TGF-ß activates NLRP3 inflammasome by an autocrine production of TGF-ß in LX-2 human hepatic stellate cells.

Inflammation contributes to the pathogenesis of liver disease, and inflammasome activation has been identified as a major contributor to the amplification of liver inflammation. Transforming growth factor-beta (TGF-ß) is a key regulator of liver physiology, contributing to all stages of liver disease. We investigated whether TGF-ß is involved in inflammasome-mediated fibrosis in hepatic stellate cells. Treatment with TGF-ß increased priming of NLRP3 inflammasome signaling by increasing NLRP3 levels and activating TAK1-NF-kB signaling. Moreover, TGF-ß increased the expression of p-Smad2/3-NOX4 in LX-2 cells and consequently increased ROS content, which is a trigger for NLRP3 inflammasome activation. Elevated expression of NEK7 and active caspase-1 was also shown in TGF-ß-induced LX-2 cells, and this level was reduced by (5Z)-oxozeaenol, a TAK inhibitor. Finally, TGF-ß-treated cells significantly increased TGF-ß secretion levels, and their production was inhibited by IL-1ß receptor antagonist treatment. In conclusion, TGF-ß may represent an endogenous danger signal to the active NLRP3 inflammasome, by which IL-1ß mediates TGF-ß expression in an autocrine manner. Therefore, targeting the NLRP3 inflammasome may be a promising approach for the development of therapies for TGF-ß-induced liver fibrosis.

Mebendazole Impedes the Proliferation and Migration of Pancreatic Cancer Cells through SK1 Inhibition Dependent Pathway.

Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortality rates and requires the development of highly efficacious medications that can improve the efficiency of existing treatment methods. In particular, in PDAC, resistance to conventional chemotherapy reduces the effectiveness of anticancer drugs, decreasing the therapeutic efficiency. Sphingosine 1-phosphate (S1P), produced by sphingosine kinase (SK), plays a vital role in cancer growth, metastasis, chemotherapy, and drug resistance. Focusing on the structural characteristics of mebendazole (MBZ), we studied whether MBZ would affect metastasis, invasion, and drug resistance in cancer by lowering S1P production through inhibition of SK activity. MBZ selectively inhibited SK1 more than SK2 and regulated the levels of sphingolipids. MBZ inhibited the proliferation and migration of cancer cells in other PDAC cell lines. To determine whether the effect of MBZ on cancer cell growth and migration is S1P-mediated, S1P was treated, and the growth and migration of cancer cells were observed. It was found that MBZ inhibited S1P-induced cancer cell growth, and MBZ showed a growth inhibitory effect by regulating the JAK2/STAT3/Bcl-2 pathway. The phosphorylation of focal adhesion kinase (FAK), a transcription factor that regulates migration, was inhibited by MBZ, so it was found that the effect of MBZ regulates the migration of cancer cells through the S1P/FAK/vimentin pathway. In conclusion, our study suggests that the anthelmintic MBZ can be used as a potential therapeutic agent for treating PDAC and for structural synthesis studies of its analogs.

Synthesis of PP2A-Activating PF-543 Derivatives and Investigation of Their Inhibitory Effects on Pancreatic Cancer Cells.

Sphingosine kinase (SK) is involved in the growth of cells, including cancer cells. However, which of its two isotypes-SK1 and SK2-is more favorable for cancer growth remains unclear. Although PF-543 strongly and selectively inhibits SK1, its anticancer effect is not high, and the underlying reason remains difficult to explain. We previously determined that the tail group of PF-543 is responsible for its low metabolic stability (MS). In this study, compounds containing aromatic or aliphatic tails in the triazole group were synthesized, and changes in the SK-inhibitory effect and anticancer activity of PF-543 were assessed using pancreatic cancer cells. The compounds with aliphatic tails showed high inhibitory effects on pancreatic cancer cells but slightly lower selectivity for SK1. A compound with an introduced aliphatic tail activated protein phosphatase 2A (PP2A), showing an effect similar to that of FTY720. Molecular docking analysis revealed that the PP2A-binding form of this newly synthesized compound was different from that noted in the case of FTY720. This compound also improved the MS of PF-543. These results indicate that the tail structure of PF-543 influences MS.

Protective Effect of Cudrania tricuspidata Extract against High-Fat Diet Induced Nonalcoholic Fatty Liver Disease through Nrf-2/HO-1 Pathway.

Nonalcoholic fatty liver disease is the most common chronic disease affecting a wide range of the world's population and associated with obesity-induced metabolic syndrome. It is possibly emerging as a leading cause of life-threatening liver diseases for which a drug with a specific therapeutic target has not been developed yet. Previously, there have been reports on the benefits of Cudrania tricuspidata (CT) for treating obesity and diabetes via regulation of metabolic processes, such as lipogenesis, lipolysis, and inflammation. In this study, we investigated the ameliorative effect of orally administered 0.25% and 0.5% (w/w) CT mixed with high-fat diet (HFD) to C57BL/6J mice for 7 weeks. It was found that body weight, fat mass, hepatic mass, serum glucose level, and liver cholesterol levels were significantly reduced after CT treatment. In CT-treated HFD-fed mice, the mRNA expression levels of hepatic lipogenic and inflammatory cytokine-related genes were markedly reduced, whereas the expression level of epididymal lipogenic genes was increased. The mRNA expression level of beta-oxidation and Nrf-2/HO-1 genes significantly increased in CT-treated obese mice livers. We propose that CT alleviates hepatic steatosis by reducing oxidative stress and inflammation.

Development and application of an antibody that binds to interleukin-1ß of various mammalian species for the treatment of inflammatory diseases.

Inflammation is provoked by host immune reactions to pathogenic or tissue injury and is arbitrated by cytokines. Among the pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) are main mediators of inflammation. The production of these pro-inflammatory cytokines is mainly triggered in macrophages by harmful stimuli including microbial pathogens, irritants, and toxic cellular components, and plays key roles in the palpation of the inflammatory response. Among the therapeutic antibodies for the treatment of inflammation, those targeting TNF-α (including adalimumab and infliximab) are frequently used in clinical settings. Although IL-1ß is a key cytokine for the onset of inflammatory diseases, such as inflammatory bowel disease (IBD) and type 2 diabetes (T2DM), few therapeutic antibodies exist for this cytokine, with the exception of canakinumab. Canakinumab binds to human IL-1ß, but does not bind to murine IL-1ß, which hampers its experimental use. Therefore, inflammation-therapeutic antibodies that bind to IL-1ß of various mammals are needed. In this study, we report the development of an antibody that bound to IL-1ß of various mammalian species and exhibited therapeutic effects in inflammatory diseases.

Allomyrina dichotoma larval extract attenuates intestinal barrier disruption by altering inflammatory response and tight junction proteins in lipopolysaccharide-induced Caco-2 cells.

Intestinal epithelial cells form a barrier between the intestinal lumen and host connective tissues and play an important role in maintaining intestinal nutrient homeostasis. This study investigated effects of Allomyrina dichotoma (rhinoceros beetle) larval extract (ADLE) on the intestinal barrier damage and explored mechanisms for reversing intestinal barrier dysfunction in lipopolysaccharide (LPS)-stimulated Caco-2, human intestinal epithelial cells. LPS reduced intestinal epithelial barrier function by increasing transepithelial electrical resistance, and this effect was significantly attenuated by ADLE treatment. ADLE also significantly countered the inhibition of tight junction-related protein expression in both LPS-induced Caco-2 cells and intestine from HFD-induced mice. Moreover, ADLE significantly decreased expression and production of inflammatory factors, such as iNOS, cox-2, nitric oxide, and cytokines induced by LPS stimulus. Reduction in phosphorylation of adenosine monophosphate-activated protein kinase was averted by ADLE treatment in LPS treated INS-1 cells. Finally, reactive oxygen stress level was decreased and ATP production was increased by ADLE treatment. ADLE appears to display gut health-promoting effects by reducing inflammation and inducing tight junction proteins in Caco-2 cells. Therefore, ADLE might be useful for preventing or treating intestine cell damage in inflammatory bowel disease.

Verification of the Necessity of the Tolyl Group of PF-543 for Sphingosine Kinase 1 Inhibitory Activity.

PF-543, the most potent sphingosine kinase (SK) inhibitor, does not demonstrate effective anticancer activity in some cancer cells, unlike other known SK1 inhibitors. PF-543 has a non-lipid structure with a unique toluene backbone; however, the importance of this structure remains unclear. Therefore, the purpose of this study was to investigate changes in SK inhibitory and anticancer activities and to explore the role of the tolyl group structure of PF-543 through various modifications. We transformed the methyl group of PF-543 into hydrogen, fluorine, and hydroxy. PF-543 derivatives in which the methyl group was substituted by hydrogen and fluorine (compound 5) demonstrated SK1 inhibitory and anticancer activities similar to PF-543. Moreover, we performed molecular modeling studies of PF-543 and compound 5. To assess the metabolic stability of PF-543 and compound 5, we determined their degree of degradation using the liver microsomes of four different animal species (human, dog, rat, and mouse). However, both PF-543 and compound 5 showed poor microsomal stability. Therefore, for the medical applications of PF-543, the structural modifications of its other parts may be necessary. Our results provide important information for the design of additional PF-543 analogs.

Synthesis and Biological Evaluation of PF-543 Derivative Containing Aliphatic Side Chain.

The PF-543 is known as a potent and selective inhibitor of sphingosine kinase (SK) 1 amongst all the SK inhibitors known to date. In a recently reported study by Pfizer on the synthesis of PF-543 derivatives and the SK inhibitory effects, the introduction of propyl moiety into sulfonyl group of PF-543 in the case of 26b revealed an excellent result of 1.7 nM of IC50 of SK1, suggesting the potential substitution of chain structure for benzenesulfonyl structure. In the present work, we aimed for identification of antitumor activity and inhibitory effects of PF-543 derivative containing aliphatic long chain (similar to known SK inhibitors) on SK1. The synthesized compound 2 exhibited an inhibitory effect on SK1 in a manner similar to that of PF-543; the PF-543 derivative manifested similar antitumor activity on HT29, HCT116 (colorectal cancer cell line), and AGS (gastric cancer cell line) cells. Also, from the docking study conducted with PF-543 and compound 2, it was apparent that the aliphatic chain in compound 2 could probably replace benzenesulfonyl structure of PF-543.

Lysophosphatidic Acid Signaling in Diabetic Nephropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid present in most tissues and body fluids. LPA acts through specific LPA receptors (LPAR1 to LPAR6) coupled with G protein. LPA binds to receptors and activates multiple cellular signaling pathways, subsequently exerting various biological functions, such as cell proliferation, migration, and apoptosis. LPA also induces cell damage through complex overlapping pathways, including the generation of reactive oxygen species, inflammatory cytokines, and fibrosis. Several reports indicate that the LPA-LPAR axis plays an important role in various diseases, including kidney disease, lung fibrosis, and cancer. Diabetic nephropathy (DN) is one of the most common diabetic complications and the main risk factor for chronic kidney diseases, which mostly progress to end-stage renal disease. There is also growing evidence indicating that the LPA-LPAR axis also plays an important role in inducing pathological alterations of cell structure and function in the kidneys. In this review, we will discuss key mediators or signaling pathways activated by LPA and summarize recent research findings associated with DN.

MicroRNA-181c Inhibits Interleukin-6-mediated Beta Cell Apoptosis by Targeting TNF-α Expression.

We have previously reported that long-term treatment of beta cells with interleukin-6 (IL-6) is pro-apoptotic. However, little is known about the regulatory mechanisms that are involved. Therefore, we investigated pro-apoptotic changes in mRNA expression in beta cells in response to IL-6 treatment. We analyzed a microarray with RNA from INS-1 beta cells treated with IL-6, and found that TNF-α mRNA was significantly upregulated. Inhibition of TNF-α expression by neutralizing antibodies significantly decreased annexin V staining in cells compared with those treated with a control antibody. We identified three microRNAs that were differentially expressed in INS-1 cells incubated with IL-6. In particular, miR-181c was significantly downregulated in IL-6-treated cells compared with control cells and the decrease of miR-181c was attenuated by STAT-3 signaling inhibition. TNF-α mRNA was a direct target of miR-181c and upregulation of miR-181c by mimics, inhibited IL-6-induced increase in TNF-α mRNA expression. Consequently, reduction of TNF-α mRNA caused by miR-181c mimics enhanced cell viability in IL-6 treated INS-1 cells. These results demonstrated that miR-181c regulation of TNF-α expression plays a role in IL-6-induced beta cell apoptosis.

Synthesis and Biological Evaluation of BODIPY-PF-543.

Sphingosine-1-phosphate (S1P) regulates the proliferation of various cells and promotes the growth of cancer cells. Sphingosine kinase (SK), which transforms sphingosine into S1P, has two isotypes: SK1 and SK2. To date, both isotypes are known to be involved in the proliferation of cancer cells. PF-543, an SK1 inhibitor developed by Pfizer, strongly inhibits SK1. However, despite its strong SK1 inhibitory effect, PF-543 shows low anticancer activity in vitro. Therefore, additional biological evidence on the anticancer activity of SK1 inhibitor is required. The present study aimed to investigate the intracellular localization of PF-543 and identify its association with anticancer activity by introducing a fluoroprobe into PF-543. Boron-dipyrromethene (BODIPY)-introduced PF-543 has a similar SK1 inhibitory effect as PF-543. These results indicate that the introduction of BODIPY does not significantly affect the inhibitory effect of SK1. In confocal microscopy after BODIPY-PF-543 treatment, the compound was mainly located in the cytosol of the cells. This study demonstrated the possibility of introducing fluorescent material into an SK inhibitor and designing a synthesized compound that is permeable to cells while maintaining the SK inhibitory effect.

Blocking lysophosphatidic acid receptor 1 signaling inhibits diabetic nephropathy in db/db mice.

Lysophosphatidic acid (LPA) is known to regulate various biological responses by binding to LPA receptors. The serum level of LPA is elevated in diabetes, but the involvement of LPA in the development of diabetes and its complications remains unknown. Therefore, we studied LPA signaling in diabetic nephropathy and the molecular mechanisms involved. The expression of autotaxin, an LPA synthesis enzyme, and LPA receptor 1 was significantly increased in both mesangial cells (SV40 MES13) maintained in high-glucose media and the kidney cortex of diabetic db/db mice. Increased urinary albumin excretion, increased glomerular tuft area and volume, and mesangial matrix expansion were observed in db/db mice and reduced by treatment with ki16425, a LPA receptor 1/3 antagonist. Transforming growth factor (TGF)ß expression and Smad-2/3 phosphorylation were upregulated in SV40 MES13 cells by LPA stimulation or in the kidney cortex of db/db mice, and this was blocked by ki16425 treatment. LPA receptor 1 siRNA treatment inhibited LPA-induced TGFß expression, whereas cells overexpressing LPA receptor 1 showed enhanced LPA-induced TGFß expression. LPA treatment of SV40 MES13 cells increased phosphorylated glycogen synthase kinase (GSK)3ß at Ser9 and induced translocation of sterol regulatory element-binding protein (SREBP)1 into the nucleus. Blocking GSK3ß phosphorylation inhibited SREBP1 activation and consequently blocked LPA-induced TGFß expression in SV40 MES13 cells. Phosphorylated GSK3ß and nuclear SREBP1 accumulation were increased in the kidney cortex of db/db mice and ki16425 treatment blocked these pathways. Thus, LPA receptor 1 signaling increased TGFß expression via GSK3ß phosphorylation and SREBP1 activation, contributing to the development of diabetic nephropathy.

Effects of Glucagon-Like Peptide-1 on Oxidative Stress and Nrf2 Signaling.

Oxidative cellular damage caused by free radicals is known to contribute to the pathogenesis of various diseases such as cancer, diabetes, and neurodegenerative diseases, as well as to aging. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein1 (Keap1) signaling pathways play an important role in preventing stresses including oxidative and inflammatory stresses. Nrf2 is a master regulator of cellular stress responses, induces the expression of antioxidant and detoxification enzymes, and protects against oxidative stress-induced cell damage. Glucagon-like peptide-1 (GLP-1) is an incretin hormone, which was originally found to increase insulin synthesis and secretion. It is now widely accepted that GLP-1 has multiple functions beyond glucose control in various tissues and organs including brain, kidney, and heart. GLP-1 and GLP-1 receptor agonists are known to be effective in many chronic diseases, including diabetes, via antioxidative mechanisms. In this review, we summarize the current knowledge regarding the role of GLP-1 in the protection against oxidative damage and the activation of the Nrf2 signaling pathway.

Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) Model, Which Is Associated with the Induction of Cell Cycle G1 Arrest.

Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.

The high-throughput solid-phase extraction of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) from Lactobacillus plantarum demonstrates efficacy against multidrug-resistant bacteria and influenza A (H3N2) virus.

Introduction: 2,5-diketopiperazines are the simplest forms of cyclic dipeptides (CDPs) and have diverse frameworks with chiral side chains that are useful for drug development. Previous research has investigated the antimicrobial properties of proline-linked CDPs and their combinations in the culture filtrate (CF) of Lactobacillus plantarum LBP-K10 using anion exchange chromatography (AEC). However, the quantity of CDPs showcasing notable anti-influenza virus activity derived from AECs was generally lower than those originating from Lactobacillus CF. Methods: To address this issue, the study aims to propose a more efficient method for isolating CDPs and to introduce the antiviral combinations of CDPs obtained using a new method. The study employed a novel technique entailing high-throughput C18-based solid-phase extraction with a methanol gradient (MeSPE). The MeSPE method involved increasing the methanol concentration from 5% to 50% in 5% increments. Results: The methanol SPE fractions (MeSPEfs) eluted with methanol concentrations between 35% and 45% evinced substantial efficacy in inhibiting the influenza A/H3N2 virus via plaque-forming assay. MeSPEf-45, the 45% MeSPEf, exhibited exceptional efficacy in preventing viral infections in Madin-Darby kidney cells, surpassing both individual CDPs and the entire set of MeSPEfs. To identify the specific antiviral components of MeSPEf-45, all MeSPEfs were further fractionated through preparative high-performance liquid chromatography (prep-HPLC). MeSPEf-45 fractions S8 and S11 presented the highest activity against multidrug-resistant bacteria and influenza A/H3N2 virus among all MeSPEfs, with 11 common fractions. Antiviral fractions S8 and S11 were identified as proline-based CDPs, specifically cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro), using gas chromatography-mass spectrometry. The combination of MeSPEf-45 fractions S8 and S11 displayed superior antibacterial and anti-influenza virus effects compared to the individual fractions S8 and S11. Discussion: High-throughput MeSPE-derived MeSPEfs and subsequent HPLC-fractionated fractions presents an innovative approach to selectively purify large amounts of potent antimicrobial CDPs from bacterial CF. The findings also show the effectiveness of physiologically bioactive combinations that utilize fractions not containing CDP. This study provides the initial evidence demonstrating the antimicrobial properties of CDPs acquired through high-throughput SPE techniques.

Antiphotoaging effects of solvent fractions isolated from Allomyrina dichotoma larvae extract.

Skin aging is affected by a variety of factors, including ultraviolet rays, oxidative stress, medications, smoking, and genetics. Among them, photo-aging accounts for about 80% of skin aging. The present study was evaluated to verify the potential of Allomyrina dichotoma larvae, which has recently been attracting attention as an edible insect, as an anti-aging substance. UVB irradiation at 100 mJ/cm2 was sufficient to induce photo-aging of fibroblasts within 24 h, which was alleviated after treatment with 70% ethanol extract of Allomyrina dichotoma larvae extract (ADLE). To obtain an extract from ADLE, which has a relatively high content of polyphenol compounds containing physiological activity, fractional solvent extraction was carried out using organic solvents such as hexane, chloroform, ethyl acetate, and butanol. Additionally, ethyl acetate and butanol fractions contributed to the inhibition of UVB-induced ROS production, cell damage, and senescence of fibroblasts. It was also confirmed that the two fractions can regulate the expression of MMP-1 and AP-1. In particular, the ethyl acetate fraction showed an excellent effect in recovering collagen decomposed by UVB. Therefore, these results suggest that ADLE has potential as a natural insect-derived biomaterial to inhibit UVB-induced photo-aging.

Eupatilin Alleviates Hyperlipidemia in Mice by Inhibiting HMG-CoA Reductase.

Artemisia princeps (family Asteraceae) is a natural product broadly used as an antioxidative, hepatoprotective, antibacterial, and anti-inflammatory agent in East Asia. In the present study, eupatilin, the main constituent of Artemisia princeps, was investigated as an antihyperlipidemic agent. Eupatilin inhibited 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HCR), an enzyme that is a therapeutic target for hyperlipidemia, in an ex vivo assay using rat liver. In addition, oral administration of eupatilin significantly lowered the serum levels of total cholesterol (TC) and triglycerides (TG) in corn oil-induced and Triton WR-1339-induced hyperlipidemic mice. These results suggest that eupatilin can alleviate hyperlipidemia by inhibiting HCR.

Gryllus bimaculatus extract ameliorates high-fat diet-induced hyperglycemia and hyperlipidemia by inhibiting hepatic lipogenesis through AMPK activation.

Insect-based food is increasingly used and is a sustainable protein source provided by eco-friendly breeding respecting the animal welfare. The cricket Gryllus bimaculatus is an approved edible insect. In this paper, the effects of G. bimaculatus extracts (AE-GBE) on hepatic insulin resistance and the underlying mechanisms were investigated in high fat diet (HFD)-fed C57BL/6J mice. Mice were fed HFD for 6 weeks and some were concomitantly given AE-GBE orally (100 mg/kg/day). AE-GBE significantly improved glucose tolerance and insulin sensitivity by attenuating hepatic lipid accumulation measured by the reduced serum and hepatic lipid contents. Moreover, AE-GBE significantly downregulated the expression of hepatic lipogenesis-related genes and activated the AMPK signaling pathway. Therefore, AE-GBE might improve fatty liver and glucose metabolism disorders as well as insulin resistance by inhibiting the expression of proteins involved in hepatic fatty acid synthesis through AMPK activation. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01117-9.

SK1 Inhibitor RB005 Induces Apoptosis in Colorectal Cancer Cells through SK1 Inhibition Dependent and Independent Pathway.

BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) is the fourth leading cause of cancer- related death globally, with a high incidence rate in economically fast-growing countries. Sphingosine- 1-phosphate (S1P) is a bioactive lipid mediator that plays critical roles in cancer cell proliferation, migration, and angiogenesis converted by the isoforms of sphingosine kinase (SK1 and SK2). SK1 is highly expressed in colorectal cancer; its inhibitors suppress the formation of S1P and increase ceramide levels having a pro-apoptotic function. RB005 is a selective SK1 inhibitor and a structural analog of PP2A activator FTY720. The purpose of this study is to test whether RB005, an SK1 inhibitor, can be used as an anticancer agent by inhibiting the growth of colon cancer cells. METHODS: We performed MTT and colony-forming assay using colon cancer cell lines HT29 and HCT116 cells to examine the cell toxicity effect of RB005. To determine whether apoptosis of RB005 in colon cancer cell line is due to SK1 inhibition or other mechanisms due to its structural similarity with FTY720, we conducted LC/MS, siRNA knockdown, and PP2A activity experiments. RESULTS: RB005 notably inhibited CRC cell growth and proliferation compared to PF543 and ABC294640 by inducing the mitochondria-mediated intrinsic apoptotic pathway. Apoptotic cell death is caused by increased mitochondrial permeability Initiated by the activation of pro-apoptotic protein BAX, increased ceramides, and activation of PP2A. Also, RB005 treatment in HT29 cells did not change the expression level of SK1, but strikingly decreased SK1 activity and S1P levels. All these events of cell death and apoptosis were less effective when SK1 was knocked down by siRNA. CONCLUSION: This result indicates that RB005 shows the in-vitro anti-CRC effect by inhibiting SK1 activity and PP2A activation, increasing proapoptotic ceramide levels following the activation of the intrinsic apoptotic pathway.

Prevention of LPS-Induced Acute Kidney Injury in Mice by Bavachin and Its Potential Mechanisms.

Acute kidney injury (AKI) is a serious complication of sepsis with a rapid onset and high mortality rate. Bavachin, an active component of Psoralea corylifolia L., reportedly has antioxidant, anti-apoptotic, and anti-inflammatory effects; however, its beneficial effects on AKI remain undetermined. We investigated the protective effect of bavachin on lipopolysaccharide (LPS)-induced AKI in mice and elucidated the underlying mechanism in human renal tubular epithelial HK-2 cells. Increased serum creatinine and blood urea nitrogen levels were observed in LPS-injected mice; however, bavachin pretreatment significantly inhibited this increase. Bavachin improved the kidney injury score and decreased the expression level of tubular injury markers, such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), in both LPS-injected mice and LPS-treated HK-2 cells. LPS-induced oxidative stress via phosphorylated protein kinase C (PKC) ß and upregulation of the NADPH oxidase (NOX) 4 pathway was also significantly decreased by treatment with bavachin. Moreover, bavachin treatment inhibited the phosphorylation of MAPKs (P38, ERK, and JNK) and nuclear factor (NF)-κB, as well as the increase in inflammatory cytokine levels in LPS-injected mice. Krüppel-like factor 5 (KLF5) expression was upregulated in the LPS-treated HK-2 cells and kidneys of LPS-injected mice. However, RNAi-mediated silencing of KLF5 inhibited the phosphorylation of NF-kB, consequently reversing LPS-induced KIM-1 and NGAL expression in HK-2 cells. Therefore, bavachin may ameliorate LPS-induced AKI by inhibiting oxidative stress and inflammation via the downregulation of the PKCß/MAPK/KLF5 axis.