Affinity-Driven Covalent Modulator of the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Cascade.

Traditional medicines provide a fertile ground to explore potent lead compounds, yet their transformation into modern drugs is fraught with challenges in deciphering the target that is mechanistically valid for its biological activity. Herein we reveal that (Z)-(+)-isochaihulactone (1) exhibited significant inhibition against multiple-drug-resistant (MDR) cancer cell lines and mice xenografts. NMR spectroscopy showed that 1 resisted an off-target thiolate, thus indicating that 1 was a target covalent inhibitor (TCI). By identifying the pharmacophore of 1 (α,ß-unsaturated moiety), a probe derived from 1 was designed and synthesized for TCI-oriented activity-based proteome profiling. By MS/MS and computer-guided molecular biology approaches, an affinity-driven Michael addition of the noncatalytic C247 residue of GAPDH was found to control the "ON/OFF" switch of apoptosis through non-canonically nuclear GAPDH translocation, which bypasses the common apoptosis-resistant route of MDR cancers.

Antimicrobial peptide GW-H1-induced apoptosis of human gastric cancer AGS cell line is enhanced by suppression of autophagy.

Gastric cancer is one of the most common malignant cancers worldwide. Due to its poor prognosis and high mortality rate, development of an effective therapeutic method is of urgent need. It has been reported that antimicrobial peptides (AMPs), also known as host-defense peptides, can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity, without harming normal cells or causing severe drug resistance. We have designed a series of novel cationic AMPs with potent antimicrobial activity against a broad spectrum of bacterial pathogens. In the current study, we evaluated their anticancer potency toward gastric cancer AGS cell line. Cell viability assay revealed that GW-H1 exhibited the lowest IC50 value (less than 20 µM). Flow cytometry showed that upon GW-H1 treatment for 0-24 h, apoptotic cell populations of AGS increased in a dose- and time-dependent manner. Western blot analysis further revealed that upon treatment for 2-6 h, apoptosis-related caspases-3, 7, 8, 9, and PARP were cleaved and activated, while autophagy-related LC3-II and beclin-1 were concomitantly increased. These results indicated that both apoptosis and autophagy were involved in the early stage of GW-H1-induced AGS cell death. However, upon treatment for 12-24 h, LC3-II began to decrease and cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provided support for future application of GW-H1 as a potential anticancer agent for gastric cancer treatment.

Rutin, a flavonoid and principal component of saussurea involucrata, attenuates physical fatigue in a forced swimming mouse model.

This study investigated the antifatigue effects of rutin, a flavonoid extracted from the ethyl acetate extract of S. involucrata. Mice were subjected to a weight-loaded forced swim test (WFST) on alternate days for 3 wk. Rutin was administered orally to the mice for 7 days in dosages of 15, 30, and 60 mg/kg body weight, and several biomarkers of physical fatigue were evaluated: swimming time, change in body weight, lipid peroxidation, lactic acid (LA), glycogen, and the activities of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). On Day 7, the rutin-treated mice had a 3-fold longer exhaustive swimming time than the control mice, as well as significantly reduced blood LA concentrations. The 15, 30, and 60 mg/kg body weight rutin-supplemented groups displayed 11.2%, 22.5%, and 37.7% reduced malondialdehyde (MDA) concentrations, respectively, in brain and muscle tissues compared with the control exercised group. Our results indicated that the administration of rutin protected the mice against the depletion of SOD and GPx activities significantly. Following 7 days of rutin treatment, we sacrificed the mice and analyzed their soleus muscle and brain for peroxisome proliferator-activated receptor-α coactivator (PGC-1α) and sirtuin 1 (SIRT1) mRNA expression. We observed that rutin treatment increased PGC-1α and SIRT1 mRNA and protein expression. The changes in these markers of mitochondrial biogenesis were associated with increased maximal endurance capacity. The application of 2D gel electrophoresis to analyze the rutin-responsive protein profiles in the WFST mouse brain further revealed the upregulation of the CB1 cannabinoid receptor-interacting protein 1, myelin basic protein, Rho GDP dissociation inhibitor (GDI) alpha, and TPI, indicating that rutin might inhibit anxiety through the upregulation of the expression of anxiety-associated proteins. Western blot analysis of MAPK expression further confirmed the antianxiety effects of rutin. Our study results thus indicate that rutin treatment ameliorates the various impairments associated with physical fatigue.

Taiwanese Green Propolis and Propolin G Protect the Liver from the Pathogenesis of Fibrosis via Eliminating TGF-ß-Induced Smad2/3 Phosphorylation.

Pathogenesis of fibrosis is a common process leading to chronic liver diseases and liver cirrhosis. New compounds for disease treatment and adjuvant therapy have been important issues in recent years. In this study, we isolated propolin G from Taiwanese green propolis (TGP) and investigated its antifibrotic effects by utilizing active hepatic stellate cells (HSCs) fibrosis model. Our results showed that TGP and propolin G inhibited α-SMA, collagen expression, and proliferation of HSC-T6 cells after TGF-ß treatment. They also reduced the accumulation of extracellular matrix (ECM) components such as collagen Iα1 (Col Iα1) through down-regulating JNK signaling. Subsequently, mRNA and protein expression of Smad2/3 but no other Smad members was specifically down-regulated in the presence of propolin G. This effect also significantly induced apoptosis-associated expression of cleaved caspase-3 and caspase-7 proteins for fibrotic cell clearance. In in vivo experiments, we found that propolin G and TGP can reduce plasma alanine aminotransferase (ALT) activation and perhaps lead to the prevention of alcohol-induced liver cirrhosis. Furthermore, TGP can significantly decrease the malondialdehyde (MDA) level but has no influence on plasma or hepatic superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, suggesting TGP protects the liver from alcohol-induced injury through antioxidant-independent pathways. In conclusion, this study provides a new perspective of propolin G and TGP on liver protection, and its application has potential for health management by daily supplement or adjuvant therapy in related diseases.

3,4-Dihydroxytoluene, a metabolite of rutin, inhibits inflammatory responses in lipopolysaccharide-activated macrophages by reducing the activation of NF-κB signaling.

BACKGROUND: Saussurea involucrata (Kar. et Kir.) (S. involucrate), is a rare traditional Chinese medicinal herb. Rutin and hispidulin as well as their metabolites are flavonoids of the flavonol type that abound in S. involucrata, which has been reported to inhibit nonoxidative advanced glycation end products which was involved in physiological inflammation. This study aims to investigate the role of 3,4-dihydroxytoluene (DHT), a metabolite of rutin, in inflammatory inhibition and its involved mechanism. METHODS: This study utilized lipopolysaccharide (LPS) stimulated murine macrophage cell line RAW 264.7 as inflammatory model. The inhibitory effects of DHT were evaluated by the expression level of several inflammation markers such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW264.7 after LPS treatment. In addition, underlying mechanisms, the activation of mitogen-activated protein kinases (MAPKs) and NF-κB, were also investigated. RESULTS: Our results showed that DHT significantly suppressed the LPS-induced production of nitric oxide (NO), iNOS, and COX-2 in a dose-dependent manner without cytotoxicity. DHT also reduced the generation of proinflammatory cytokines majorly in tumor necrosis factor (TNF)-α and minor in interleukin (IL)-1ß and IL-6. In addition, LPS-stimulated I-κBα phosphorylation and degradation followed by translocation of the nuclear factor κB (NF-kB)-p65 from the cytoplasm to the nucleus were attenuated after DHT treatment. CONCLUSIONS: Combined, the results suggest that DHT might exert anti-inflammatory effects in vitro in LPS stimulated RAW 264.7 macrophages and is potential in adjuvant treatment in inflammation disease.

Potential Therapeutic Role of Hispidulin in Gastric Cancer through Induction of Apoptosis via NAG-1 Signaling.

Gastric cancer is one of the most common malignant cancers due to poor prognoses and high mortality rates worldwide. However, an effective chemotherapeutic drug without side effects remains lacking. Saussurea involucrata (SI) Kar. et Kir., also known as snow lotus, grows in mountainous rocky habitats at 2600 m elevation in the Tian Shan and A'er Tai regions of China. The ethyl acetate extract of SI had been shown to inhibit proliferation and induce apoptosis in various tumor cells. In this study, we demonstrated that Hispidulin, active ingredients in SI, inhibits the growth of AGS gastric cancer cells. After Hispidulin treatment, NAG-1 remained highly expressed, whereas COX-2 expression was downregulated. Flow cytometric analysis indicated that Hispidulin induces G1/S phase arrest and apoptosis in time- and concentration-dependent manners. G1/S arrest correlated with upregulated p21/WAF1 and p16 and downregulated cyclin D1 and cyclin E, independent of p53 pathway. In addition, Hispidulin can elevate Egr-1 expression and ERK1/2 activity, whereas ERK1/2 inhibitor markedly attenuated NAG-1 mediated apoptosis. Taken together, Hispidulin can efficiently activate ERK1/2 signaling followed by NAG-1 constitutive expression and trigger cell cycle arrest as well as apoptosis in cancer cell. It can be a potential compound for combination therapy of gastric cancer in the future.

Rutin, a Flavonoid That Is a Main Component of Saussurea involucrata, Attenuates the Senescence Effect in D-Galactose Aging Mouse Model.

Saussurea involucrata (Kar. et Kir.), known as the snow lotus, grows in the Tian Shan and A'er Tai areas of China. It has recently been reported that the ethyl acetate extract of S. involucrata (SI-2) can inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells. This study investigated the protective effect of ethyl acetate extract of S. involucrata (SI-2) or rutin, a flavonoid extracted from ethyl acetate extract of S. involucrata (SI-2), on D-galactose- (D-gal-) induced brain injury in mice. Administering SI-2 or rutin (30 mg/kg/d and 30 mg/kg/d) for 6 weeks, concomitant with D-gal injection, significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, the result showed that the percentages of cleaved caspase-3 and PARP in the D-gal-treated mice were much higher than those in the control. Pretreatment using SI-2 or rutin decreased the expression of cyclooxygenase-2 via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. Furthermore, our results also showed that oral administration of rutin to these mice significantly improved behavioral performance in a step-through passive avoidance task and these results suggest that SI-2 or rutin exerts potent antiaging effects on D-gal in mice via antioxidative mechanisms.

Novel cationic antimicrobial peptide GW-H1 induced caspase-dependent apoptosis of hepatocellular carcinoma cell lines.

Due to its malignancy, the development of effective therapeutic strategies for hepatocellular carcinoma (HCC) is of urgent needs. Natural antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), not only act as direct antimicrobial agents, but also represent important regulators of the innate immune system. It has been reported that cationic AMPs may exhibit cancer-selective toxicity. We have designed a series of novel AMPs with potent antimicrobial activity against a broad spectrum of bacterial pathogens. In the current study, we evaluate the antitumor potency of these AMPs toward HCC cell lines J5, Huh7, and Hep3B. Selected AMPs inhibit the viability of HCC cells in a dose-dependent fashion, while the normal 3T3 cells were significantly less susceptible to these AMPs. GW-H1 treatment (20µM) of J5 cells for 24-72h resulted in the induction of apoptosis, as revealed by flow cytometry (increased sub-G1 populations), and western blot analysis for the appearance of activated caspase-3, -7 and -9 cleavages. Two-dimensional gel electrophoresis was applied to further analyze the AMP-responsive protein profiles of HCC, down-regulation of Hsp27, phophoglycerate kinase 1 and triosephosphate isomerase indicated that GW-H1 may induce apoptosis, and further inhibit progression and metastasis of J5 HCC cells. FITC-labeled GW-H1 was found to attach to cell membrane initially, then translocated into the cytoplasm, and eventually membranous organelles or nucleus. GW-H1 induced a marked growth suppression of J5 xenografts in nude mice in a dose dependent manner. These findings provided support for future application of GW-H1 as potential therapeutic agent for HCC.

Microrheology of human synovial fluid of arthritis patients studied by diffusing wave spectroscopy.

The viscoelastic properties of synovial fluid (SF) are critical to its functions of lubrication and shock-absorption of joints in human body; a change in the viscoelastic properties, even of only a few percents, is often concomitant with arthritis. In this work, the elastic modulus G '(f) and the viscous modulus G ''(f) of SF from patients suffering from three kinds of joint diseases, namely, osteoarthritis (OA), rheumatoid arthritis (RA), and gouty arthritis (GA), were determined as a function of frequency "f " (in the low frequency range from f ∼ 0.1 to 10 Hz) by Diffusing Wave Spectroscopy (DWS) and correlated with the white blood cell (WBC) count. A strong correlation was observed, showing a higher WBC count corresponding to lower elastic and viscous moduli, G ' and G ''; further details depend on inflammatory vs. non-inflammatory, and on the severity of inflammation. Different types of arthritis lead to different degrees of decreasing viscoelasticity. Identical measurements were carried out with a commercial visco-supplementation (or artificial SF) to serve as reference. In general, the reduction in both G ' and G '' was most severe in the case of GA and least severe in the case of OA. Besides, in all cases, the reduction in G ' was more prominent than the reduction in G '', indicating that in general, the deterioration in the elasticity of SF by inflammation is more severe than that in the viscosity. This simple method for quantitative physical characterization of synovial fluid may serve as a useful complementary metric to the conventional biochemical analysis in clinical diagnosis of arthritis.

Freshwater clam extract inhibits inflammatory responses in LPS-activated macrophages by reducing the activation of mitogen-activated protein kinases and NF-kappaB.

Recent studies demonstrated that freshwater clam (Corbicula fluminea) has lipid-lowering and hepatoprotective activities, but its effect on immune responses has not yet been addressed. Here we showed that ethanol extracts of C. fluminea (ECF) reduced nitrite oxide, interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in lipopolysaccharide-activated macrophages. Further, ECF was fractionated into n-hexane, ethyl acetate, ethanol, and water soluble fractions. Of these, the ethyl acetate soluble fraction (EACF) had the highest capacity to inhibit pro-inflammatory mediators expression. The underlying mechanisms for the anti-inflammatory activity of EACF were demonstrated as down-regulation of ERK1/2, JNK1/2, and p38 phosphorylation and NF-kappaB activity. Using gas chromatography-mass spectrometric analysis EACF was found to be composed mainly of fatty acids and steroids. Our results provide evidence that freshwater clam has anti-inflammatory activity, and support the possibility for the development of freshwater clam as a health supplement or adjuvant therapeutic agent for either preventing or treating inflammation related diseases.

Synergistic anti-tumor activity of isochaihulactone and paclitaxel on human lung cancer cells.

Drug resistance frequently develops in tumors during chemotherapy. Therefore, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Here, we show that isochaihulactone (K8) enhanced paclitaxel-induced apoptotic death in human lung cancer cells, and the enhancing effect was related to increased NSAID-activated gene-1 (NAG-1) expression. CalcuSyn software was used to evaluate the synergistic interaction of K8 and paclitaxel on human lung cancer cells; the synergistic effect of K8 in combination with paclitaxel was increased more than either of these drugs alone. Furthermore, the activity of ERK1/2 was enhanced by the combination of K8 and paclitaxel, and an ERK1/2 inhibitor dramatically inhibited NAG-1 expression in human lung cancer cells. Therefore, this synergistic apoptotic effect in human lung cancer cells may be directly associated with K8-induced NAG-1 expression through ERK1/2 activation. Moreover, over-expression of NAG-1 enhanced K8/paclitaxel-induced apoptosis in human lung cancer cells. In addition, treatment of nude mice with K8 combined with paclitaxel induced phospho-ERK1/2 and NAG-1 expression in vivo. Targeting of NAG-1 signaling could enhance therapeutic efficacy in lung cancer. Our results reveal that activation of NAG-1 by K8 enhanced the therapeutic efficacy of paclitaxel in human lung cancer cells via the ERK1/2 signaling pathway.

RC-RNase-induced cell death in estrogen receptor positive breast tumors through down-regulation of Bcl-2 and estrogen receptor.

RC-RNase exerts anti-cancer effects on many tumors. However, the mechanisms by which RC-RNase induces cytotoxicity in different tumor cells are unclear. Currently, estrogen receptor (ER)-positive and negative breast tumors are treated with RC-RNase. Our data demonstrate that RC-RNase induces cell death on ER-positive but not on ER-negative breast tumors. This study also shows that down-regulation of ER and Bcl-2 is found on RC-RNase-treated ER-positive breast tumors. Additionally, Bcl-2 overxpression can prevent ER-positive breast tumors from cell death treated with RC-RNase. In summary, this study demonstrates that RC-RNase-induced cell death of ER-positive breast tumors is through regulation of ER and Bcl-2.

Isochaihulactone protects PC12 cell against H(2)O(2) induced oxidative stress and exerts the potent anti-aging effects in D-galactose aging mouse model.

AIM: to investigate the effect of isochaihulactone (also known as K8), a lignan compound of Bupleurum scorzonerifolium, on H(2)O(2)-induced cytotoxicity in neuronally differentiated PC12 cells (nPC12). METHODS: viability of neuronal PC12 cells was measured using MTT assay. Protein expression was determined by Western blot. Apoptotic cells was determined using TUNEL assay. D-galactose aging mice were used as a model system to study the anti-oxidant effects of isochaihulactone in vivo. RESULTS: pretreatment with isochaihulactone (5-10 micromol/L) increased cell viability and decreased membrane damage, generation of reactive oxygen species and degradation of poly (ADP-ribose) polymerase in H(2)O(2)-treated nPC12 cells and also decreased the expression of cyclooxygenase-2, via downregulation of NF-kappaB, resulting in a decrease in lipid peroxidation. The results suggest that isochaihulactone is a potential antioxidant agent. In a murine aging model, in which chronic systemic exposure to D-galactose (D-gal) causes the acceleration of senescence, administration of isochaihulactone (10 mgxkg(-1)xd(-1), sc) for 7 weeks concomitant with D-gal injection significantly increased superoxide dismutase and glutathione peroxidase activities and decreased the MDA level in plasma. Furthermore, H&E staining to quantify cell death within hippocampus showed that percentage of pyknotic nuclei in the D-gal-treated mice were much higher than in control. CONCLUSION: the results suggest that isochaihulactone exerts potent anti-aging effects against D-gal in mice possibly via antioxidative mechanisms.

Hispidulin potently inhibits human glioblastoma multiforme cells through activation of AMP-activated protein kinase (AMPK).

Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor. Despite recent therapeutic advances in other cancers, the treatment of GBM remains ineffective and essentially palliative. The current focus lies in the finding of components that activate the AMP-activated protein kinase (AMPK), one key enzyme thought to be activated during the caloric restriction (CR). In the present study, we found that treatment of hispidulin, a flavone isolated from Saussurea involucrate Kar. et Kir., resulted in dose-dependent inhibition of GBM cellular proliferation. Interestingly, we show that hispidulin activated AMPK in GBM cells. The activation of AMPK suppressed downstream substrates, such as the mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and resulted in a general decrease in mRNA translation. Moreover, hispidulin-activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Furthermore, hispidulin blocked the progression of the cell cycle at the G1 phase and induced apoptosis by inducing p53 expression and further upregulating p21 expression in GBM cells. On the basis of these results, we demonstrated that hispidulin has the potential to be a chemopreventive and therapeutic agent against human GBM.

Extended O6-methylguanine methyltransferase promoter hypermethylation following n-butylidenephthalide combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on inhibition of human hepatocellular carcinoma cell growth.

Epigenetic alteration of DNA methylation plays an important role in the regulation of gene expression associated with chemosensitivity of human hepatocellular (HCC) carcinoma cells. With the aim of improving the chemotherapeutic efficacy for HCC, the effect of the naturally occurring compound n-butylidenephthalide (BP), which is isolated from a chloroform extract of Angelica sinensis, was investigated. In both HepG2 and J5 HCC cell lines, a synergistic antiproliferative effect was observed when a low dosage of BP was combined with the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU is an alkylating agent, and it prompts us to examine one of DNA repair genes, O(6)-methylguanine methyltransferase (MGMT). It was evident from methylation-specific polymerase chain reaction (PCR) analysis that BP/BCNU combined treatment caused a time- and concentration-dependent enhancement of MGMT promoter methylation. Overexpression of MGMT could abolish BP-induced growth inhibition in the J5 tumor cell line as measured by colony formation assay. When BP was combined with BCNU and administered, it showed significant antitumor effects in both HepG2 and J5 xenograft tumors as compared with the use of only one of these drugs. The BCNU-induced apoptosis and inhibited MGMT protein expression in HCC cells, both in vitro and in vivo, resulting from the combination treatment of BP and BCNU suggest a potential clinical use of this compound for improving the prognosis for HCCs.

n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity.

AIM: To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells. METHODS: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining. RESULTS: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT. CONCLUSION: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.