Colorectal Cancer Therapy Using a Pediococcus pentosaceus SL4 Drug Delivery System Secreting Lactic Acid Bacteria-Derived Protein p8.

Despite decades of research into colorectal cancer (CRC), there is an ongoing need for treatments that are more effective and safer than those currently available. Lactic acid bacteria (LAB) show beneficial effects in the context of several diseases, including CRC, and are generally regarded as safe. Here, we isolated a Lactobacillus rhamnosus (LR)-derived therapeutic protein, p8, which suppressed CRC proliferation. We found that p8 translocated specifically to the cytosol of DLD-1 cells. Moreover, p8 down-regulated expression of Cyclin B1 and Cdk1, both of which are required for cell cycle progression. We confirmed that p8 exerted strong anti-proliferative activity in a mouse CRC xenograft model. Intraperitoneal injection of recombinant p8 (r-p8) led to a significant reduction (up to 59%) in tumor mass when compared with controls. In recent years, bacterial drug delivery systems (DDSs) have proven to be effective therapeutic agents for acute colitis. Therefore, we aimed to use such systems, particularly LAB, to generate the valuable therapeutic proteins to treat CRC. To this end, we developed a gene expression cassette capable of inducing secretion of large amounts of p8 protein from Pediococcus pentosaceus SL4 (PP). We then confirmed that this protein (PP-p8) exerted anti-proliferative activity in a mouse CRC xenograft model. Oral administration of PP-p8 DDS led to a marked reduction in tumor mass (up to 64%) compared with controls. The PP-p8 DDS using LAB described herein has advantages over other therapeutics; these advantages include improved safety (the protein is a probiotic), cost-free purification, and specific targeting of CRC cells.

Anti-inflammatory effects of apocynin on dextran sulfate sodium-induced mouse colitis model.

BACKGROUND AND AIM: Various drugs have been developed for inflammatory bowel disease (IBD), but still there are limitations in the treatment due to the insufficient responses and significant adverse effects of immunosuppressant. Apocynin is an NADPH-oxidase inhibitor with established safety profiles. We aimed to investigate the protective efficacy of apocynin in IBD using chemical-induced mouse colitis model. METHOD: We induced experimental colitis by administrating 5% dextran sulfate sodium (DSS) to 8-week old BALB/c mouse for 11 days. Apocynin (400 mg/kg) or sulfasalazine (150 mg/kg) were administeredduring7 days. We monitored bodyweight daily and harvested colon and spleen at day 11 to check weight and length. We also examined histopathologic change and pro-, anti-inflammatory cytokines and enzymes from harvested colons (iNOS, COX-2, TNF-α, MCP-1, p-NrF2, and HO-1). RESULT: Apocynin significantly alleviated weight reduction induced by DSS treatment (21.64 ± 0.55 for Apocynin group vs. 20.33 ± 0.90 for DSS group, p = 0.005). Anti-inflammatory efficacy of apocynin was also shown by the recovery of colon weight and length. Histopathologic examination revealed significantly reduced inflammatory foci and erosions by apocynin treatment. Colonic expression of iNOS, COX-2, TNF-α, and MCP-1 was decreased significantly in the apocynin treated group. Anti-inflammatory mediators Nrf2 and HO-1 were activated significantly in apocynin treated mouse. CONCLUSION: Apocynin showed significant anti-inflammatory efficacy against chemically induced colonic inflammation. This study also revealed the unique action of apocynin compared to the currently prescribed drug, sulfasalazine. Given its excellent safety profile and potent efficacy with novel action mechanism, apocynin can be a new therapeutic molecule for the IBD treatment, which can be added to the currently available drugs.

Sequestration of sorcin by aberrant forms of tau results in the defective calcium homeostasis.

Neurofi brillary tangles (NFTs) of microtubule-associated protein tau are a pathological hallmark of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress has been known to be involved in the pathogenesis of AD. However, the exact role of ER stress in tau pathology has not yet been clearly elucidated. In present study, the possible relationship between tau pathology and ER stress was examined in terms of sorcin, which is a calcium binding protein and plays an important role in calcium homeostasis. Our previous yeast two hybrid study showed that sorcin is a novel tau interacting protein. Caspase-3-cleaved tau (T4C3) showed significantly increased tau-sorcin interaction compared to wild type tau (T4). Thapsigargin-induced ER stress and co-expression of constitutively active GSK3ß (GSK3ß-S9A) also exhibited significantly increased tau-sorcin interactions. T4C3-expressing cells showed potentiated thapsigargin-induced apoptosis and disruption of intracellular calcium homeostasis compared to T4-expressing cells. Overexpression of sorcin signifi cantly attenuated thapsigargin-induced apoptosis and disruption of calcium homeostasis. In contrary, siRNA-mediated knock-down of sorcin showed significantly increased thapsigargin-induced apoptosis and disruption of calcium homeostasis. These data strongly suggest that sequestration of sorcin by aberrant forms of tau compromises the function of sorcin, such as calcium homeostasis and cellular resistance by ER stress, which may consequently result in the contribution to the progression of AD.

Quercetin-3-O-ß-D-Glucuronide Suppresses Lipopolysaccharide-Induced JNK and ERK Phosphorylation in LPS-Challenged RAW264.7 Cells.

Quercetin, a flavonol, has been reported to exhibit a wide range of biological properties including anti-oxidant and anti-inflammatory activities. However, pharmacological properties of quercetin-3-O-ß-D-glucuronide (QG), a glycoside derivative of quercetin, have not been extensively examined. The objective of this study is to elucidate the anti-inflammatory property and underlying mechanism of QG in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells in comparison with quercetin. QG significantly suppressed LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE2, and pro-inflammatory protein expressions of iNOS and COX-2. To elucidate the underlying mechanism of the anti-inflammatory property of QG, involvement of MAPK signaling pathways was examined. QG significantly attenuated LPS-induced activation of JNK and ERK in concentration-dependent manners with a negligible effect on p38. In conclusion, the present study demonstrates QG exerts anti-inflammatory activity through the suppression of JNK and ERK signaling pathways in LPS-challenged RAW264.7 macrophage cells.

Isorhamnetin-3-O-Glucuronide Suppresses JNK and p38 Activation and Increases Heme-Oxygenase-1 in Lipopolysaccharide-Challenged RAW264.7 Cells.

Preclinical Research Isorhanmetin (ISH) exhibits a wide range of biological properties including anticancer, anti-oxidant and anti-inflammatory activities. However, the pharmacological properties of isorhamnetin-3-O-glucuronide (IG), a glycoside derivative of ISH, have not been extensively examined. The objective of this study was to examine the anti-inflammatory properties of IG and its underlying mechanism in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells in comparison with its aglycone, ISH. IG suppressed LPS-induced extracellular secretion of the proinflammatory mediators, nitric oxide (NO) and PGE2 , and proinflammatory protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2. IG also increased expression of heme oxygenase-1 (HO-1). IG attenuated LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 in a concentration-dependent manner with negligible suppression of extracellular signal-regulated kinases (ERK) phosphorylation. In conclusion, this study demonstrates that IG exerts anti-inflammatory activity by increasing HO-1 expression and by suppressing JNK and p38 signaling pathways in LPS-challenged RAW264.7 macrophage cells. Drug Dev Res 77 : 143-151, 2016. © 2016 Wiley Periodicals, Inc.

3,4,5-trihydroxycinnamic acid inhibits lipopolysaccharide (LPS)-induced inflammation by Nrf2 activation in vitro and improves survival of mice in LPS-induced endotoxemia model in vivo.

NF-E2-related factor 2 (Nrf2) has been demonstrated to be a key transcription factor regulating the anti-inflammatory genes including heme oxygenase-1 (HO-1) in experimental sepsis models. Based on the fact that 3,4,5-trihydorxycinnamic acid (THC) has been reported to possess anti-inflammatory properties in BV2 microglial cells, the possible effects of THC and its underlying mechanism was examined against lipopolysaccharide (LPS)-induced RAW 264.7 cell culture and septic mouse models. Pretreatment of RAW 264.7 cells with THC significantly attenuated LPS-induced NO, PGE2 production, and expression of iNOS and COX-2. THC also significantly suppressed LPS-induced release of pro-inflammatory cytokines and degradation of IκB-α. Increased phosphorylation of Nrf2 and nuclear translocation of Nrf2 were observed with THC treatment with consequent expression of HO-1. The data demonstrated that multiple signaling pathways including Akt, p38, and PKC are involved in the THC-induced activation of Nrf2/HO-1 pathway. Treatment of THC resulted in significantly increased survival of LPS-induced septic mice. THC also significantly ameliorated LPS-induced septic features such as hypothermia and increased vascular leakage. In accordance with the data from cell culture model, THC exhibited increased expression of HO-1 in kidney and decreased serum level of pro-inflammatory mediators such as TNF-α, IL-1ß, and NO. Taken together, the present study for the first time demonstrates that THC inhibits inflammation in LPS-induced RAW264.7 cells by Nrf2 activation and improves survival of mice in LPS-induced endotoxemia model.

3,4,5-Trihydroxycinnamic Acid Inhibits LPS-Induced iNOS Expression by Suppressing NF-κB Activation in BV2 Microglial Cells.

Although various derivatives of caffeic acid have been reported to possess a wide variety of biological activities such as neuronal protection against excitotoxicity and anti-inflammatory property, the biological activity of 3,4,5-trihydroxycinnamic acid (THC), a derivative of hydroxycinnamic acids, has not been clearly examined. The objective of the present study is to evaluate the anti-inflammatory effects of THC on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. THC significantly suppressed LPS-induced excessive production of nitric oxide (NO) and expression of iNOS, which is responsible for the production of iNOS. THC also suppressed LPS-induced overproduction of pro-inflammatory cytokines such as IL-1ß and TNF-α in BV2 microgilal cells. Furthermore, THC significantly suppressed LPS-induced degradation of IκB, which retains NF-κB in the cytoplasm. Therefore, THC attenuated nuclear translocation of NF-κB, a major pro-inflammatory transcription factor. Taken together, the present study for the first time demonstrates that THC exhibits anti-inflammatory activity through the suppression of NF-κB transcriptional activation in LPS-stimulated BV2 microglial cells.

Suppression of aggregate formation of mutant huntingtin potentiates CREB-binding protein sequestration and apoptotic cell death.

Although aggregates of mutant huntingtin are a pathological hallmark of Huntington's disease (HD), the role of inclusions in the pathogenesis remains inconclusive. Sequestration of CBP into mutant huntingtin has been reported to play a significant role in the pathogenesis of HD. However, whether aggregate formation of mutant huntingtin is necessary for the sequestration of CBP is not fully elucidated. In the present study, YFP was linked into either N- or C-terminus of exon 1 huntingtin to modulate the aggregation propensity of huntingtin. Efficient aggregation was observed with C-terminally YFP-tagged huntingtin (MT-YFP) whereas N-terminally YFP-tagged mutant huntingtin (YFP-MT) exhibited significantly attenuated aggregation frequency. The sequestration of CBP and apoptosis were significantly increased with YFP-MT. Microarray study showed transcriptional changes favoring apoptosis. Furthermore, expression of PGC1-α was significantly decreased with YFP-MT. The data strongly demonstrate that microscopically non-aggregate form of mutant huntingtin might exert essential pathogenic role of mutant huntingtin in HD.

Thrombin-induced Migration and Matrix Metalloproteinase-9 Expression Are Regulated by MAPK and PI3K Pathways in C6 Glioma Cells.

Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme.

Suppression of autophagy and activation of glycogen synthase kinase 3beta facilitate the aggregate formation of tau.

Neurofibrillary tangle (NFT) is a characteristic hallmark of Alzheimer's disease. GSK3ß has been reported to play a major role in the NFT formation of tau. Dysfunction of autophagy might facilitate the aggregate formation of tau. The present study examined the role of GSK3ß-mediated phosphorylation of tau species on their autophagic degradation. We transfected wild type tau (T4), caspase-3-cleaved tau at Asp421 (T4C3), or pseudophosphorylated tau at Ser396/Ser404 (T4-2EC) in the presence of active or enzyme-inactive GSK3ß. Trehalose and 3-methyladenine (3-MA) were used to enhance or inhibit autophagic activity, respectively. All tau species showed increased accumulation with 3-MA treatment whereas reduced with trehalose, indicating that tau undergoes autophagic degradation. However, T4C3 and T4-2EC showed abundant formation of oligomers than T4. Active GSK3ß in the presence of 3-MA resulted in significantly increased formation of insoluble tau aggregates. These results indicate that GSK3ß-mediated phosphorylation and compromised autophagic activity significantly contribute to tau aggregation.