Loss of fragile WWOX gene leads to senescence escape and genome instability.

Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox-/-) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox-/- MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16Ink4a promoter hypermethylation, aberrant p53/p21Cip1/Waf1 signaling axis and accelerated p27Kip1 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21Cip1/Waf1 in late-passage Wwox-/- MEFs. Treatment of N-acetyl-L-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox-/- MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.

Polysaccharide of Ganoderma lucidum Ameliorates Cachectic Myopathy Induced by the Combination Cisplatin plus Docetaxel in Mice.

Cachexia is a lethal muscle-wasting syndrome associated with cancer and chemotherapy use. Mounting evidence suggests a correlation between cachexia and intestinal microbiota, but there is presently no effective treatment for cachexia. Whether the Ganoderma lucidum polysaccharide Liz-H exerts protective effects on cachexia and gut microbiota dysbiosis induced by the combination cisplatin plus docetaxel (cisplatin + docetaxel) was investigated. C57BL/6J mice were intraperitoneally injected with cisplatin + docetaxel, with or without oral administration of Liz-H. Body weight, food consumption, complete blood count, blood biochemistry, and muscle atrophy were measured. Next-generation sequencing was also performed to investigate changes to gut microbial ecology. Liz-H administration alleviated the cisplatin + docetaxel-induced weight loss, muscle atrophy, and neutropenia. Furthermore, upregulation of muscle protein degradation-related genes (MuRF-1 and Atrogin-1) and decline of myogenic factors (MyoD and myogenin) after treatment of cisplatin and docetaxel were prevented by Liz-H. Cisplatin and docetaxel treatment resulted in reducing comparative abundances of Ruminococcaceae and Bacteroides, but Liz-H treatment restored these to normal levels. This study indicates that Liz-H is a good chemoprotective reagent for cisplatin + docetaxel-induced cachexia. IMPORTANCE Cachexia is a multifactorial syndrome driven by metabolic dysregulation, anorexia, systemic inflammation, and insulin resistance. Approximately 80% of patients with advanced cancer have cachexia, and cachexia is the cause of death in 30% of cancer patients. Nutritional supplementation has not been shown to reverse cachexia progression. Thus, developing strategies to prevent and/or reverse cachexia is urgent. Polysaccharide is a major biologically active compound in the fungus Ganoderma lucidum. This study is the first to report that G. lucidum polysaccharides could alleviate chemotherapy-induced cachexia via reducing expression of genes that are known to drive muscle wasting, such as MuRF-1 and Atrogin-1. These results suggest that Liz-H is an effective treatment for cisplatin + docetaxel-induced cachexia.

The Establishment of a Noninvasive Bioluminescence-Specific Viral Encephalitis Model by Pseudorabies Virus-Infected NF-κBp-Luciferase Mice.

Encephalitis is a rare brain inflammation that is most commonly caused by a viral infection. In this study, we first use an in vivo imaging system (IVIS) to determine whether NF-κBp-luciferase expression could be detected in the brain of pseudorabies virus (PRV)-infected NF-κBp-luciferase mice and to evaluate proinflammatory mediators in a well-described mouse model of PRV encephalitis. In in vitro studies, we used murine microglia (BV-2) cells to demonstrate the PRV-induced encephalitis model entailing the activation of microglia cells. The results indicate that PRV-induced neuroinflammation responses through the induction of IL-6, TNF-α, COX-2, and iNOS expression occurred via the regulation of NF-κB expression in BV-2 cells. In in vivo studies, compared with MOCK controls, the mice infected with neurovirulent PRV exhibited significantly elevated NF-κB transcription factor activity and luciferase protein expression only in the brain by IVIS. Mild focal necrosis was also observed in the brain. Further examination revealed biomarkers of inflammation, including inducible cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and tumor necrosis factor (TNF)-α and interleukin (IL)-6, both of which constituted proinflammatory cytokines. PRV infection stimulated inflammation and COX-2 and iNOS expression of IL-6 and TNF-α. The presented results herein suggest that PRV induces iNOS and COX-2 expression in the brain of NF-κBp-luciferase mice via NF-κB activation. In conclusion, we used NF-κBp-luciferase mice to establish a specific virus-induced encephalitis model via PRV intranasal infection. In the future, this in vivo model will provide potential targets for the development of new therapeutic strategies focusing on NF-κB inflammatory biomarkers and the development of drugs for viral inflammatory diseases.

Ornithine decarboxylase functions in both autophagy and apoptosis in response to ultraviolet B radiation injury.

We present a mechanism for how ornithine decarboxylase (ODC) regulates the crosstalk between autophagy and apoptosis. In cancer cells, low-intensity ultraviolet B (UVBL ) induces autophagy while high-intensity UVB (UVBH ) induces apoptosis. Overexpression of ODC decreases UVBL -induced autophagy by inhibiting Atg5-Atg12 conjugation and suppressing the expression of autophagy markers LC3, Atg7, Atg12, and BECN1 proteins. In contrast, when ODC-overexpressing cells are exposed to UVBH radiation, the levels of LC3-II, Atg5-Atg12 conjugate, BECN1, Atg7, and Atg12 increase, while the apoptosis marker cleaved-PARP proteins decrease, indicating that ODC overexpression induced UVBH -induced autophagy but inhibited UVBH -induced cellular apoptosis. Additionally, when exposed to UVBH radiation, silencing BECN1, Atg5, and Atg12 genes results in a decrease in the level of LC3-II proteins but an increase in the level of cleaved-PARP proteins, and apoptotic bodies were significantly increased while autophagosomes were significantly decreased. These findings imply that ODC inhibits apoptosis in cells via the autophagy pathway. The role of Atg12 in ODC-overexpressing cells exposed to UVBH radiation is investigated using site-directed mutagenesis. Our results indicate that the Atg12-D111S mutant has increased cell survival. The Atg12-ΔG186 mutant impairs autophagy and enhances apoptosis. We demonstrate that when ODC-overexpressing cells are silenced for the Atg12 protein, autophagy and apoptosis are strongly affected, and ODC-induced autophagy protects against UVBH -induced apoptosis via the Atg12 protein.

Periodontitis is associated with incidental valvular heart disease: A nationwide population-based cohort study.

AIM: Periodontitis and valvular heart disease (VHD) are common diseases. Both diseases are related to chronic inflammation and share many common risk factors. Previous periodontal studies had focused mainly on atherosclerotic cardiovascular disease. This study aimed to determine whether periodontitis is associated with the development of VHD. MATERIALS AND METHODS: This was a retrospective nationwide cohort study using Taiwan's Longitudinal Health Insurance Database. Using ICD-9-CM coding, both the periodontitis and non-periodontitis groups were matched. RESULTS: There were 8483 cases and 4919 cases of VHD diagnosed in the periodontitis group and non-periodontitis group, respectively. The cumulative incidence of VHD was significantly higher in the periodontitis group (log-rank test, p < .001), with the incidence density of 6.44 (95% CI, 6.31-6.58) per 1000 person-years in the periodontitis group compared to 4.65 (95% CI, 4.52-4.78) in the non-periodontitis group. The relative risk for VHD was 1.39 (95% CI, 1.34-1.44). After multivariate analysis, periodontitis was independently associated with a risk for VHD (HR, 1.38; 95% CI, 1.33-1.42, p < .001). Intensive treatment of periodontitis significantly lowered the risk for VHD (HR, 0.68; 95% CI, 0.60-0.77, p < .001). CONCLUSIONS: Periodontitis was significantly associated with the development of VHD. Treatment of periodontitis reduced the risk for VHD.

Early Gut Microbiota Changes in Preterm Infants with Bronchopulmonary Dysplasia: A Pilot Case-Control Study.

OBJECTIVE: Bronchopulmonary dysplasia (BPD) is a complex chronic lung disease that primarily affects premature or critically ill infants. This pilot study investigated early changes in gut microbiota composition in BPD patients and explored the potential risk factors associated with these changes. STUDY DESIGN: Preterm infants admitted to our neonatal intensive care unit with a gestational age of 26 to 32 weeks were prospectively surveyed and eligible for stool collection on days 7 and 28 of postnatal age between February 2016 and June 2017. A 16S rRNA sequencing approach was applied to compare the gut microbiota composition between the BPD group and controls. Multiple linear regression analysis was used to identify the predictor variables. RESULTS: Eight subjects in the BPD group and 10 subjects in the preterm group were analyzed during the observation period. Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes were the four dominant bacteria phyla of intestinal microflora. A significantly lower diversity of gut microbiota was observed in the BPD group compared with the preterm group on day 28 (number of observed operational taxonomic units, p = 0.034; abundance-based coverage estimator, p = 0.022; Shannon index, p = 0.028). Multiple linear regression analysis revealed that high Neonatal Therapeutic Intervention Scoring System score (≧19) at 24 hours was statistically significant in predicting the proportion of aerobic with facultative anaerobic bacteria on day 28 (p = 0.002). CONCLUSION: Infants with BPD are prone to develop gut dysbiosis in early life. A higher severity of illness and treatment intensity may indicate a higher risk of disrupting an anaerobic environment in the gut during the first month of life. KEY POINTS: · BPD patients are prone to develop gut dysbiosis.. · Lower diversity of gut microbiota.. · Higher risk of disrupting anaerobic environment..

Lactobacillus Attenuate the Progression of Pancreatic Cancer Promoted by Porphyromonas Gingivalis in K-rasG12D Transgenic Mice.

Accumulating evidence suggests that there is a link between the host microbiome and pancreatic carcinogenesis, and that Porphyromonas gingivalis (P. gingivalis) increases the risk of developing pancreatic cancer. The aim of the current study was to clarify the role of P. gingivalis in the pathogenesis of pancreatic cancer and the potential immune modulatory effects of probiotics. The six-week-old LSL-K-rasG12D; Pdx-1-cre (KC) mice smeared P. gingivalis on the gums, causing pancreatic intraepithelial neoplasia (PanIN) after four weeks to be similar to the extent of lesions in untreated KC mice at 24 weeks. The oral inoculation of P. gingivalis of six-week-old LSL-K-rasG12D; Pdx-1-cre (KC) mice caused significantly pancreatic intraepithelial neoplasia (PanIN) after treatment four weeks is similar to the extent of lesions in untreated KC mice at 24 weeks. The pancreas weights of P. gingivalis plus probiotic-treated mice were significantly lower than the mice treated with P. gingivalis alone (P = 0.0028). The histological expressions of Snail-1, ZEB-1, collagen fibers, Galectin-3, and PD-L1 staining in the pancreas were also notably lower. In addition, probiotic administration reduced the histological expression of Smad3 and phosphorylated Smad3 in P. gingivalis treated KC mice. We demonstrated that oral exposure to P. gingivalis can accelerate the development of PanIN lesions. Probiotics are likely to have a beneficial effect by reducing cancer cell proliferation and viability, inhibiting PanIN progression, and cancer cell metastasis (Epithelial-mesenchymal transition, EMT). The transforming growth factor-ß signaling pathway may be involved in the tumor suppressive effects of probiotics.

The synergistic tumor growth-inhibitory effect of probiotic Lactobacillus on transgenic mouse model of pancreatic cancer treated with gemcitabine.

Pancreatic cancer is one of the most lethal and chemo-resistant cancers worldwide. Growing evidence supports the theory that the gut microbiota plays an essential role in modulating the host response to anti-cancer therapy. The present study aimed to explore the effect of probiotics as an adjuvant during chemotherapy for pancreatic cancer. An LSL-KrasG12D/--Pdx-1-Cre mouse model of pancreatic ductal adenocarcinoma (PDAC) was created to study the effects of using four-week multi-strain probiotics (Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89) as an adjuvant therapy for controlling cancer progression. At 12 weeks of age, pancreatitis was induced in the mice by two intraperitoneal injection with caerulein (25 µg/kg 2 days apart). Over the next 4 weeks the mice were treated with intraperitoneal injections of gemcitabine in combination with the oral administration of probiotics. The pancreas was then harvested for analysis. Following caerulein treatment, the pancreases of the LSL-KrasG12D/--Pdx-1-Cre transgenic mice exhibited more extensive pancreatic intraepithelial neoplasia (PanIN) formation. Combined treatment with gemcitabine and probiotics revealed a lower grade of PanIN formation and a decrease in the expression of vimentin and Ki-67. Mice that received gemcitabine in combination with probiotics had lower aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Notably, the use of high-dose probiotics alone without gemcitabine also had an inhibitory effect on PanIN changes and serum liver enzyme elevation. These findings suggest that probiotics are able to make standard chemotherapy more effective and could help improve the patient's tolerance of chemotherapy.

Constipation might be associated with risk of allergic rhinitis: A nationwide population-based cohort study.

BACKGROUND: Allergic rhinitis (AR) is a burdensome respiratory disorder whose etiology and pathophysiology remain controversial and most likely multifactorial. Accumulated evidence indicates that gut dysbiosis contributes to AR via the gut-airway axis. Constipation could result in alteration of the intestinal microflora. The clinical impact of constipation on AR has not been studied. We aimed to evaluate the risk of AR in constipated patients using a nationwide longitudinal population-based cohort. METHODS: We identified 57786 patients with constipation and 57786 matched controls between 1999 and 2013 from the Longitudinal Health Insurance Database, which is a subset of Taiwanese National Health Insurance Research Database. Propensity score analysis was used for matching age, sex, comorbidities, and medications at a ratio of 1:1. Multiple Cox regression and subgroup analyses were used to estimate the adjusted hazard ratio of AR. RESULTS: The incidence of AR was 32.2 per 1,000 person-years in constipated patients, which was twice that of non-constipated patients. After adjustment for patients' age, gender, comorbidities, and medications, patients with constipation had a 2.3-fold risk of AR compared to those without constipation (adjusted hazard ratio [aHR]: 2.30; 95% CI, 2.23-2.37). In subgroup analyses, patients aged 20-39 years had a 2.24-fold higher risk of AR in the constipation cohort (aHR; 95% CI, 2.12-2.36). Patients aged <20, 40-64, and ≥65 years had a 2.09, 2.05, and 2.07-fold risk of AR in the constipation cohort, respectively (aHR; 95% CI, 1.98-2.20, 1.94-2.18, and 1.92-2.23). Also, patients with constipation had a higher likelihood of AR, regardless of sex, and with or without comorbidities including hyperlipidemia, hypertension, chronic kidney disease, chronic liver disease, diabetes, chronic obstructive pulmonary disease, rheumatoid arthritis, dyspepsia, irritable bowel syndrome, and anxiety. CONCLUSION: Constipation might be associated with an increased risk of incidental AR. It seems that physicians should keep a higher index of suspicion for AR in people with constipation. The patency issue of gut could not be ignored in patients with AR.

BMI1-Mediated Pemetrexed Resistance in Non-Small Cell Lung Cancer Cells Is Associated with Increased SP1 Activation and Cancer Stemness.

Lung cancer is the leading cause of cancer death worldwide and the therapeutic strategies include surgery, chemotherapy and radiation therapy. Non-small cell lung cancers (NSCLCs) account for around 85% of cases of lung cancers. Pemetrexed is an antifolate agent that is currently used as the second line chemotherapy drug in the treatment of advanced NSCLC patients with a response rate of 20-40%. The search for any combination therapy to improve the efficacy of pemetrexed is required. The existence of cancer stem cells (CSCs) is considered as the main reason for drug resistance of cancers. In this study, we first found that pemetrexed-resistant NSCLC cells derived from A549 cells displayed higher CSC activity in comparison to the parental cells. The expression of CSC related proteins, such as BMI1 or CD44, and the epithelial-mesenchymal transition (EMT) signature was elevated in pemetrexed-resistant NSCLC cells. We next discovered that the overexpression of BMI1 in A549 cells caused the pemetrexed resistance and inhibition of BMI1 by a small molecule inhibitor, PTC-209, or transducing of BMI1-specific shRNAs suppressed cell growth and the expression of thymidylate synthase (TS) in pemetrexed-resistant A549 cells. We further identified that BMI1 positively regulated SP1 expression and treatment of mithramycin A, a SP1 inhibitor, inhibited cell proliferation, as well as TS expression, of pemetrexed-resistant A549 cells. Furthermore, overexpression of BMI1 in A549 cells also caused the activation of EMT in and the enhancement of CSC activity. Finally, we demonstrated that pretreatment of PTC-209 in mice bearing pemetrexed-resistant A549 tumors sensitized them to pemetrexed treatment and the expression of Ki-67, BMI1, and SP1 expression in tumor tissues was observed to be reduced. In conclusion, BMI1 expression level mediates pemetrexed sensitivity of NSCLC cells and the inhibition of BMI1 will be an effective strategy in NSCLC patients when pemetrexed resistance has developed.

Targeting a cysteine protease from a pathobiont alleviates experimental arthritis.

BACKGROUND: Several lines of evidence suggest that the pathobiont Porphyromonas gingivalis is involved in the development and/or progression of auto-inflammatory diseases. This bacterium produces cysteine proteases, such as gingipain RgpA, endowed with the potential to induce significant bone loss in model systems and in patients. OBJECTIVE: We sought to gain further insight into the role of this pathobiont in rheumatoid arthritis (RA) and to identify novel therapeutic targets for auto-inflammatory diseases. METHODS: We profiled the antibody response to RgPA-specific domains in patient sera. We also tested the potential protective effects of RgpA domains in an experimental arthritis model. RESULTS: Pre-immunization of rats with purified recombinant RgpA domains alleviated arthritis in the joints of the rodents and reduced bone erosion. Using a functional genomics approach at both the mRNA and protein levels, we report that the pre-immunizations reduced arthritis severity by impacting a matrix metalloprotease characteristic of articular injury, a chemokine known to be involved in recruiting inflammatory cells, and three inflammatory cytokines. Finally, we identified an amino acid motif in the RgpA catalytic domain of P. gingivalis that shares sequence homology with type II collagen. CONCLUSION: We conclude that pre-immunization against gingipain domains can reduce the severity of experimentally induced arthritis. We suggest that targeting gingipain domains by pre-immunization, or, possibly, by small-molecule inhibitors, could reduce the potential of P. gingivalis to translocate to remote tissues and instigate and/or exacerbate pathology in RA, but also in other chronic inflammatory diseases.

Wwox deficiency leads to neurodevelopmental and degenerative neuropathies and glycogen synthase kinase 3ß-mediated epileptic seizure activity in mice.

Human WWOX gene resides in the chromosomal common fragile site FRA16D and encodes a tumor suppressor WW domain-containing oxidoreductase. Loss-of-function mutations in both alleles of WWOX gene lead to autosomal recessive abnormalities in pediatric patients from consanguineous families, including microcephaly, cerebellar ataxia with epilepsy, mental retardation, retinal degeneration, developmental delay and early death. Here, we report that targeted disruption of Wwox gene in mice causes neurodevelopmental disorders, encompassing abnormal neuronal differentiation and migration in the brain. Cerebral malformations, such as microcephaly and incomplete separation of the hemispheres by a partial interhemispheric fissure, neuronal disorganization and heterotopia, and defective cerebellar midline fusion are observed in Wwox-/- mice. Degenerative alterations including severe hypomyelination in the central nervous system, optic nerve atrophy, Purkinje cell loss and granular cell apoptosis in the cerebellum, and peripheral nerve demyelination due to Schwann cell apoptosis correspond to reduced amplitudes and a latency prolongation of transcranial motor evoked potentials, motor deficits and gait ataxia in Wwox-/- mice. Wwox gene ablation leads to the occurrence of spontaneous epilepsy and increased susceptibility to pilocarpine- and pentylenetetrazol (PTZ)-induced seizures in preweaning mice. We determined that a significantly increased activation of glycogen synthase kinase 3ß (GSK3ß) occurs in Wwox-/- mouse cerebral cortex, hippocampus and cerebellum. Inhibition of GSK3ß by lithium ion significantly abolishes the onset of PTZ-induced seizure in Wwox-/- mice. Together, our findings reveal that the neurodevelopmental and neurodegenerative deficits in Wwox knockout mice strikingly recapitulate the key features of human neuropathies, and that targeting GSK3ß with lithium ion ameliorates epilepsy.

Lung Tumorigenesis Alters the Expression of Slit2-exon15 Splicing Variants in Tumor Microenvironment.

Slit2 expression is downregulated in various cancers, including lung cancer. We identified two Slit2 splicing variants at exon15-Slit2-WT and Slit2-ΔE15. In the RT-PCR analyses, the Slit2-WT isoform was predominantly expressed in all the lung cancer specimens and in their normal lung counterparts, whereas Slit2-ΔE15 was equivalently or predominantly expressed in 41% of the pneumothorax specimens. A kRasG12D transgenic mice system was used to study the effects of tumorigenesis on the expressions of the Slit2-exon15 isoforms. The results revealed that a kRasG12D-induced lung tumor increased the Slit2-WT/Slit2-ΔE15 ratio and total Slit2 expression level. However, the lung tumors generated via a tail vein injection of lung cancer cells decreased the Slit2-WT/Slit2-ΔE15 ratio and total Slit2 expression level. Interestingly, the lipopolysaccharide (LPS)-induced lung inflammation also decreased the Slit2-WT/Slit2-ΔE15 ratio. Since Slit2 functions as an anti-inflammatory factor, the expression of Slit2 increases in kRasG12D lungs, which indicates that Slit2 suppresses immunity during tumorigenesis. However, an injection of lung cancer cells via the tail vein and the LPS-induced lung inflammation both decreased the Slit2 expression. The increased Slit2 in the tumor microenvironment was mostly Slit2-WT, which lacks growth inhibitory activity. Thus, the results of our study suggested that the upregulation of Slit2-WT, but not Slit2-ΔE15, in a cancer microenvironment is an important factor in suppressing immunity while not interfering with cancer growth.

The microRNA expression profiles in extracellular vesicles from HeLa cancer cells in response to cationic lipid- or polyethylenimine-mediated gene delivery.

Intercellular communication is known to be involved in various stages of tumour development and metastasis through the secretion of extracellular vesicles (EVs) containing messengers such as microRNAs (miRNAs). Therefore, this study explored miRNA profiles in cancer cell-derived EVs after non-viral gene delivery in order to better understand the molecular information of intercellular communication in cancer cells after gene delivery. Two commonly used non-viral vectors (Lipofectamine 2000 and jet polyethylenimine) were used for the delivery of gene fluorescent protein plasmid in HeLa cancer cells. EVs were extracted and the contents of their RNA were subjected to the next-generation sequencing. In order to illustrate the common characteristics of non-viral vectors in the cancer cells, two overlapped up-regulated miRNAs (hsa-miR-143-3p and hsa-miR-193b-3p) were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction in the secreted EVs in response to both lipoplexes and polyplexes. The prediction of target genes and molecular pathways involved in these two miRNAs were determined, and the protein expressions related to the pathways of cell death and stress in HeLa cells were identified. Hsa-miR-143-3p and hsa-miR-193b-3p were found to be up-regulated by the use of different non-viral vectors and can thus serve as potential targets of non-viral cancer gene therapy.

The Chinese medicine JC-001 enhances the chemosensitivity of Lewis lung tumors to cisplatin by modulating the immune response.

BACKGROUND: JC-001 is a Chinese medicine that can modulate the immunity in Hepa 1-6 tumor-bearing mice, and we questioned whether JC-001 can serve as efficient adjuvant chemotherapy. We aimed to identify a novel approach for enhancing cis-diamminedichloroplatinum (II) (CDDP)-based chemotherapy by immunomodulation. METHODS: The anti-tumor activity in vitro was determined based on foci formation and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A LLC1 tumor xenograft model was used to analyze the activity of tumor rejection in vivo. The tumors were analyzed through hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) staining and cytokine arrays. RESULTS: JC-001 suppressed foci formation and reduced the viability of Lewis lung carcinoma (LLC1) cells in vitro. JC-001 suppressed LLC1 tumor growth in immunodeficient BALB/c nude mice and in immunocompetent C57BL/6 mice to an even greater extent. Furthermore, JC-001 up-regulated interferon-γ expression in the tumor microenvironment, enhanced the Th1 response in tumor-bearing mice, and increased the chemosensitivity of LLC1 tumors to CDDP chemotherapy. The results of our study suggest that JC-001 is associated with low cytotoxicity and can significantly suppress tumor growth by enhancing the Th1 response. CONCLUSION: JC-001 is a Chinese medicine with potential clinical applications in CDDP-based chemotherapeutic regimens.

Phellinus linteus Mycelium Alleviates Myocardial Ischemia-Reperfusion Injury through Autophagic Regulation.

The incidence of myocardial ischemia-reperfusion (IR) injury is rapidly increasing around the world and this disease is a major contributor to global morbidity and mortality. It is known that regulation of programmed cell death including apoptosis and autophagy reduces the impact of myocardial IR injury. In this study, the cardioprotective effects and underlying mechanisms of Phellinus linteus (Berk. and Curt.) Teng, Hymenochaetaceae (PL), a type of medicinal mushroom, were examined in rats subjected to myocardial IR injury. The left main coronary artery of rats was ligated for 1 h and reperfused for 3 h. The arrhythmia levels were monitored during the entire process and the infarct size was evaluated after myocardial IR injury. Furthermore, the expression levels of proteins in apoptotic and autophagic pathways were observed. Pretreatment with PL mycelium (PLM) significantly reduced ventricular arrhythmia and mortality due to myocardial IR injury. PLM also significantly decreased myocardial infarct size and plasma lactate dehydrogenase level after myocardial IR injury. Moreover, PLM administration resulted in decreased caspase 3 and caspase 9 activation and increased Bcl-2/Bax ratio. Phosphorylation level of AMPK was elevated while mTOR level was reduced. Becline-1 and p62 levels decreased. These findings suggest that PLM is effective in protecting the myocardium against IR injury. The mechanism involves mediation through suppressed pro-apoptotic signaling and regulation of autophagic signaling, including stimulation of AMPK-dependent pathway and inhibition of beclin-1-dependent pathway, resulting in enhancement of protective autophagy and inhibition of excessive autophagy.

The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells.

Despite the greater potential for clinical applications of autophagic microRNA (miRNA) delivery, the vector-related effects of such delivery on cells have not been fully explored. In this study, autophagic mmu-miR-494-3p (miR-494) in mouse embryonic fibroblast (MEF) cells was selected as a cargo miRNA, and two commonly used non-viral carriers (Lipofectamine 2000 (Lipo) and polyethylenimine 25K (PEI)), were used as delivery vectors to mechanistically elucidate its vector-related effects. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with Lipo (miR-494 lipoplexes) were lower than those of the complexes of miR-494 with PEI (miR-494 polyplexes) in MEF cells. The indicator of autophagic activity (LC3 (microtubule-associated protein 1 light chain 3)-II/LC3-I ratio) in cells treated with miR-494 lipoplexes was higher than that in cells treated with miR-494 polyplexes. Lipo alone and PEI alone induced slight increases in the quantitative levels of miR-494 in cells, but Lipo resulted in higher gene and protein expressions of target Igf1, higher LC3-II/LC3-I ratios, and higher autophagosome formation than PEI. We also demonstrated that the delivery of miR-494 by Lipo was more involved in apoptotic caspase-3 pathways than such delivery by PEI. By applying knock-out atg5 gene in MEF cells, we found that autophagy played a protective role in cell survival and also affected cellular uptake, the quantitative level of miR-494, and target gene Igf1 regulation of delivery systems. Taken together, these results indicate that there are different degrees of responses in MEF cells for autophagic miR-494 delivery through the use of Lipo or PEI vectors that also induce autophagy in cells. Therefore, Lipo and PEI vectors cannot be treated as inert molecules, and their effects must be known and evaluated when they are used in autophagic miRNA delivery systems. Most importantly, understanding these vector-related effects on cells will be helpful in achieving optimal delivery of autophagic miRNAs.

Antitumor Activity of the Chinese Medicine JC-001 Is Mediated by Immunomodulation in a Murine Model of Hepatocellular Carcinoma.

JC-001 is a Chinese medicine that has been used to treat liver disease; however, its significance in cancer treatment has not been characterized. In this study, we used an immunocompetent tumor model to characterize the antitumor activity of JC-001. A total of 48 Hepa 1-6 tumor-bearing C57BL/6 mice were randomly grouped into 4 groups and treated with H2O or JC-001 via oral administration. After hepatoma cell lines, including HepG2, Hep3B, SK-Hep-1, and Hepa 1-6, underwent 96 hours of JC-001 treatment, a low cytotoxic effect was observed. In contrast, no direct cytotoxic effect of JC-001 on a normal human liver cell line, THLE-3, was observed under the same incubation conditions. Using a murine tumor model, we found that tumor growth could be inhibited by JC-001 in C57BL/6 mice but not in immunodeficient mice. Histopathological analysis of tumors from C57BL/6 mice revealed immune cell infiltration in tumors from the JC-001-treated group, as observed by hematoxylin and eosin staining; in addition, Ki67, hypoxia-inducible factor-1-α, and high mobility group box 1 expression levels were suppressed in the tumors. Both the coculture assay and murine spleen mRNA quantitative PCR analyses demonstrated that JC-001 could suppress Th17 immunity. Our data suggest that JC-001 is a Chinese medicine with low cytotoxicity that can significantly suppress tumor growth by immune regulation. This herbal remedy has great potential for future clinical application in hepatoma therapy.

Promotion of metastasis of thyroid cancer cells via NRP-2-mediated induction.

Tumor-node-metastasis is one of the leading causes of morbidity and mortality in thyroid cancer patients. Upregulation of vascular endothelial growth factor-C (VEGF-C) increases the migratory ability of thyroid cancer cells to lymph nodes. Expression of neuropilin-2 (NRP-2), the co-receptor of VEGF-C, has been reported to be correlated with lymph node metastasis in human thyroid cancer. The present study investigated the role of VEGF-C/NRP-2 signaling in the regulation of metastasis of two different types of human thyroid cancer cells. The results indicated that the VEGF-C/NRP-2 axis significantly promoted the metastatic activities of papillary thyroid carcinoma cells through the activation of the mitogen-activated protein kinase (MAPK) kinase (MEK)/extracellular signal-regulated kinase and p38 MAPK signaling cascades. However, neither MEK or p38 MAPK inhibitors produced significant inhibition of the migratory activity and invasiveness regulated by the VEGF-C/NRP-2 axis in follicular thyroid carcinoma cells. Finally, VEGF-C/NRP-2-mediated invasion and migration of thyroid cancer cells required the expression of NRP-2. The present results demonstrate that the promotion of metastasis by VEGF-C is mainly due to the upregulation of NRP-2 in thyroid cancer cells, and this metastatic activity regulated by the VEGF-C/NRP-2 axis provides further insight into the process of tumor metastasis.

Lumbrokinase attenuates myocardial ischemia-reperfusion injury by inhibiting TLR4 signaling.

Lumbrokinase, a novel antithrombotic agent, purified from the earthworm Lumbricus rubellus, has been clinically used to treat stroke and cardiovascular diseases. However, inflammatory responses associated with the cardioprotective effect of lumbrokinase remain unknown. In this study, the signaling pathways involved in lumbrokinase-inhibited expressions of inflammation mediators were investigated in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left main coronary artery of anesthetized rats was subjected to 1h occlusion and 3h reperfusion. The animals were treated with/without lumbrokinase and the severities of I-R-induced arrhythmias and infarction were compared. Lumbrokinase inhibited I-R-induced arrhythmias and reduced mortality, as well as decreased the lactate dehydrogenase levels in carotid blood. Lumbrokinase also inhibited the enhancement of I-R induced expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), and matrix metalloproteinase (MMP)-9 through toll-like receptor 4 (TLR4) signaling pathway. Moreover, our results demonstrated that stimulation with lumbrokinase decreases the phosphorylation of JNK, IκB, and NF-κB. These findings suggested that lumbrokinase is a potent cardioprotective drug in rats with I-R injury. The cardioprotective effects of lumbrokinase may be correlated with its inhibitory effect on the I-R-induced expressions of COX-2, iNOS and MMP-9, mediated by TLR4 signaling through JNK and NF-κB pathways.