Concurrent Ingestion of Alkaline Water and L-Glutamine Enhanced Salivary α-Amylase Activity and Testosterone Concentration in Boxing Athletes.

Athletes often take sport supplements to reduce fatigue and immune disturbances during or after training. This study evaluated the acute effects of concurrent ingestion of alkaline water and L-glutamine on the salivary immunity and hormone responses of boxers after training. Twelve male boxing athletes were recruited in this study. During regular training, the participants were randomly divided into three groups and asked to consume 400 mL of alkaline water (Group A), 0.15 g/kg body weight of L-glutamine with 400 mL of water (Group G), and 0.15 g/kg of L-glutamine with 400 mL of alkaline water (Group A+G) at the same time each day for three consecutive weeks. Before and immediately after the training, saliva, heart rates, and the rate of perceived exertion were investigated. The activity of α-amylase and concentrations of lactoferrin, immunoglobulin A (IgA), testosterone, and cortisol in saliva were measured. The results showed that the ratio of α-amylase activity/total protein (TP) significantly increased after training in Group A+G but not in Group A or G, whereas the ratios of lactoferrin/TP and IgA/TP were unaffected in all three groups. The concentrations of salivary testosterone after training increased significantly in Group A+G but not in Group A or G, whereas the salivary cortisol concentrations were unaltered in all groups. In conclusion, concurrent ingestion of 400 mL of alkaline water and 0.15 g/kg of L-glutamine before training enhanced the salivary α-amylase activity and testosterone concentration of boxers, which would be beneficial for post-exercise recovery.

Supplementation of L-glutamine enhanced mucosal immunity and improved hormonal status of combat-sport athletes.

BACKGROUND: Maintaining proper immune function and hormone status is important for athletes to avoid upper respiratory tract infection (URTI) and insufficient recovery, which is detrimental to sport performance and health. The aim of this study was to evaluate whether three-week supplementation of L-glutamine could benefit the mucosal immunity and hormonal status of combat-sport athletes as well as their rates of upper respiratory tract infection (URTI) and subjective feelings of well-being after intensive training. METHODS: Twenty-one combat-sport athletes from the National Taiwan University of Sport were recruited in this study. After intensive training, two groups of the participants were asked to consume powder form of 0.3 g/kg body weight of L-glutamine (GLU group) or maltodextrin (PLA group) with drinking water in a randomized design at the same time every day during 3 weeks. Saliva samples were collected to measure immunoglobulin A (IgA), nitric oxide (NO), testosterone (T) and cortisol (C) before and after three-week supplementation; moreover, Hooper's index questionnaires were completed for wellness assessment. The incidence and duration of URTI were recorded by using a health checklist throughout the entire study period. RESULTS: Supplementation of L-glutamine significantly enhanced the concentrations of IgA and NO in saliva; additionally, the incidence of URTI was significantly reduced. Regarding hormones, T concentration was significantly decreased in the PLA group, whereas C concentration was significantly increased, resulting in a significant decrease of T/C ratio. In contrast, the GLU group showed a significant increase of T/C ratio, while the mood scores of the Hooper's index questionnaire were higher in the PLA group. CONCLUSIONS: Three-week supplementation of L-glutamine after intensive training enhanced the mucosal immunity, improved hormonal status and reduced the rate of URTI of combat-sport athletes while feelings of well-being were also enhanced. Therefore, L-glutamine would be beneficial for the sports performance and recovery of athletes.

In Silico and In Vitro studies of taiwan chingguan yihau (NRICM101) on TNF-α/IL-1ß-induced human lung cells.

COVID-19 pandemic has been a global outbreak of coronavirus (SARS-CoV-2 virus) since 2019. Taiwan Chingguan Yihau (NRICM101) is the first traditional Chinese medicine (TCM) classic herbal formula and is widely used for COVID-19 patients in Taiwan and more than 50 nations. This study is to investigate in silico target fishing for the components of NRICM101 and to explore whether NRICM101 inhibits cytokines-induced normal human lung cell injury in vitro. Our results showed that network prediction of NRICM101 by a high throughput target screening platform showed that NRICM101 has multiple functions that may affect cytokine regulation to prevent human lung cell injury. In addition, NRICM101 revealed protective effects against TNF-α/IL-1ß-induced normal human lung HEL 299 cell injury through JNK and p38MAPK kinase signaling. Next-generation sequencing (NGS) analysis of NRICM101 on TNF-α/IL-1ß-injured HEL 299 cells indicated that inflammatory pathway, cell movement of macrophages, cellular infiltration by macrophages, and Th1/Th2 immuno-regulation pathways were included. Thus, NRICM101 is a therapeutic agent, and it can improve COVID-19 syndrome to confer beneficial effects through multiple targeting and multiple mechanisms.

Approaches towards fighting the COVID­19 pandemic (Review).

The coronavirus disease 2019 (COVID­19) outbreak, which has caused >46 millions confirmed infections and >1.2 million coronavirus related deaths, is one of the most devastating worldwide crises in recent years. Infection with COVID­19 results in a fever, dry cough, general fatigue, respiratory symptoms, diarrhoea and a sore throat, similar to those of acute respiratory distress syndrome. The causative agent of COVID­19, SARS­CoV­2, is a novel coronavirus strain. To date, remdesivir has been granted emergency use authorization for use in the management of infection. Additionally, several efficient diagnostic tools are being actively developed, and novel drugs and vaccines are being evaluated for their efficacy as therapeutic agents against COVID­19, or in the prevention of infection. The present review highlights the prevalent clinical manifestations of COVID­19, characterizes the SARS­CoV­2 viral genome sequence and life cycle, highlights the optimal methods for preventing viral transmission, and discusses possible molecular pharmacological mechanisms and approaches in the development of anti­SARS­CoV­2 therapeutic agents. In addition, the use of traditional Chinese medicines for management of COVID­19 is discussed. It is expected that novel anti­viral agents, vaccines or an effective combination therapy for treatment/management of SARS­CoV­2 infection and spread therapy will be developed and implemented in 2021, and we would like to extend our best regards to the frontline health workers across the world in their fight against COVID­19.

Ursolic acid promotes apoptosis, autophagy, and chemosensitivity in gemcitabine-resistant human pancreatic cancer cells.

Gemcitabine (GEM) resistance in pancreatic adenocarcinoma mediated by the receptor for advanced glycation end products (RAGE) has been demonstrated. Therefore, investigating the safety and the potential of new auxiliary methods for pancreatic cancer treatment is urgent. Ursolic acid (UA), a natural pentacyclic triterpenoid found in apple peels, rosemary, and thyme, has been reported to have anticancer capacity. This study aimed to reveal the underlying mechanisms of UA in cell death and drug enhancement, especially in GEM-resistant pancreatic cancer cells. First, GEM-resistant cells (MIA Paca-2GEMR cells) were established by incrementally increasing GEM culture concentrations. UA treatment reduced cell viability through cell cycle arrest and endoplasmic reticulum (ER) stress, resulting in apoptosis and autophagy in a dose-dependent manner in MIA Paca-2 and MIA Paca-2GEMR cells. High RAGE expression in MIA Paca-2GEMR cells was suppressed by UA treatment. Interestingly, knocking down RAGE expression showed similar UA-induced effects in both cell lines. Remarkably, UA had a drug-enhancing effect by decreasing cell viability and increasing cell cytotoxicity when combined with GEM treatment. In conclusions, UA triggered ER stress, subsequently regulating apoptosis- and autophagy-related pathways and increasing GEM chemosensitivity in pancreatic cancer cells by inhibiting the expression of RAGE.

The hepatoprotective activities of Kalimeris indica ethanol extract against liver injury in vivo.

Kalimeris indica (L.) Sch. Bip. is a traditional Chinese medicine (TCM) and a portion of food used for cooking in China. It has been demonstrated that an ethanol extract of K. indica has an anti-inflammatory effect by inhibition of nitric oxide (NO) production on murine macrophage RAW264.7 cells after lipopolysaccharide (LPS) induction. In this study, the hepatoprotective effects of the total phenolics of K. indica (TPK), the total triterpenes of K. indica (TTK), and the total flavones of K. indica (TFK) from ethanol extracts of K. indica were evaluated in Bacille Calmette-Guerin (BCG)/LPS-induced liver injury in vivo. The treatments of TPK, TTK, and TFK improved liver injury in mice. Additionally, all treatments significantly not only reduced the hepatic malondialdehyde (MDA) content and hepatic total nitric oxide synthase (tNOS) but also induced the hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. The treatments of TPK and TTK significantly reduced the hepatic inducible nitric oxide synthase (iNOS). The treatments of TPK, TTK, and TFK reduced the serum total bilirubin (T-Bil), and only TFK treatment reduced the serum alanine aminotransferase (ALT). Our results suggest that TPK, TTK, and TFK from ethanol extracts of K. indica might play an essential protective role against BCG/LPS-induced liver injury in vivo.

Autophagy and apoptotic machinery caused by Polygonum cuspidatum extract in cisplatin­resistant human oral cancer CAR cells.

Polygonum cuspidatum (Hu Zhang) is a traditional Chinese medicine (TCM) and has been revealed to exert anticancer, anti­angiogenesis, anti­human immunodeficiency virus (HIV), anti­hepatitis B virus, anti­microbial, anti­inflammatory, and neuro­protective bio­activities. However, the effect of P. cuspidatum extract (PCE) on drug­resistant human oral cancer cells regarding cell death is not fully understood yet. The present study was undertaken to explore the induction of autophagic and apoptotic cell death and to investigate their underlying molecular mechanisms in PCE­treated cisplatin­resistant human oral cancer CAR cells. Our results revealed that PCE was determined via HPLC analytic method, and it was revealed that resveratrol may be a major compound in PCE. The data also demonstrated that PCE reduced CAR cell viability in a concentration­ and time­dependent response via an MTT assay. PCE had an extremely low toxicity in human normal gingival fibroblasts (HGF). Autophagic and apoptotic cell death was found after PCE treatment by morphological determination. PCE was revealed to induce autophagy as determined using acridine orange (AO), LC3­GFP, monodansylcadaverine (MDC) and LysoTracker Red staining in CAR cells. In addition, PCE was revealed to induce apoptosis in CAR cells via 4',6­diamidino­2­phenylindole (DAPI)/terminal deoxynucleotidyl transferase dUTP nick­end labeling (TUNEL) double staining. PCE significantly stimulated caspase­9 and ­3 activities as revealed using caspase activity assays. PCE markedly increased the protein levels of Atg5, Atg7, Atg12, Beclin­1, LC3, Bax and cleaved caspase­3, while it decreased the protein expression of Bcl­2 in CAR cells as determined by western blotting. In conclusion, our findings are the first to suggest that PCE may be potentially efficacious for the treatment of cisplatin­resistant human oral cancer.

Baicalin, Baicalein, and Lactobacillus Rhamnosus JB3 Alleviated Helicobacter pylori Infections in Vitro and in Vivo.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, and gastric cancer. The flavonoid compounds baicalin and baicalein found in many medicinal plants exhibit an anti-inflammatory effect. The administration of Lactobacillus strains reducing the risk of H. pylori infection is well accepted. In this study, the therapeutic effects against H. pylori infection of baicalin, baicalein, and L. rhamnosus JB3 (LR-JB3), isolated from a dairy product, were investigated. Compared to baicalin, baicalein exhibited stronger anti-H. pylori activity and cytotoxicity on human gastric cancer epithelial AGS cells. Baicalin and baicalein both suppressed the vacA gene expression of H. pylori and interfered with the adhesion and invasion ability of H. pylori to AGS cells, as well as decreased H. pylori-induced interleukin (IL)-8 expression. In the mice infection model, high dosages of baicalin and baicalein inhibited H. pylori growth in the mice stomachs. Serum IL-1ß levels and H. pylori-specific serum IgM and IgA levels in mice treated with baicalin and baicalein were decreased. Moreover, a synergistic therapeutic effect of baicalein and LR-JB3 on eradicating H. pylori infections was observed. Thus, administrating baicalin, baicalein, or LR-JB3 for an H. pylori infection could offer similar therapeutic effects to administering antibiotics while not disturbing the balance of gut microbiota. This study revealed the effects of baicalin, baicalein, and LR-JB3 on attenuating the virulence of H. pylori. The synergistic effect with baicalein and LR-JB3 provides the experimental rationale for testing the reliability, safety, and efficacy of this approach in higher animals and perhaps ultimately in humans to eradicate H. pylori infections. PRACTICAL APPLICATION: Baicalin and baicalein exert health promotion and avoidance of H. pylori infections by interfering with H. pylori growth and virulence. Lactobacillus rhamnosus JB3 was used to reduce the gastric inflammation caused by H. pylori infection.

Synergistic inhibitory effects of cetuximab and curcumin on human cisplatin-resistant oral cancer CAR cells through intrinsic apoptotic process.

Cetuximab, an epidermal growth factor receptor (EGFR)-targeting monoclonal antibody (mAb), is a novel targeted therapy for the treatment of patients with oral cancer. Cetuximab can be used in combination with chemotherapeutic agents to prolong the overall survival rates of patients with oral cancer. Curcumin is a traditional Chinese medicine, and it has been demonstrated to have growth-inhibiting effects on oral cancer cells. However, information regarding the combination of cetuximab and curcumin in drug-resistant oral cancer cells is lacking, and its underlying mechanism remains unclear. The purpose of the present study was to explore the oral anticancer effects of cetuximab combined with curcumin on cisplatin-resistant oral cancer CAR cell apoptosis in vitro. The results demonstrated that combination treatment synergistically potentiated the effect of cetuximab and curcumin on the suppression of cell viability and induction of apoptosis in CAR cells. Cetuximab and curcumin combination induced apoptosis and dramatically increased caspase-3 and caspase-9 activities compared with singular treatment. Combination treatment also markedly suppressed the protein expression levels of EGFR and mitogen-activated protein kinases (MAPKs) signaling (phosphorylation of ERK, JNK and p38). The results demonstrated that co-treatment with cetuximab and curcumin exerts synergistic oral anticancer effects on CAR cells through the suppression of the EGFR signaling by regulation of the MAPK pathway.

Recent progress in natural dietary non-phenolic bioactives on cancers metastasis.

From several decades ago to now, cancer continues to be the leading cause of death worldwide, and metastasis is the major cause of cancer-related deaths. For health benefits, there is a great desire to use non-chemical therapy such as nutraceutical supplementation to prevent pathology development. Over 10,000 different natural bioactives or phytochemicals have been known that possessing potential preventive or supplementary effects for various diseases including cancer. Previously, the in vitro and in vivo anti-invasive and anti-metastatic activities of phenolic acids, monophenol, polyphenol and their derivatives and flavonoids and their derivatives have been reviewed. However, a vast number of natural dietary compounds other than phenolics have been demonstrated to potentially possess the ability to inhibit the invasion and metastasis of various cancers. In this review, we summarize the studies in recent decade on in vitro and in vivo effects and molecular mechanisms of natural bioactives, excluding the phenolics in food, in cancer invasion and metastasis. By combining this review of non-phenolics with the previous phenolics reviews, the puzzle for the contribution of natural dietary bioactives on cancer invasive or/and metastatic progress will be almost complete and more clear.

Effects of Lycium barbarum (goji berry) on dry eye disease in rats.

Lycium barbarum (goji berry) has long been used as a food and traditional herbal medicine. This study aimed to investigate the beneficial effect of the goji berry on dry eye disease in rats. Male Sprague­Dawley rats with induced dry eye disease were randomly assigned to four groups: Vehicle (control), low­dose goji berry extract [GBE; 250 mg/kg/body weight (bw)], median­dose GBE (350 mg/kg/bw), and high­dose GBE (500 mg/kg/bw). Three methods, Schirmer's test, tear break­up time (BUT) measurement and keratoconjunctival fluorescein staining, were used to evaluate the effect of GBE on symptoms of dry eye disease experienced by the rats. The results of the present study revealed that both the Schirmer's test score and tear BUT significantly increased following 1 week of GBE administration. Furthermore, the severity of the keratoconjunctival staining decreased significantly. In addition, the results suggested that administration of GBE may ameliorate dry eye disease symptoms in a dose­dependent manner. There were no mortalities and no apparent abnormal histopathology changes in the liver or kidney tissues of rats administered GBE for 21 consecutive days. Polysaccharides and betaine present in GBE may have important effects in alleviating dry eye disease induced by oxidative stress and inflammation. In conclusion, the goji berry is a safe, functional food with beneficial effects in alleviating dry eye disease.

The Suppressive Effects of Geniposide and Genipin on Helicobacter pylori Infections In Vitro and In Vivo.

Geniposide and genipin have been found in Gardenia jasminoides Ellis, a traditional Chinese medicine that exhibits multiple biological functions. However, no report showing the effects of geniposide and genipin on gastric protection in Helicobacter pylori infections in vitro and in vivo has been done. In this study, we clarified how geniposide and genipin suppress H. pylori-mediated inflammation in gastric AGS cells and C57BL/6 mice. Our results demonstrated that genipin shows a strong ability to inhibit H. pylori growth and is able to reduce vacA and cagA gene expression of H. pylori in infected AGS cells. Genipin also attenuates the abilities of adhesion and invasion of H. pylori to AGS cells. An attenuation of interleukin (IL)-8 and IFN-γ production caused by genipin was observed to inhibit cell inflammatory responses. In the in vivo experiments, geniposide and genipin both showed suppressive effects on the vacA gene expression in mice after H. pylori infection. The serum levels of IFN-γ, IL-1ß, immunoglobulin A, and Immunoglobulin M were decreased by geniposide and genipin in infected mice. The inflammatory maker COX2 was downregulated in H. pylori-infected mice after exposure to geniposide and genipin. Together, geniposide and genipin effectively exert a healthy promotion to reduce H. pylori infections in vivo by interfering with the growth and virulence of H. pylori as well as attenuating the gastric inflammation caused by an H. pylori infection. PRACTICAL APPLICATION: Geniposide and genipin have a healthy promotion to eradicate H. pylori infections by interfering with the growth and virulence of H. pylori and to attenuate the gastric inflammation caused by an H. pylori infection.

Hypouricemic effects of Mesona procumbens Hemsl. through modulating xanthine oxidase activity in vitro and in vivo.

Uric acid is a metabolite obtained from purine by xanthine oxidase activity (XO) and high levels of serum uric acid leads to hyperuricemia and gout. Mesona procumbens Hemsl. has been used as a healthy beverage and a traditional remedy. In this study, the hypouricemic effects of M. procumbens extracts were evaluated in vitro and in vivo. The 50% ethanol extract of M. procumbens (EE50) showed the strongest inhibitory effect on monosodium urate (MSU)-induced XO activity in THP-1 cells. However, the phenolics and flavonoids in EE50 may not serve as inhibitors of XO. EE50 prevented an increase in the serum uric acid level in potassium oxonate (PO)-challenged ICR mice and streptozocin (STZ)-induced SD rats. EE50 down-regulated STZ-induced liver XO activity, and it restored renal OAT1 and urate transporter expression. STZ-induced renal interleukin-1ß (IL-1ß) and the tumor necrosis factor-α (TNF-α) level were inhibited by EE50 treatment. EE50 exhibits the hypouricemic effect via down-regulation of XO activity, suggesting that EE50 has potential to improve hyperuricemia and its complications.

Casticin Induced Apoptosis in A375.S2 Human Melanoma Cells through the Inhibition of NF-[Formula: see text]B and Mitochondria-Dependent Pathways In Vitro and Inhibited Human Melanoma Xenografts in a Mouse Model In Vivo.

Casticin, a polymethoxyflavone occurring in natural plants, has been shown to have anticancer activities. In the present study, we aims to investigate the anti-skin cancer activity of casticin on melanoma cells in vitro and the antitumor effect of casticin on human melanoma xenografts in nu/nu mice in vivo. A flow cytometric assay was performed to detect expression of viable cells, cell cycles, reactive oxygen species production, levels of [Formula: see text] and caspase activity. A Western blotting assay and confocal laser microscope examination were performed to detect expression of protein levels. In the in vitro studies, we found that casticin induced morphological cell changes and DNA condensation and damage, decreased the total viable cells, and induced G2/M phase arrest. Casticin promoted reactive oxygen species (ROS) production, decreased the level of [Formula: see text], and promoted caspase-3 activities in A375.S2 cells. The induced G2/M phase arrest indicated by the Western blotting assay showed that casticin promoted the expression of p53, p21 and CHK-1 proteins and inhibited the protein levels of Cdc25c, CDK-1, Cyclin A and B. The casticin-induced apoptosis indicated that casticin promoted pro-apoptotic proteins but inhibited anti-apoptotic proteins. These findings also were confirmed by the fact that casticin promoted the release of AIF and Endo G from mitochondria to cytosol. An electrophoretic mobility shift assay (EMSA) assay showed that casticin inhibited the NF-[Formula: see text]B binding DNA and that these effects were time-dependent. In the in vivo studies, results from immuno-deficient nu/nu mice bearing the A375.S2 tumor xenograft indicated that casticin significantly suppressed tumor growth based on tumor size and weight decreases. Early G2/M arrest and mitochondria-dependent signaling contributed to the apoptotic A375.S2 cell demise induced by casticin. In in vivo experiments, A375.S2 also efficaciously suppressed tumor volume in a xenotransplantation model. Therefore, casticin might be a potential therapeutic agent for the treatment of skin cancer in the future.

Beneficial Effects of Camellia Oil (Camellia oleifera Abel.) on Hepatoprotective and Gastroprotective Activities.

Epidemiological studies have shown that increased dietary intake of natural antioxidants is beneficial for health because of their bioactivities, including antioxidant and anti-inflammation actions. Camellia oil made from tea seed (Camellia oleifera Abel.) is commonly used as an edible oil and a traditional medicine in Taiwan and China. Until now, the camellia oil has been widely considered as a dietary oil for heath. In this review, we summarize the protective effects of camellia oil with antioxidant activity against oxidative stress leading to hepatic damage and gastrointestinal ulcers. The information in this review leads to the conclusion that camellia oil is not only an edible oil but also a vegetable oil with a potential function for human health.

Ethanol extract of Hedyotis diffusa willd upregulates G0/G1 phase arrest and induces apoptosis in human leukemia cells by modulating caspase cascade signaling and altering associated genes expression was assayed by cDNA microarray.

The authors' previous study has shown that water extract of Hedyotis diffusa Willd (HDW) promoted immune response and exhibited anti-leukemic activity in BALB/c leukemic mice in vivo. In this study, the anti-proliferation effects of ethanol extract of H. diffusa Willd (EEHDW) on lung cancer cell lines (A549, H1355, and LLC), leukemia cell lines (HL-60, WEHI-3), and a mouse melanoma cell line (B16F10) in vitro were investigated. The results demonstrated that EEHDW suppressed the cell proliferation of A549, H1355, HL-60, WEHI-3, and B16F10 cells as well as reduced cell viability in a concentration-dependent manner. We found that EEHDW inhibited the cell proliferation of HL-60 cells in concentration-dependent manner. In addition, EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and sub-G1 population (apoptotic cells). EEHDW provoked DNA condensation and DNA damage in HL-60 cells. The activities of caspase-3, caspase-8, and caspase-9 were elevated in EEHDW-treated HL-60 cells. DNA microarray to investigate and display the gene levels related to cell growth, signal transduction, apoptosis, cell adhesion, cell cycle, DNA damage and repair, transcription and translation was also used. These findings suggest that EEHDW may be a potential herbal medicine and therapeutic agent for the treatment of leukemia.

Suppressions of Migration and Invasion by Cantharidin in TSGH-8301 Human Bladder Carcinoma Cells through the Inhibitions of Matrix Metalloproteinase-2/-9 Signaling.

Cancer metastasis becomes an initial cause of cancer death in human population. In many cancers, it has been shown that the high levels of matrix metalloproteinase (MMP)-2 and/or MMP-9 are associated with the invasive phenotypes of cancer cells. In this study, we investigated the effects of cantharidin, a derivative of blister beetles which is one of the traditional Chinese medicines, on the adhesion, migration, and invasion of human bladder cancer TSGH-8301 cells. Cantharidin effectively suppressed TSGH-8301 cell adhesion, migration, and invasion in a concentration-dependent manner. Results from Western blotting, RT-PCR, and gelatin zymography assays indicated that cantharidin blocked the protein levels, gene expression (mRNA), and activities of MMP-2 and -9 in TSGH-8301 cells. Cantharidin also significantly suppressed the protein expressions of p-p38 and p-JNK1/2 in TSGH-8301 cells. Taken together, cantharidin was suggested to present antimetastatic potential via suppressing the levels of MMP-2 and MMP-9 expression that might be mediated by targeting the p38 and JNK1/2 MAPKs pathway in TSGH-8301 human bladder cancer cells.

Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells.

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho­mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.

Triggering apoptotic death of human malignant melanoma a375.s2 cells by bufalin: involvement of caspase cascade-dependent and independent mitochondrial signaling pathways.

Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and nitric oxide (NO) formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨ(m) and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways.

Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.