ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance.

Accumulating evidence demonstrates that the activity regulation of ELK3, a member of the E26 transformation-specific oncogene family, is critical to regulating cell proliferation, migration, and survival in human cancers. However, the molecular mechanisms of how ELK3 induces chemoresistance in prostate cancer (PCa) have not been elucidated. In this study, we found that SPOP and ELK3 are an interacting partner. The interaction between SPOP and ELK3 resulted in increased ELK3 ubiquitination and destruction, assisted by checkpoint kinase-mediated ELK3 phosphorylation. Notably, the modulation of SPOP-mediated ELK3 protein stability affected the c-Fos-induced cell proliferation and invasion of PCa cells. The clinical involvement of the SPOP-ELK3 axis in PCa development was confirmed by an immunohistochemical assay on 123 PCa tissues, with an inverse correlation between increased ELK3 and decreased SPOP being present in ~80% of the specimens. This observation was supported by immunohistochemistry analysis using a SPOP-mutant PCa specimen. Finally, docetaxel treatment induced cell death by activating checkpoint kinase- and SPOP-mediated ELK3 degradation, while SPOP-depleted or SPOP-mutated PCa cells showed cell death resistance. Notably, this observation was correlated with the protein levels of ELK3. Taken together, our study reveals the precise mechanism of SPOP-mediated degradation of ELK3 and provides evidence that SPOP mutations contribute to docetaxel resistance in PCa.

Induction of Autophagy by Extract from Corydalis heterocarpa for Skin Anti-Aging.

The extracts of Corydalis heterocarpa, a salt-tolerant plant, exhibit diverse physiological properties, including anti-inflammatory, anticancer, and antiadipogenic effects. However, the anti-aging effects of C. heterocarpa extract (CHE) on human skin cells have not yet been investigated. In the present study, we determined that CHE inhibited senescence-associated ß-galactosidase (SA-ß-gal)-stained senescent human dermal fibroblasts (HDFs). Furthermore, CHE markedly suppressed the expression of major regulatory proteins involved in senescence, including p53, p21, and caveolin-1. Interestingly, CHE promoted autophagic flux, as confirmed by the formation of microtubule-associated protein 1 light chain 3B (LC3B) puncta and lysosomal activity. Notably, using RNA sequencing (RNA-seq), we showed that CHE selectively regulated the gene expression of leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1), an important regulator of autophagy. The adenosine-monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway, which is essential for autophagy regulation, was also modulated by CHE. LRSAM1 depletion not only inhibited LC3B expression but also decreased the autophagy flux induced by CHE. Moreover, the knockdown of LRSAM1 suppressed the reversal of CHE-induced senescence in old HDFs. Collectively, our study has revealed the rejuvenating effects and molecular mechanisms of CHE, suggesting that CHE may be a promising anti-aging agent.

Effects of poly (ADP-ribose) polymerase inhibitor treatment on the repair process of ischemic acute kidney injury.

Excessive activation of poly (ADP-ribose) polymerase (PARP) contributes to ischemic acute kidney injury (AKI). PARP inhibition has been shown to be beneficial in renal ischemia-reperfusion injury (IRI) in the early phase, but its role in the repair process remains unclear. The effects of JPI-289, a novel PARP inhibitor, during the healing phase after renal IRI were investigated. IRI was performed on 9-week-old male C57BL/6 mice. Saline or JPI-289 100 mg/kg was intraperitoneally administered once at 24 h or additionally at 48 h after IRI. Hypoxic HK-2 cells were treated with JPI-289. Renal function and fibrosis extent were comparable between groups. JPI-289 treatment caused more prominent tubular atrophy and proinflammatory intrarenal leukocyte phenotypes and cytokines/chemokines changes at 12 weeks after unilateral IRI. JPI-289 treatment enhanced gene expressions associated with collagen formation, toll-like receptors, and the immune system in proximal tubules and endothelial cells after IRI. JPI-289 treatment at 3 or 6 h after hypoxia facilitated proliferation of hypoxic HK-2 cells, whereas further treatment after 24 h suppressed proliferation. Delayed inhibition of PARP after renal IRI did not facilitate the repair process during the early healing phase but rather may aggravate renal tubular atrophy during the late healing phase in ischemic AKI.

Ginsenoside Rb2 suppresses cellular senescence of human dermal fibroblasts by inducing autophagy.

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated ß-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased ß-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

Clinical value of urinary cytokines/chemokines as prognostic markers in patients with crescentic glomerulonephritis.

Crescentic glomerulonephritis (CrGN) usually requires urgent immunosuppressive treatment. However, aggressive immunosuppressive treatment is often difficult because of the patients' medical conditions or comorbidities. Prognostic markers including urinary cytokines/chemokines as noninvasive biomarkers were explored in CrGN patients. This prospective cohort study included 82 patients with biopsy-confirmed CrGN from 2002 to 2015 who were followed up for 5 years. Urine and serum cytokines/chemokines on the day of kidney biopsy were analyzed in 36 patients. The median age was 65 years and 47.6% were male. Baseline estimated glomerular filtration rate (eGFR) and interstitial fibrosis and tubular atrophy (IFTA) scores were identified as significant prognostic factors. Among patients with cytokines/chemokines measurement, increased IL-10 level was identified as an independent predictor of good prognosis, and increased levels of urinary MCP-1 and fractalkine tended to be associated with good prognosis after adjusting for baseline eGFR and IFTA score. However, semiquantitative analysis of intrarenal leukocytes did not show prognostic value predicting renal outcome or correlation with urinary cytokines/chemokines. This study supports the clinical importance of baseline eGFR and IFTA scores and suggests potential usefulness of urinary IL-10, MCP-1, and fractalkine as prognostic markers for predicting renal outcomes in patients with CrGN.

Repair phase modeling of ischemic acute kidney injury: recovery vs. transition to chronic kidney disease.

The repair mechanism after ischemic acute kidney injury (AKI) involves complex immunologic processes, which determine long-term renal outcomes. Through investigating two murine ischemia-reperfusion injury (IRI) models: bilateral IRI (BIRI) and unilateral IRI (UIRI), we aimed to determine an appropriate murine model that could simulate the recovery phase of ischemic AKI. Changes in renal function, phenotypes of kidney mononuclear cells, renal fibrosis, and intrarenal cytokine/chemokine expression were serially analyzed up to 12 weeks after IRI. Plasma creatinine and BUN concentrations increased and remained elevated in the BIRI group until 7 days but decreased to comparable levels with the sham control group at 2 weeks after surgery and thereafter, whereas plasma creatinine and BUN concentrations remained unchanged in the UIRI group. Intrarenal total leukocytes, and effector memory and activated phenotypes of CD4 and CD8 T cells markedly increased in the postischemic kidneys in both IRI groups. Expression of proinflammatory cytokines/chemokines and TGF-ß1 was enhanced in the postischemic kidneys of both IRI groups with a higher degree in the UIRI group. Importantly, intrarenal immunologic changes of the BIRI group persisted until 6 weeks despite full functional recovery. The postischemic kidneys of the UIRI group showed earlier and more pronounced proinflammatory conditions as well as more severe atrophic and fibrotic changes compared to the BIRI group. These findings support the utility of longer follow-ups of BIRI and UIRI models for investigating the adaptive repair process, which facilitates recovery of ischemic AKI and maladaptive repair process may result in AKI to CKD transition, respectively.

Bean Extract-Based Gargle for Efficient Diagnosis of Active COVID-19 Infection Using Rapid Antigen Tests.

The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. IMPORTANCE An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.

Early postoperative urinary MCP-1 as a potential biomarker predicting acute rejection in living donor kidney transplantation: a prospective cohort study.

We investigated the clinical relevance of urinary cytokines/chemokines reflecting intrarenal immunologic micromilieu as prognostic markers and the optimal measurement timing after living donor kidney transplantation (LDKT). This prospective cohort study included 77 LDKT patients who were followed for ≥ 5 years. Patients were divided into control (n = 42) or acute rejection (AR, n = 35) group. Early AR was defined as AR occurring within 3 months. Serum and urine cytokines/chemokines were measured serially as follows: intraoperative, 8/24/72 h, 1 week, 3 months, and 1 year after LDKT. Intrarenal total leukocytes, T cells, and B cells were analyzed with immunohistochemistry followed by tissueFAXS. Urinary MCP-1 and fractalkine were also analyzed in a validation cohort. Urinary MCP-1 after one week was higher in the AR group. Urinary MCP-1, fractalkine, TNF-α, RANTES, and IL-6 after one week were significantly higher in the early AR group. Intrarenal total leukocytes and T cells were elevated in the AR group compared with the control group. Urinary fractalkine, MCP-1, and IL-10 showed positive correlation with intrarenal leukocyte infiltration. Post-KT 1 week urinary MCP-1 showed predictive value in the validation cohort. One-week post-KT urinary MCP-1 may be used as a noninvasive diagnostic marker for predicting AR after LDKT.

Dietary Modification Alters the Intrarenal Immunologic Micromilieu and Susceptibility to Ischemic Acute Kidney Injury.

The versatility of the intrarenal immunologic micromilieu through dietary modification and the subsequent effects on susceptibility to ischemic acute kidney injury (AKI) are unclear. We investigated the effects of high-salt (HS) or high-fat (HF) diet on intrarenal immunologic micromilieu and development of ischemic AKI using murine ischemic AKI and human kidney-2 (HK-2) cell hypoxia models. Four different diet regimens [control, HF, HS, and high-fat diet with high-salt (HF+HS)] were provided individually to groups of 9-week-old male C57BL/6 mice for 1 or 6 weeks. After a bilateral ischemia-reperfusion injury (BIRI) operation, mice were sacrificed on day 2 and renal injury was assessed with intrarenal leukocyte infiltration. Human kidney-2 cells were treated with NaCl or lipids. The HF diet increased body weight and total cholesterol, whereas the HF+HS did not. Although the HF or HS diet did not change total leukocyte infiltration at 6 weeks, the HF diet and HF+HS diet increased intrarenal CD8 T cells. Plasma cells increased in the HF and HS diet groups. The expression of proinflammatory cytokines including TNF-α, IFN-γ, MCP-1, and RANTES was increased by the HF or HS diet, and intrarenal VEGF decreased in the HS and HF+HS diet groups at 6 weeks. Deterioration of renal function following BIRI tended to be aggravated by the HF or HS diet. High NaCl concentration suppressed proliferation and enhanced expression of TLR-2 in hypoxic HK-2 cells. The HF or HS diet can enhance susceptibility to ischemic AKI by inducing proinflammatory changes to the intrarenal immunologic micromilieu.

Cordyceps militaris Exerts Anticancer Effect on Non-Small Cell Lung Cancer by Inhibiting Hedgehog Signaling via Suppression of TCTN3.

This study aimed to investigate the effect of Cordyceps militaris extract on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells and determine the underlying mechanisms. We performed a CCK-8 assay to detect cell proliferation, detection of morphological changes through transmission electron microscopy (TEM), annexin V-FITC/PI double staining to analyze apoptosis, and immunoblotting to measure the protein expression of apoptosis and hedgehog signaling-related proteins, with C militaris treated NSCLC cells. In this study, we first found that C militaris reduced the viability and induced morphological disruption in NSCLC cells. The gene expression profiles indicated a reprogramming pattern of genes and transcription factors associated with the action of TCTN3 on NSCLC cells. We also confirmed that the C militaris-induced inhibition of TCTN3 expression affected the hedgehog signaling pathway. Immunoblotting indicated that C militaris-mediated TCTN3 downregulation induced apoptosis in NSCLC cells, involved in the serial activation of caspases. Moreover, we demonstrated that the C militaris negatively modulated GLI1 transcriptional activity by suppressing SMO/PTCH1 signaling, which affects the intrinsic apoptotic pathway. When hedgehog binds to the PTCH1, SMO dissociates from PTCH1 inhibition at cilia. As a result, the active GLI1 translocates to the nucleus. C militaris clearly suppressed GLI1 nuclear translocation, leading to Bcl-2 and Bcl-xL down-regulation. These results suggested that C militaris induced NSCLC cell apoptosis, possibly through the downregulation of SMO/PTCH1 signaling and GLI1 activation via inhibition of TCTN3. Taken together, our findings provide new insights into the treatment of NSCLC using C militaris.

Stereoisomer-specific ginsenoside 20(S)-Rg3 reverses replicative senescence of human diploid fibroblasts via Akt-mTOR-Sirtuin signaling.

BACKGROUND: The replicative senescence of human dermal fibroblasts (HDFs) is accompanied by growth arrest. In our previous study, the treatment of senescent HDFs with Rg3(S) lowered the intrinsic reactive oxygen species (ROS) levels and reversed cellular senescence by inducing peroxiredoxin-3, an antioxidant enzyme. However, the signaling pathways involved in Rg3(S)-induced senescence reversal in HDFs and the relatedness of the stereoisomer Rg3(R) in corresponding signaling pathways are not known yet. METHODS: We performed senescence-associated ß-galactosidase and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP) as well as the mitochondrial DNA copy number, nicotinamide adenine dinucleotide (NAD)+/1,4-dihydronicotinamide adenine dinucleotide (NADH) ratio, and NAD-dependent sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of phosphatidylinositol 3-kinase/Akt, 5' adenosine monophosphate-activated protein kinase (AMPK), and sirtuin 1/3, including cell cycle regulatory proteins, were examined by immunoblot analysis. RESULTS: Ginsenoside Rg3(S) reversed the replicative senescence of HDFs by restoring the ATP level and NAD+/NADH ratio in downregulated senescent HDFs. Rg3(S) recovered directly the cellular levels of ROS and the NAD+/NADH ratio in young HDFs inactivated by rotenone. Rg3(S) mainly downregulated phosphatidylinositol 3-kinase/Akt through the inhibition of mTOR by cell cycle regulators like p53/p21 in senescent HDFs, whereas Rg3(R) did not alter the corresponding signaling pathways. Rg3(S)-activated sirtuin 3/PGC1α to stimulate mitochondrial biogenesis. CONCLUSION: Cellular molecular analysis suggests that Rg3(S) specifically reverses the replicative senescence of HDFs by modulating Akt-mTOR-sirtuin signaling to promote the biogenesis of mitochondria.

Cordyceps militaris induces apoptosis in ovarian cancer cells through TNF-α/TNFR1-mediated inhibition of NF-κB phosphorylation.

BACKGROUND: Cordyceps militaris (L.) Fr. (C. militaris) exhibits pharmacological activities, including antitumor properties, through the regulation of the nuclear factor kappa B (NF-κB) signaling. Tumor Necrosis Factor (TNF) and TNF-α modulates cell survival and apoptosis through NF- κB signaling. However, the mechanism underlying its mode of action on the NF-κB pathway is unclear. METHODS: Here, we analyzed the effect of C. militaris extract (CME) on the proliferation of ovarian cancer cells by confirming viability, morphological changes, migration assay. Additionally, CME induced apoptosis was determined by apoptosis assay and apoptotic body formation under TEM. The mechanisms of CME were determined through microarray, immunoblotting and immunocytochemistry. RESULTS: CME reduced the viability of cells in a dose-dependent manner and induced morphological changes. We confirmed the decrease in the migration activity of SKOV-3 cells after treatment with CME and the consequent induction of apoptosis. Immunoblotting results showed that the CME-mediated upregulation of tumor necrosis factor receptor 1 (TNFR1) expression induced apoptosis of SKOV-3 cells via the serial activation of caspases. Moreover, CME negatively modulated NF-κB activation via TNFR expression, suggestive of the activation of the extrinsic apoptotic pathway. The binding of TNF-α to TNFR results in the disassociation of IκB from NF-κB and the subsequent translocation of the active NF-κB to the nucleus. CME clearly suppressed NF-κB translocation induced by interleukin (IL-1ß) from the cytosol into the nucleus. The decrease in the expression levels of B cell lymphoma (Bcl)-xL and Bcl-2 led to a marked increase in cell apoptosis. CONCLUSION: These results suggest that C. militaris inhibited ovarian cancer cell proliferation, survival, and migration, possibly through the coordination between TNF-α/TNFR1 signaling and NF-κB activation. Taken together, our findings provide a new insight into a novel treatment strategy for ovarian cancer using C. militaris.

Cordycepin inhibits human ovarian cancer by inducing autophagy and apoptosis through Dickkopf-related protein 1/ß-catenin signaling.

Cordycepin, the major active component from Cordyceps militaris, has been reported to significantly inhibit some types of cancer; however, its effects on ovarian cancer are still not well understood. In this study, we treated human ovarian cancer cells with different doses of cordycepin and found that it dose-dependently reduced ovarian cancer cell viability, based on Cell counting kit-8 reagent. Immunoblotting showed that cordycepin increased Dickkopf-related protein 1 (Dkk1) levels and inhibited ß-catenin signaling. Atg7 knockdown in ovarian cancer cells significantly inhibited cordycepin-induced apoptosis, whereas ß-catenin overexpression abolished the effects of cordycepin on cell death and proliferation. Furthermore, we found that Dkk1 overexpression by transfection downregulated the expression of c-Myc and cyclin D1. siRNA-mediated Dkk1 silencing downregulated the expression of Atg8, beclin, and LC3 and promoted ß-catenin translocation from the cytoplasm into the nucleus. These results suggest that cordycepin inhibits ovarian cancer cell growth, possibly through coordinated autophagy and Dkk1/ß-catenin signaling. Taken together, our findings provide new insights into the treatment of ovarian cancer using cordycepin.

Nectandrin B-mediated activation of the AMPK pathway prevents cellular senescence in human diploid fibroblasts by reducing intracellular ROS levels.

Nectandrin B (NecB) is a bioactive lignan compound isolated from Myristica fragrans (nutmeg), which functions as an activator of AMP-activated protein kinase (AMPK). Because we recently found that treatment with NecB increased the cell viability of old human diploid fibroblasts (HDFs), the underlying molecular mechanism was investigated. NecB treatment in old HDFs reduced the activity staining of senescence-associated ß-galactosidase and the levels of senescence markers, such as the Ser15 phosphorylated p53, caveolin-1, p21waf1, p16ink4a, p27kip1, and cyclin D1. NecB treatment increased that in S phase, indicating a enhancement of cell cycle entry. Interestingly, NecB treatment ameliorated age-dependent activation of AMPK in old HDFs. Moreover, NecB reversed the age-dependent expression and/or activity changes of certain sirtuins (SIRT1-5), and cell survival/death-related proteins. The transcriptional activity of Yin-Yang 1 and the expression of downstream proteins were elevated in NecB-treated old HDFs. In addition, NecB treatment exerted a radical scavenging effect in vitro, reduced cellular ROS levels, and increased antioxidant enzymes in old HDFs. Moreover, NecB-mediated activation of the AMPK pathway reduced intracellular ROS levels. These results suggest that NecB-induced protection against cellular senescence is mediated by ROS-scavenging through activation of AMPK. NecB might be useful in ameliorating age-related diseases and extending human lifespan.

Degradation of Kidney and Psoas Muscle Proteins as Indicators of Post-Mortem Interval in a Rat Model, with Use of Lateral Flow Technology.

We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3ß were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase α, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and ß-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.

Effect of rutin from tartary buckwheat sprout on serum glucose-lowering in animal model of type 2 diabetes.

This study investigates the anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model. The rutin content in tartary buckwheat sprout (TBS) is five times higher than that found in common buckwheat sprout (CBS) as evident from high-performance liquid chromatography analysis. Administration of either rutin or TBS ethanolic extract to diabetes mice decreased the serum glucose level significantly. Rutin down-regulated the expression levels of protein-tyrosine phosphatase 1B; it is negative regulator of insulin pathway, both transcriptionally and translationally in myocyte C2C12 in a dose-dependent manner. In conclusion, rutin can play a critical role in down-regulation of serum glucose level in type 2 diabetes.

Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer.

OBJECTIVE: To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer. METHODS: The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis. RESULTS: The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed. CONCLUSION: Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via nitric oxide production in human diploid fibroblasts.

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of the small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose- and time-dependent manner and resulted in senescence-associated ß-galactosidase (SA-ß-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16(ink4a), and p21(waf1), were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy, and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-ß-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.