Mitochondria-mediated Pathway Research Articles

Human Keratinocyte UVB-Protective Effects of a Low Molecular Weight Fucoidan from Sargassum horneri Purified by Step Gradient Ethanol Precipitation.

Ultraviolet B (UVB) radiation-induced oxidative skin cell damage is a major cause of photoaging. In the present study, a low molecular weight fucoidan fraction (SHC4) was obtained from Sargassum horneri by Celluclast-assisted extraction, followed by step gradient ethanol precipitation. The protective effect of SHC4 was investigated in human keratinocytes against UVB-induced oxidative stress. The purified fucoidan was characterized by Fourier-transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR), agarose gel-based molecular weight analysis and monosaccharide composition analysis. SHC4 had a mean molecular weight of 60 kDa, with 37.43% fucose and 28.01 ± 0.50% sulfate content. The structure was mainly composed of α-L-Fucp-(1→4) linked fucose units. SHC4 treatment dose-dependently reduced intracellular reactive oxygen species (ROS) levels and increased the cell viability of UVB exposed HaCaT keratinocytes. Moreover, SHC4 dose-dependently inhibited UVB-induced apoptotic body formation, sub-G1 accumulation of cells and DNA damage. Inhibition of apoptosis was mediated via the mitochondria-mediated pathway, re-establishing the loss of mitochondrial membrane potential. The UVB protective effect of SHC4 was facilitated by enhancing intracellular antioxidant defense via nuclear factor erythroid 2–related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Further studies may promote the use of SHC4 as an active ingredient in cosmetics and nutricosmetics.

Endosulfan induces cardiotoxicity through apoptosis via unbalance of pro-survival and mitochondrial-mediated apoptotic pathways

Although the associations between endosulfan and adverse cardiovascular health have been reported, the toxic effects and underlying mechanism of endosulfan on the heart are not well understood. In this study, we examined the cardiotoxicity induced by endosulfan using Wistar rats and human cardiomyocytes (AC16) cells. Wistar rats were divided into control group (received corn oil alone) and three concentrations of endosulfan groups (1, 5 and 10 mg/kg·bw) by gavage. The AC16 cells were treated with three various concentrations (0, 1.25, 5, and 20 μg/mL) of endosulfan. The results showed that endosulfan induced cytotoxicity through damaging myocardial structure, decreasing the viability of cardiomyocytes, and elevating the serum levels of cardiac troponin I, heart fatty acid binding protein, aspartate aminotransferase, and reactive oxygen species (p < 0.05). Moreover, measurement of mitochondrial function showed that endosulfan could significantly decrease adenosine triphosphate levels and cytochrome c oxidase IV expression in AC16 cells (p < 0.05). In addition, endosulfan obviously inhibited Bcl-2 expression, activated the expressions of cytochrome c/Caspase-9/Caspase-3 signaling pathway, and induced the apoptosis of AC16 cells (p < 0.05). Furthermore, endosulfan significantly increased the expression of Bim, and inhibited the expressions of PI3K/Akt/FoxO3a signaling pathways in cardiomyocytes (p < 0.05). These results suggest that endosulfan may induce cardiotoxicity by inducing myocardial apoptosis resulting from activation of mitochondria-mediated apoptosis pathway and inhibition of pro-survival signaling pathways, which might be helpful in elucidating the mechanism of cardiac dysfunction induced by endosulfan.

Sirtuins family as a target in endothelial cell dysfunction: implications for vascular ageing.

The vascular endothelium is a protective barrier between the bloodstream and the vasculature that may be disrupted by different factors such as the presence of diseased states. Diseases like diabetes and obesity pose a great risk toward endothelial cell inflammation and oxidative stress, leading to endothelial cell dysfunction and thereby cardiovascular complications such as atherosclerosis. Sirtuins are NAD+-dependent histone deacetylases that are implicated in the pathophysiology of cardiovascular diseases, and they have been identified to be important regulators of endothelial cell function. A handful ofrecent studies suggest that disbalance in the regulation of endothelial sirtuins, mainly sirtuin 1 (SIRT1), contributes to endothelial cell dysfunction. Herein, we summarize how SIRT1 and other sirtuins may contribute to endothelial cell function and how presence of diseased conditions may alter their expressions to cause endothelial dysfunction. Moreover, we discuss how the beneficial effects of exercise on the endothelium are dependent on SIRT1. These mainly include regulation of signalingpathways related to endothelial nitric oxide synthase phosphorylation and nitric oxide production, mitochondrial biogenesis and mitochondria-mediated apoptotic pathways, oxidative stress and inflammatory pathways. Sirtuins as modulators of the adverse conditions in the endothelium hold a promising therapeutic potential for health conditions related to endothelial dysfunctionand vascular ageing.

Evaluation of anticancer activities of Poria cocos ethanol extract in breast cancer: In vivo and in vitro, identification and mechanism

Ethnopharmacological relevancePoria cocos Wolf (P. cocos), a well-known traditional East-Asian medicinal and edible fungus, is one of the most important components in Chinese medicine formulas like “Guizhi fuling wan” to treat hyperplasia of mammary glands and breast cancer. Aiming of studyIn this study, we attempted to verify the anticancer efficacy of the ethanol extract of P. cocos (PC) on the breast cancer as well as to investigate its most active compound and its underlying molecular mechanism in vivo and in vitro. Materials and methodsThe key anti-cancer components were separated and purified through chromatography and identified by spectral analyses. The in vivo anti-breast cancer efficacy and side effects of PC were evaluated in BALB/c nude mice that have been subcutaneously injected with breast cancer cells MDA-MB-231. Cytotoxicity, apoptosis and cell cycle arrest of PC were evaluated in vitro by cell viability assays and flow cytometry. The protein levels were examined via western blotting. ResultsPachymic acid (PA), separated and identified as the most active compound, induced the significant cytotoxicity on breast cancer cells MDA-MB-231(IC50 value, 2.13 ± 0.24 μg/mL) and was not active against the normal breast epithelium cells MCF-10A. The in vivo experiment revealed that PC could significantly inhibit the tumor development and the final mean tumor weight of the mice in the PC group (0.51 ± 0.12g) was significantly lower than that in the model group (1.22 ± 0.45g). Notably, compared to the first-line anticancer drug cisplatin, PC showed less side effects on the function of the vital organs and the muscle strength of the mice. Among in vitro study, PC significantly inhibited the cell growth of MDA-MB-231 by inducing cell apoptosis and cell cycle arrested at G0/G1 phase in a dose-dependent manner. The expression of cell cycle-associated cyclin D1, cyclin E, CDK2, and CDK4 were downregulated, while p53 and p21 expression were upregulated following the PA treatment. In addition, PA downregulated the apoptotic regulator Bcl-2, increased the expression of pro-apoptotic protein Bax, and promoted the release of cytochrome c and the activation of cleaved caspase-3, -9 and caspase -8 via mitochondria-mediated and death receptor-mediated signaling pathways. ConclusionThis study verified the anticancer efficacy of PC on breast cancer in vivo and in vitro through induction of cell apoptosis and G0/G1 cell cycle arrest. The data also suggested that PA could be developed as an efficacious agent for breast cancer treatment with less side effects.

Fucoidan Purified from Sargassum polycystum Induces Apoptosis through Mitochondria-Mediated Pathway in HL-60 and MCF-7 Cells.

Fucoidans are biocompatible, heterogeneous, and fucose rich sulfated polysaccharides biosynthesized in brown algae, which are renowned for their broad-spectrum biofunctional properties. As a continuation of our preliminary screening studies, the present work was undertaken to extract polysaccharides from the edible brown algae Sargassum polycystum by a modified enzyme assisted extraction process using Celluclast, a food-grade cellulase, and to purify fucoidan by DEAE-cellulose anion exchange chromatography. The apoptotic and antiproliferative properties of the purified fucoidan (F5) were evaluated on HL-60 and MCF-7 cells. Structural features were characterized by FTIR and NMR analysis. F5 indicated profound antiproliferative effects on HL-60 leukemia and MCF-7 breast cancer cells with IC50 values of 84.63 ± 0.08 µg mL−1 and 93.62 ± 3.53 µg mL−1 respectively. Further, F5 treatment increased the apoptotic body formation, DNA damage, and accumulation of HL-60 and MCF-7 cells in the Sub-G1 phase of the cell cycle. The effects were found to proceed via the mitochondria-mediated apoptosis pathway. The Celluclast assisted extraction is a cost-efficient method of yielding fucoidan. With further studies in place, purified fucoidan of S. polycystum could be applied as functional ingredients in food and pharmaceuticals.

Aviculin Isolated from Lespedeza cuneata Induce Apoptosis in Breast Cancer Cells through Mitochondria-Mediated Caspase Activation Pathway.

The global incidence of breast cancer has increased. However, there are many impediments to the development of safe and effective anticancer drugs. The aim of the present study was to evaluate the effect of aviculin isolated from Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae) on MCF-7 human breast cancer cells and determine the underlying mechanism. Using the bioassay-guided isolation by water soluble tetrazolium salt (WST-1)-based Ez-Cytox assay, nine compounds (four lignan glycosides (1–4), three flavonoid glycosides (5–7), and two phenolic compounds (8 and 9)) were isolated from the ethyl acetate (EA) fraction of the L. cuneata methanolic extract. Of these, aviculin (2), a lignan glycoside, was the only compound that reduced metabolic activity on MCF-7 cells below 50% (IC50: 75.47 ± 2.23 μM). The underlying mechanism was analyzed using the annexin V Alexa Fluor 488 binding assay and Western blotting. Aviculin (2) was found to induce apoptotic cell death through the intrinsic apoptosis pathway, as indicated by the increased expression of initiator caspase-9, executioner caspase-7, and poly (ADP-ribose) polymerase (PARP). Aviculin (2)-induced apoptotic cell death was accompanied by an increase in the Bax/Bcl-2 ratio. These findings demonstrated that aviculin (2) could induce breast cancer cell apoptosis through the intrinsic apoptosis pathway, and it can therefore be considered an excellent candidate for herbal treatment of breast cancer.

Inhibition of cell growth and induction of apoptosis by bilobalide in FaDu human pharyngeal squamous cell carcinoma

Bilobalide isolated from the leaves of Ginkgo biloba has several pharmacological activities such as neuroprotective, anti-inflammatory, and anticonvulsant. However, the effect of bilobalide on cancer has not been clearly established. The main purpose of this study was to investigate the effect of bilobalide on cell growth and apoptosis induction in FaDu human pharyngeal squamous cell carcinoma. This was examined by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay, nuclear 4′,6-diamidino-2-phenylindole dihydrochloride staining, DNA fragmentation analysis, and immunoblotting. Bilobalide inhibited the growth of FaDu cells in dose- and time-dependent manners. Treatment with bilobalide resulted in nuclear condensation and DNA fragmentation in FaDu cells. Furthermore, it promoted the proteolytic cleavage of procaspase-3/-7/-8/-9 with increase in the amount of cleaved caspase-3/-7/-8/-9. Bilobalide-induced apoptosis in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting revealed that the antiapoptotic mitochondrial protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated by bilobalide in FaDu cells. Bilobalide significantly increased Bax/Bcl-2 ratio. These results suggest that bilobalide inhibits cell proliferation and induces apoptosis in FaDu human pharyngeal squamous cell carcinoma via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway.

Dexmedetomidine Attenuates Glutamate-Induced Cytotoxicity by Inhibiting the Mitochondrial-Mediated Apoptotic Pathway

BackgroundGlutamate (GLU) is the most excitatory amino acid in the central nervous system and plays an important role in maintaining the normal function of the nervous system. During cerebral ischemia, massive release of GLU leads to neuronal necrosis and apoptosis. It has been reported that dexmedetomidine (DEX) possesses anti-oxidant and anti-apoptotic properties. The objective of this study was to investigate the effects of DEX on GLU-induced neurotoxicity in PC12 cells.Material/MethodsPC12 cells were treated with 20 mM GLU to establish an ischemia-induced injury model. Cell viability was accessed by MTT assay. MDA content and SOD activity were analyzed by assay kits. Apoptosis rate, ROS production, intracellular Ca2+ concentration, and MMP were evaluated by flow cytometry. Western blot analysis was performed to analyze expressions of caspase-3, caspase-9, cyt-c, bax, and bcl-2.ResultsPC12 cells treated with GLU exhibited reduced cell viability and increased apoptosis rates, which were ameliorated by pretreatment with DEX. DEX significantly increased SOD activity, reduced content of MDA, and decreased production of ROS in PC12 cells. In addition, DEX clearly reduced the level of intracellular Ca2+ and attenuated the decline of MMP. Moreover, DEX notably reduced expressions of caspase-3, caspase-9, cyt-c, and bax and increased expression of bcl-2.ConclusionsOur findings suggest that DEX can protect PC12 cells against GLU-induced cytotoxicity, which may be attributed to its anti-oxidative property and reduction of intracellular calcium overload, as well as its ability to inhibit the mitochondria-mediated apoptotic pathway.

Mitochondrial apoptosis and curtailment of hypoxia-inducible factor-1α/fatty acid synthase: A dual edge perspective of gamma linolenic acid in ER+ mammary gland cancer.

Gamma linolenic acid is a polyunsaturated fatty acid having selective anti-tumour properties with negligible systemic toxicity. In the present study, the anti-cancer potential of gamma linolenic acid and its effects on mitochondrial as well as hypoxia-associated marker was evaluated. The effect of gamma linolenic acid was scrutinised against ER + MCF-7 cells by using fluorescence microscopy, JC-1 staining, dot plot assay and cell cycle analysis. The in vitro results were also confirmed using carcinogen (n-methyl-n-nitrosourea) induced in vivo model. The early and late apoptotic signals in the conjugation with mitochondrial depolarisation were found once scrutinised through mitochondrial membrane potential and life death staining after gamma linolenic acid treatment. Gamma linolenic acid arrested the cell cycle in G0/G1 phase with the majority of cell populations in the early apoptotic stage. The translocation of phosphatidylserine was studied through annexin-V FITC dot plot assay. The markers of cellular proliferation (decreased alveolar bud count, histopathological architecture restoration and loss of tumour micro-vessels) were diminished after gamma linolenic acid treatment. Gamma linolenic acid ameliorates the biological effects of n-methyl-n-nitrosourea persuading the mitochondrial mediated death pathway and impeding the hypoxic microenvironment to make a halt in palmitic acid synthesis. SIGNIFICANCE: The present study elaborates the effect of gamma linolenic acid on mammary gland cancer by following mitochondrial-mediated death apoptosis pathway. Gamma linolenic acid also inhibits cell-wall synthesis by the curtailment of HIF-1α and FASN level in mammary gland cancer.

Thioredoxin-1 blocks methamphetamine-induced injury in brain through inhibiting endoplasmic reticulum and mitochondria-mediated apoptosis in mice

Methamphetamine (METH) has been reported to induce endoplasmic reticulum (ER) stress and neuronal apoptosis in the central nervous system (CNS) during the development of addiction. Thioredoxin-1 (Trx-1) is a redox regulating protein and plays an important role in inhibiting apoptosis and protects neurons from cytotoxicity through ER and mitochondria-mediated pathways. Our previous study has been reported that Trx-1 protects mice from METH-induced rewarding effect. However, whether Trx-1 plays the role in resisting METH injury is still unclear. Here, we aim to investigate whether Trx-1 participates in the regulation of METH-induced CNS injury via ER stress and mitochondria-mediated pathways. Our study first repeated the conditioned place preference expression induced by METH. Then we detected and found that METH increased the expression of N-methyl-d-asparate (NMDA) receptor subunit 2B (NR2B) and the level of glutamate (Glu) in the ventral tegmental area (VTA) and nucleus accumbens (NAc), while Trx-1 overexpression suppressed the increases. We further examined ER stress-related proteins and mitochondrial apoptosis pathway in the VTA and NAc, and found that METH increased the expressions of glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Bax, as same time decreased the expressions of procaspase12, Bcl-2, and procaspase3, while Trx-1 overexpression blocked these changes. These results indicate that Trx-1 blocks METH-induced injury by suppressing ER stress and mitochondria-mediated apoptosis in the VTA and NAc via targeting glutamatergic system.

Ginsenoside Rb 1: A novel therapeutic agent in Staphylococcusaureus-induced Acute Lung Injury with special reference to Oxidative stress and Apoptosis

Acute lung injury (ALI) is considered as an uncontrolled inflammatory response that can leads to acute respiratory distress syndrome (ARDS), which limits the therapeutic strategies. Ginsenosides Rb1 (Rb1), an active ingredient obtained from Panax ginseng, possesses a broad range of pharmacological and medicinal properties, comprising the anti-inflammatory, anti-oxidant, and anti-tumor activities. Therefore, the purpose of the present study was to investigate the protective effects of Rb1 against S. aureus-induced (ALI) through regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and mitochondrial-mediated apoptotic pathways in mice (in-vivo), and RAW264.7 cells (in-vitro). For that purpose, forty Kunming mice were randomly assigned into four treatment groups; (1) Control group (phosphate buffer saline (PBS); (2) S. aureus group; (3) S. aureus + Rb1 (20 mg/kg) group; and (4) Rb1 (20 mg/kg) group. The 20 μg/mL dose of Rb1 was used in RAW264.7 cells. In the present study, we found that Rb1 treatment reduced ALI-induced oxidative stress via suppressing the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) and increase the antioxidant enzyme activities of superoxidase dismutase 1 (SOD1), Catalase (CAT), and glutathione peroxidase 1 (Gpx1). Similarly, Rb1 markedly increased messenger RNA (mRNA) expression of antioxidant genes (SOD1, CAT and Gpx1) in comparison with ALI group. The histopathological results showed that Rb1 treatment ameliorated ALI-induced hemorrhages, hyperemia, perivascular edema and neutrophilic infiltration in the lungs of mice. Furthermore, Rb1 enhanced the antioxidant defense system through activating the Nrf2 signaling pathway. Our findings showed that Rb1 treated group significantly up-regulated mRNA and protein expression of Nrf2 and its downstream associated genes down-regulated by ALI in vivo and in vitro. Moreover, ALI significantly increased the both mRNA and protein expression of mitochondrial-apoptosis-related genes (Bax, caspase-3, caspase-9, cytochrome c and p53), while decreased the Bcl-2. In addition, Rb1 therapy significantly reversed the mRNA and protein expression of these mitochondrial-apoptosis-related genes, as compared to the ALI group in vivo and in vitro. Taken together, Rb1 alleviates ALI-induced oxidative injury and apoptosis by modulating the Nrf2 and mitochondrial signaling pathways in the lungs of mice.

Helveticoside Exhibited p53-dependent Anticancer Activity Against Colorectal Cancer

Investigation into the anti-cancer activities of natural products and their derivatives represents an efficient approach to develop safe and effective chemotherapeutic agents for the treatment of colorectal cancer. Helveticoside is a biologically active component of the seed extract of Descurainia sophia. This compound has been reported to regulate the genes related to cell proliferation and apoptosis in lung cancer cells, however its anticancer activity has not been fully explored yet. Cell viability was evaluated by MTT and Trypan blue exclusion assay; cell apoptosis was measured by flow cytometry; mitochondrial membrane potential was determined by using JC1-mitochondrial membrane potential assay kit; protein levels were determined by western blot assay; invivo tumor growth was assessed in a xenograft nude mice model. The current study demonstrated the invitro anti-cancer activity of helveticoside against colorectal cancer using colorectal cancer cells SW480 and HCT116. Moreover, induction of apoptosis was found to mediate the cytotoxic action of helveticoside on SW480 and HCT116cells. Based on the decrease in the mitochondrial membrane potential, upregulation of Bax, downregulation of Bcl-2 and cleavage of caspase-3 and 9, apoptosis was induced by helveticoside via mitochondria-mediated intrinsic apoptotic signaling pathways in colorectal cancer cells. Besides, using p53-knockout SW480cells, the cytotoxic action of helveticoside was found to be p53-dependent. More importantly, administration of helveticoside inhibited the growth of HCT116cells derived-colorectal cancer xenograft in mice via activation of apoptosis. Helveticoside might be a potential candidate for the development of novel chemotherapeutic agents for the treatment of colorectal cancer, while the potential toxic effects of helveticoside may be worthy of further investigations.

Ketamine treatment affects hippocampal but not cortical mitochondrial function in prepubertal rats.

Previous reports have shown that ketamine triggered apoptosis in immature developing brain involving mitochondrial-mediated pathways. However, no data for ketamine effects on hippocampal and cortical mitochondrial function are available in prepubertal rats. Twenty-one-day-old Sprague-Dawley rats received ketamine (40mg/kg i.p.) for 3days and were killed 24hr after thelast injection. Hippocampal mitochondria from ketamine-treated rats showed decreased malate-glutamate state 4 and 3 respiratory rates and an inhibition in complex I and IV activities. Hippocampal mitochondrial membrane depolarization and mitochondrial permeability transition induction were observed. This was not reflected in an increment of H2 O2 production probably due to increased Mn-SOD and catalase activities, 24hr after treatment. Interestingly, increased H2 O2 production rates and cardiolipin oxidation were found in hippocampal mitochondria shortly after ketamine treatment (45min). Unlike the hippocampus, ketamine did not affect mitochondrial parameters in the brain cortex, being the area less vulnerable to suffer ketamine-induced oxidative damage. Results provide evidences that exposure of prepubertal rats to ketamine leads to an induction of mitochondrial ROS generation at early stages of treatment that was normalized by the triggering of antioxidant systems. Although hippocampal mitochondria from prepubertal rats were capable of responding to the oxidative stress, they remain partially dysfunctional.

Astaxanthin Protects Ochratoxin A-Induced Oxidative Stress and Apoptosis in the Heart via the Nrf2 Pathway.

This study assessed the protective mechanism of astaxanthin (ASX) against ochratoxin A- (OTA-) induced cardiac injury in mice. Four groups of mice were established: control group (0.1 mL olive oil + 0.1 mL NaHCO2), OTA group (0.1 mL OTA 5 mg/kg body weight), ASX group (0.1 mL ASX 100 mg/kg body weight), and ASX + OTA group (0.1 mL ASX 100 mg/kg body weight, 2 h later, 0.1 mL OTA 5 mg/kg body weight). The test period lasted for 27 days (7 days of dosing, 2 days of rest). Electrocardiogram, body weight, heart weight, tissue pathology, oxidative markers (malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH)), biochemical markers (creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH)), electron microscopy, TUNEL, and Western blot tests were used to examine the effects of OTA on myocardial injury and ASX detoxification. The results showed that OTA exposure significantly decreased both body weight and heart weight. OTA induced a decrease in heart rate in mice and decreased tissue concentrations of SOD, CAT, and GSH, while increasing serum concentrations of cardiac enzymes (CK, CK-MB, and LDH) and tissue MDA. ASX improved heart rate, cardiac enzymes, and antioxidant levels in mice. The results of tissue pathology and TUNEL assay showed that ASX protects against OTA-induced myocardial injury. In addition, Western blot results showed that the OTA group upregulated Keap1, Bax, Caspase3, and Caspase9, while it downregulated Nrf2, HO-1, and Bcl-2 protein expression. ASX played a protective role by changing the expression of Keap1, Nrf2, HO-1, Bax, Bcl-2, Caspase3, and Caspase9 proteins. These results indicate that the protective mechanism of ASX on the myocardium works through the Keap1-Nrf2 signaling pathway and mitochondria-mediated apoptosis pathway. This study provides a molecular rationale for the mechanism underlying OTA-induced myocardial injury and the protective effect of ASX on the myocardium.

MicroRNA-214 protects L6 skeletal myoblasts against hydrogen peroxide-induced apoptosis

MicroRNAs (miRNAs) have been reported as key gene regulators, and they control many fundamental biological processes. Previously, we demonstrated that miR-214 had a protective effect against myocardial apoptosis and myocardial fibrosis. In this study, we sought to investigate the expression of miR-214 in L6 skeletal myoblast (SKM), the regulatory effect of miR-214 on hydrogen peroxide (H2O2) induced cell apoptosis and the underlying mechanisms of the antiapoptotic effect. MiR-214 expression was up-regulated by H2O2 in a dose and time-dependent manner in L6 SKMs. To investigate the regulatory effects of miR-214 on L6 SKM, both gain-of-function and loss-of-function approaches were applied. The results showed that miR-214 improved cell survival and inhibited cell apoptosis, and blockage of miR-214 abrogated the protective effect on cell survival and resistance to apoptosis. Phosphatase and tensin homolog (PTEN) was negatively regulated by miR-214, and PTEN inhibitor obviously reversed the effect of miR-214 blockage on enhancing cell apoptosis. In addition, miR-214 up-regulated antiapoptotic protein Bcl-2, down-regulated proapoptotic protein Bax, prevented release of cytochrome c and inhibited caspase-3 activation. In summary, H2O2-induced injury increases miR-214 expression in L6 SKM, and miR-214 contributes to the protection of L6 SKM against apoptosis via lowering PTEN and subsequently inhibiting the mitochondrial-mediated caspase-dependent apoptotic signaling pathway.

Anticancer effects of a traditional Thai herbal recipe Benja Amarit extracts against human hepatocellular carcinoma and colon cancer cell by targeting apoptosis pathways

Ethnopharmacological relevanceA preparation of Benja Amarit (BJA) has been effectively used in folk medicine to treat diseases related to the liver and colon and forms of cancer for hundreds of years in Thailand. However, there has not been any research on BJA with regard to its anticancer activity against human hepatocellular carcinoma and colon cancer cells. Aim of the studyThis study was to obtain the scientific supports for the traditional usage in anticancer potential of BJA extracts on hepatocellular carcinoma and colon cancer. Materials and methodsThe cytotoxic effects were determined using MTT assay. Apoptosis was quantitated by annexin V-FITC/PI staining. Caspases activities were measured by using specific substrates and colorimetric analysis. The protein expressions were determined by Western blot analysis. Reactive oxygen species (ROS) generation, mitochondrial transmembrane potential, and calcium ion levels were measured by specific fluorescence probes and flow cytometry. The chick embryo chorioallantoic membrane model has been used to study the in vivo anticancer activity. The phytochemical identification was performed by GC-MS and LC-MS. ResultsNotably, 95% (BJA-95) and 50% (BJA-50) ethanolic extract of BJA inhibited hepatocellular carcinoma and colon cancer cell viability in a dose-dependent manner. While, the water extract of BJA (BJA-W) was not found to be toxic to both kinds of cancer cell lines. BJA extract induced both the extrinsic and intrinsic or mitochondria-mediated apoptosis pathways. Moreover, BJA-95 caused ROS generation and endoplasmic reticulum stress-mediated apoptosis. The extract exhibited the growth inhibitory effects on cancer cells in vivo. Phytochemical analysis revealed that the major active compounds were piperine, xanthotoxol and dihydrogambogic acid. ConclusionThis study is the first to demonstrate anticancer efficiency of BJA extracts on human cancer cells. We consider BJA extract to be a potentially alternative cancer treatment and to be a promising candidate in the future development of antitumor agents.

MiRNA-574-5p downregulates ZNF70 and influences the progression of human esophageal squamous cell carcinoma through reactive oxygen species generation and MAPK pathway activation.

There is growing evidence shown that microRNAs (miRNAs) are associated with cancer and can play a role in human cancers as oncogenes or tumor suppressor genes. miRNA-574-5p is a candidate oncogene in various types of cancer, but little is known about biological functions of miR-574-5p in esophageal squamous cell carcinoma (ESCC). In this study, we observe that the expression of miR-574-5p is not only increased in human ESCC tissues but also remarkably increased in cell lines correlates with ZNF70. In vitro, we explored the role of miR-574-5p in ESCC progression via transfection of the miR-574-5p inhibitor into ECA-109 cells. The results show miR-574-5p serve as a tumor promoter regulating cells proliferation and apoptosis in ESCC through mitochondrial-mediated reactive oxygen species (ROS) generation and MAPK pathways. Furthermore, ZNF70 has been proved to as a functional target for miR-574-5p to regulate cells poliferation and apoptosis. In summary, these results suggest that miR-574-5p serves as tumor promoter to promote proliferation and inhibit apoptosis of ESCC cells by targeting ZNF70 via mitochondrial-mediated ROS generation and MAPK pathways. The miR-574-5p/ZNF70 pathway provides a new insight into the molecular mechanisms that the occurrence and development of ESCC and it provides a novel therapeutic target for ESCC.

Suppression of tumor growth and metastasis by ethanol extract of Angelica dahurica Radix in murine melanoma B16F10 cells.

The roots of Angelica dahurica have long been used as a traditional medicine in Korea to treat various diseases such as toothache and cold. In this study, we investigated the effect of ethanol extract from the roots of this plant on metastatic melanoma, a highly aggressive skin cancer, in B16F10 melanoma cells and B16F10 cell inoculated-C57BL/6 mice. Our results showed that the ethanol extracts of Angelicae dahuricae Radix (EEAD) suppressed cell growth and induced apoptotic cell death in B16F10 cells. EEAD also activated the mitochondria-mediated intrinsic apoptosis pathway, with decreased mitochondrial membrane potential, and increased production of intracellular reactive oxygen species and ration of Bax/Bcl-2 expression. Furthermore, EEAD reduced the migration, invasion, and colony formation of B16F10 cells through the reduced expression and activity of matrix metalloproteinase (MMP)-2 and -9. In addition, in vivo results demonstrated that oral administration of EEAD inhibited lactate dehydrogenase activity, hepatotoxicity, and nephrotoxicity without weight loss in B16F10 cell inoculated-mice. Importantly, EEAD was able to markedly suppress lung hypertrophy, the incidence of B16F10 cells lung metastasis, and the expression of tumor necrosis factor-alpha in lung tissue. Taken together, our findings suggest that EEAD may be useful for managing metastasis and growth of malignant cancers, including melanoma.

Characterization and anti-tumor activity of saponin-rich fractions of South Korean sea cucumbers (Apostichopus japonicus).

In this study, the saponin-rich fractions of five individual (two Red and three Black) sea cucumbers (Apostichopus japonicus) in South Korea were investigated for their antiproliferative effect against HL-60, B16F10, MCF-7, and Hep3B tumor cell lines. The red sea cucumber saponin-rich fraction (SSC) from Jeju Island (JRe) decreased the growth of HL-60 with an IC50 value of 23.55 ± 3.40μg/mL, which represented the strongest anticancer activity among the extracts. Further, SSC downregulated B-cell lymphoma extra-large (Bcl-xL), while upregulating, to different degrees, Bcl-2-associated X protein (Bax), caspase-9, caspase-3, PARP cleavage, and apoptotic bodies in cancer cells. Evidence for SSC inducing apoptosis via the mitochondria-mediated pathway was found. The contents of SSCs were determined using ultra high-performance liquid chromatography coupled with a quadrupole orbitrap mass spectrometry to comparatively evaluate the regional influence. In West Sea, the total SSC content of A. japonicus was 15.5mg/g, representing the highest content, while A. japonicus in the South Sea yielded the lowest content at 8mg/g. The major saponin constituent in SSC was identified as Holotoxin A1, which may the anti-tumor compound in A. japonicus.

Structural characterization of an acid polysaccharide from Pinellia ternata and its induction effect on apoptosis of Hep G2 cells

In the present study, a polysaccharide fraction (PTP) was isolated and purified from the tubers of Pinellia ternata. We researched its structure and anti-tumor activity, and further studied its molecular mechanism of inducing apoptosis of Hep G2 cells. The results indicated that PTP was an acid heteropolysaccharide and the average molecular weight of PTP identified by HPGPC was 3.06 × 106 Da. Ion chromatography (IC) determined that PTP was mainly composed of Ara:Gal:Glu:Man:GlcA:GalA in a molar ratio of 6.98:16.56:7.25:2.04:1:4.16. Combined with the results of FT-IR and NMR spectroscopy, it was found that PTP is a pyranose containing α-configuration and β-configuration, mainly consist of β-D-Gal, α-D-Glu, α-D-Ara and β-D-Man. By analyzing the results of MTT, cell cycle, Annexin V-FITC/PI double staining and cell morphology observation, we concluded that PTP induced dose-dependent apoptosis of Hep G2 cells via S phase arrest. In addition, mitochondrial membrane potential detection and Western blot further indicated that PTP was capable of inducing apoptosis in Hep G2 cells through an endogenous mitochondria-mediated apoptotic pathway.