 and affects the KLF10 expression level in cells. Thus these findings as a whole indicate that RAF-1 phosphorylation and PIN1 isomerization together regulate KLF10 stability and further affect the role of KLF10 in tumor progression.

A 16-amino-acid peptide was isolated from the leaves of sweet potato. The peptide caused a rapid alkalinization response in tomato suspension culture media, a characteristic of defense peptides in plants. No post-translational modification was observed on the peptide according to MALDI-MS analysis. We have named the peptide Ipomoea batatas anti-cancer peptide (IbACP). IbACP also was shown with the ability to dose-dependently inhibit Panc-1, a pancreatic cancer line, cell proliferation. The morphological observations of the Panc-1 cells by phase contrast microscopy showed significant changes after treatment with IbACP. Moreover, caspase-3 and PARP [poly(ADP-ribose) polymerase] were activated by IbACP treatment, followed by cell death. An increase in the levels of cleaved caspase-3 and -9 was also detected by an immunoblot assay after treatment with IbACP. In addition, genomic DNA fragmentation and decreased cellular proliferation were induced when IbACP was supplied to the Panc-1 cells, further demonstrating its biological relevance. The combined data indicates that IbACP peptide may have an important role in the regulation of cellular proliferation by inducing and promoting apoptosis through the mitochondrial apoptotic pathway. This report also showed that IbACP peptide contains potent anti-cancer effects and may play an important role in herbal medicine development.

Kruppel-like family is a group of zinc-finger transcription factors which play key regulatory roles in cellular growth, development, differentiation and vascularization. Recent studies have shown that one of the members, KLF10, is specifically involved in the process of angiogenesis by acting as a key transcriptional regulator of TGF-β1 in pro-angiogenic cells differentiation and function. KLF10−/− mice also displayed impaired blood flow recovery after hindlimb ischemia. However, the mechanism of KLF10 induced angiogenesis is still not well understood. From ChIP-chip, which have been adopt to elucidate the novel target genes and signaling cascades of KLF10, COX-1 (also named as PTGS1) is one of the target genes that may be regulated by Klf10 through promoter binding. In order to investigate the function of KLF10/COX-1 axis, promoter activity, EMSA, ChIP-PCR and tube formation assays were serially performed. Our results demonstrated that KLF10 acts as a transcriptional activator on COX-1 promoter where overexpression of KLF10 induces COX-1 protein expression and mRNA expression in endothelial cells. It has been known that COX-1 is the key enzyme in prostaglandin biosynthesis which regulated angiogenesis in endothelial cells. Using tube formation assay, we further demonstrated that KLF10 overexpressed endothelial cells formed better organized three-dimensional tube structure in contrast to the control group did. To specifically investigate the role for KLF10 in angiogenesis, the its deficient mice exhibited decreased light transmission which represents the extend of platelet aggregation slowing down. Taken together, our results indicate an important role for KLF10 in angiogenesis through the activation of COX-1.

Aim: Deregulation of transforming growth factor (TGF)-beta function is a common feature of pancreatic cancer, rendering it unresponsive to radiotherapy. Kruppel-like factor 10 (KLF10) is an important transcription factor mediating TGF-beta 1 signaling. The aim of this study is to evaluate the mechanisms of Klf10 in regulating radio-sensitivity of pancreatic cancer. Methods: The expression of Klf10 and radio-sensitivity of genetically modulated and radiation selected pancreatic cancer cells were demonstrated by immunoblots and clonogenic assays. Chip-PCR and luciferase promoter assay were used to demonstrate down-stream targets interaction with Klf10. The level of autophagy, apoptosis and DNA damage after radiation was evaluated. Klf10 modulated tumor response to radiation was measured in orthotopic model. Klf10 expression was correlated with pathologic tumor regression in pancreatic cancer patients. Results: Decreasing Klf10 expression was found in Panc-1 cells with increasing radiation resistance. Over-expressing Klf10 suppressed surviving fraction of MiaPaCa cells after radiation. The observation was associated with increased apoptosis, DNA damage, and decreased autophagy. The expression of ultraviolet irradiation resistance-associated gene (UVRAG) was demonstrated to be transcriptionally suppressed by Klf10. In vitro studies demonstrated Klf10 regulated pancreatic cancer radiation sensitivity via UVRAG. In the orthotopic model, up-regulating Klf10 genetically or pharmacologically sensitized pancreatic tumors to radiation. Analysis from pancreatic cancer patients, receiving neoadjuvant chemo-radiotherapy before surgery, showed a significant correlation of high Klf10 expression with good pathologic tumor regression. Conclusions: Our results suggest Klf10 regulates radio-sensitivity of pancreatic cancer cells by transcriptionally suppressing UVRAG expression which modulates autophagy, apoptosis and DNA damage repair after radiation. Klf10 is a potential biomarker of tumor response to radiotherapy and a therapeutic target to pancreatic cancer. Disclosure: All authors have declared no conflicts of interest.