Anti-Cancer Effect and the Underlying Mechanisms of Gypenosides on Human Colorectal Cancer SW-480 Cells

Background

Gypenosides (Gyp), the main components from Gynostemma pentaphyllum Makino, are widely used in traditional Chinese medicine. The present study aimed to investigate the anti-cancer effect and the underlying mechanisms of Gyp on human colorectal cancer cells SW-480.

Materials and Methods

The inhibitory effect of Gyp on SW-480 cells was evaluated by MTT assay. Apoptotic cell death was detected by nuclear Hoechst 33342 staining and DNA fragmentation analysis. Apoptosis was analyzed using Annexin V-PE/7-amino-actinomycin D staining. Cell membrane integrity was evaluated with flow cytometry following PI staining. Changes of mitochondrial membrane potential (Δψ _m) were detected through flow cytometry analysis of rhodamine 123 (Rh123). The role of reactive oxygen species (ROS) in Gyp induced cell death was investigated by intracellular ROS generation and general ROS scavenger. Wound-healing assay was carried out to investigate Gyp-inhibited migration of SW-480 cells in vitro. Additionally, the alterations in F-actin microfilaments were analyzed by FITC-labeled phalloidin toxin staining and the morphological changes were evaluated under scanning electron microscope (SEM).

Results

After the Gyp treatment, the plasma membrane permeability of SW-480 cell was increased, Δψ _m was decreased significantly, the level of intracellular ROS level was increased, DNA fragmentation and apoptotic morphology were observed. Cells treated with Gyp exert serious microfilament network collapse as well as the significant decrease in the number of microvilli. Gyp induced the changes of cell viability, cell migration, intracellular ROS generation and nuclear morphology were alleviated obviously by NAC.

Conclusion

The results in this study implied that ROS play an important role in Gyp induced cell toxicity and apoptosis, and the mitochondria damage may be upstream of ROS generation post Gyp treatment. The findings of the present study provide new evidences for anti-tumor mechanisms by which Gyp induces apoptosis in vitro.     

Gypenosides Synergistically Enhances the Anti-Tumor Effect of 5-Fluorouracil on Colorectal Cancer In Vitro and In Vivo: A Role for Oxidative Stress-Mediated DNA Damage and p53 Activation

Objective

5-Fluorouracil (5-Fu) has been widely used as a first-line drug for colorectal cancer (CRC) treatment but limited by drug resistance and severe toxicity. The chemo-sensitizers that augment its efficiency and overcome its limitation are urgently needed. Gypenosides (Gyp), the main components from Gynostemma pentaphyllum (Thunb.) Makino, has shown potential anti-tumor property with little side-effect. Here, we carefully explored the chemo-sensitization of Gyp to potentiate the anti-tumor effect of 5-Fu in vitro and in vivo.

Methodology / Principal Findings

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium assay and colony formation test reveal that Gyp could significantly enhance the 5-Fu-caused SW-480,SW-620 and Caco2 cells viability loss. Calcusyn analysis shows that Gyp acts synergistically with 5-Fu. Annexin V-PE/7-AAD staining indicates 5-Fu + Gyp could induce SW-480 cell apoptosis. The activations of caspase 3, caspase 9 and poly (ADP-ribose) polymerase (PARP) were involved in the process. Gyp was also found to up-regulate 5-Fu-caused phospho-p53 expression and thus augment 5-Fu-induced G0/G1 phase arrest. Gyp elevated intracellular ROS level, significantly enhanced 5-Fu-triggered DNA damage response as evidenced by flow cytometry, comet assay and the expression of Ser139-Histone H2A.X. Inhibition of ROS and p53 respectively reversed the cell death induced by 5-Fu + Gyp, suggesting the key roles of ROS and p53 in the process. Moreover, 5-Fu and Gyp in combination exhibits much superior tumor volume and weight inhibition on CT-26 xenograft mouse model in comparison to 5-Fu or Gyp alone. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation. Preliminary toxicological results show that 5-Fu + Gyp treatment is relatively safe.

Conclusions

As a potential chemo-sensitizer, Gyp displays a splendid synergistic effect with 5-Fu to inhibit cancer cell proliferation and tumor growth. By using 5-Fu and Gyp in combination would be a promising therapeutic strategy for CRC treatment.     

TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production

Background

Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a key regulator of signal cascades of TNF-α receptor and TLR4, and can induce NF-κB activation for preventing cell apoptosis and eliciting inflammation response.

Results

TAK1 inhibitor (TAKI) can decrease the cell viability of murine bone marrow-derived macrophages (BMDM), RAW264.7 and BV-2 cells, but not dermal microvascular endothelial cells, normal human epidermal keratinocytes, THP-1 monocytes, human retinal pigment epithelial cells, microglia CHME3 cells, and some cancer cell lines (CL1.0, HeLa and HCT116). In BMDM, TAKI-induced caspase activation and cell apoptosis were enhanced by lipopolysaccharide (LPS). Moreover, TAKI treatment increased the cytosolic and mitochondrial reactive oxygen species (ROS) production, and ROS scavengers NAC and BHA can inhibit cell death caused by TAKI. In addition, RIP1 inhibitor (necrostatin-1) can protect cells against TAKI-induced mitochondrial ROS production and cell apoptosis. We also observed the mitochondrial membrane potential loss after TAKI treatment and deterioration of oxygen consumption upon combination with LPS. Notably TNF-α neutralization antibody and inhibitor enbrel can decrease the cell death caused by TAKI.

Conclusions

TAKI-induced cytotoxicity is cell context specific, and apoptosis observed in macrophages is dependent on the constitutive autocrine action of TNF-α for RIP1 activation and ROS production.     

Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation

Background

Hypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.

Methods

Human hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.

Results

Exposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.

Conclusions

CD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.     

Effects of tobacco smoking during pregnancy on oxidative stress in the umbilical cord and mononuclear blood cells of neonates

Background

Although cigarette smoke is known to be a complex mixture of over 4000 substances that can lead to damage through active or passive smoking, its mechanisms and biochemical consequences in pregnancy and neonates are not yet fully understood. Therefore, in the present study, we propose to study the impact of smoking during gestation on the viability of blood mononuclear cells (MNC) from umbilical cords of newborns to assess the degree of oxidative stress and cell viability. After childbirth, the cord blood and the umbilical cord were immediately collected in public hospitals in Greater Vitoria, ES, Brazil. Flow cytometry was used to analyze the cord blood followed by biochemical and histological tests to analyze possible changes in the umbilical cord.

Results

Pregnant smokers had a reduction of MNC viability from the umbilical cord (10%), an increase in the production of reactive oxygen species (ROS) and an increase in cell apoptosis (~2-fold) compared to pregnant non-smokers. In the umbilical cord, it was observed an increase of advanced oxidation protein products - AOPP (~2.5-fold) and a loss of the typical architecture and disposition of endothelial cells from the umbilical artery.

Conclusions

These data suggest that maternal cigarette smoking during pregnancy (even in small amounts) may compromise the viability of MNC cells and damage the umbilical cord structure, possibly by excessive ROS bioavailability.     

Lysophosphatidic acid induces MDA-MB-231 breast cancer cells migration through activation of PI3K/PAK1/ERK signaling

Background

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.

Methodology/Principal Findings

In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.

Conclusions/Significance

Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.     

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

Background

Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.

Results

The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.

Conclusion

Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

LIF-Free Embryonic Stem Cell Culture in Simulated Microgravity

Background

Leukemia inhibitory factor (LIF) is an indispensable factor for maintaining mouse embryonic stem (ES) cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required.

Methodology/Principal Findings

We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF.

Conclusions/Significance

Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.     

GRP78 knockdown enhances apoptosis via the down-regulation of oxidative stress and Akt pathway after epirubicin treatment in colon cancer DLD-1 cells

Introduction

The 78-kDa glucose-regulated protein (GRP78) is induced in the cancer microenvironment and can be considered as a novel predictor of responsiveness to chemotherapy in many cancers. In this study, we found that intracellular reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation were higher in GRP78 knockdown DLD-1 colon cancer cells compared with scrambled control cells.

Methodology/Principal Findings

Treatment with epirubicin in GRP78 knockdown DLD-1 cells enhanced apoptosis and was associated with decreased production of intracellular ROS. In addition, apoptosis was increased by the antioxidants propyl gallate (PG) and dithiothreitol (DTT) in epirubicin-treated scrambled control cells. Epirubicin-treated GRP78 knockdown cells resulted in more inactivated Akt pathway members, such as phosphorylated Akt and GSK-3β, as well as downstream targets of β-catenin expression. Knockdown of Nrf2 with small interfering RNA (siRNA) increased apoptosis in epirubicin-treated GRP78 knockdown cells, which suggested that Nrf2 may be a primary defense mechanism in GRP78 knockdown cells. We also demonstrated that epirubicin-treated GRP78 knockdown cells could decrease survival pathway signaling through the redox activation of protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase that negatively regulates the Akt pathway.

Conclusions

Our results indicate that epirubicin decreased the intracellular ROS in GRP78 knockdown cells, which decreased survival signaling through both the Akt pathway and the activation of PP2A. Together, these mechanisms contributed to the enhanced level of epirubicin-induced apoptosis that was observed in the GRP78 knockdown cells.     

Epigallocatechin-3-gallate (EGCG) inhibits the migratory behavior of tumor bronchial epithelial cells

Background

Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior.

Methods

The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG.

Results

We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin.

Conclusion

Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.     

Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Background

Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer.

Methodology/Principal Findings

In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR.

Conclusion/Significance

Our study provides evidence that FeNG, a redox active metal chelate may be a promising new therapeutic agent against drug resistance cancers.     

Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines

Background

We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in pathogenesis of diffuse large B cell lymphoma (DLBCL). In an attempt to identify newer therapeutic agents, we investigated the role of Resveratrol (trans-3,4′, 5-trihydroxystilbene), a naturally occurring polyphenolic compound on a panel of diffuse large B-cell lymphoma (DLBCL) cells in causing inhibition of cell viability and inducing apoptosis.

Methodology/Principal Findings

We investigated the action of Resveratrol on DLBCL cells and found that Resveratrol inhibited cell viability and induced apoptosis by inhibition of constitutively activated AKT and its downstream targets via generation of reactive oxygen species (ROS). Simultaneously, Resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; and interestingly, co-treatment of DLBCL with sub-toxic doses of TRAIL and Resveratrol synergistically induced apoptosis via utilizing DR5, on the other hand, gene silencing of DR5 abolished this effect.

Conclusion/Significance

Altogether, these data suggest that Resveratrol acts as a suppressor of AKT/PKB pathway leading to apoptosis via generation of ROS and at the same time primes DLBCL cells via up-regulation of DR5 to TRAIL-mediated apoptosis. These data raise the possibility that Resveratrol may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.     

Global expression of cell surface proteins in embryonic stem cells

Background

Recent studies have shown that embryonic stem (ES) cells globally express most genes in the genome at the mRNA level; however, it is unclear whether this global expression is propagated to the protein level. Cell surface proteins could perform critical functions in ES cells, so determining whether ES cells globally express cell surface proteins would have significant implications for ES cell biology.

Methods and Principal Findings

The surface proteins of mouse ES cells were purified by biotin labeling and subjected to proteomics analysis. About 1000 transmembrane or secreted cell surface proteins were identified. These proteins covered a large variety if functional categories including signal transduction, adhesion and transporting. More over, mES cells promiscuously expressed a wide variety of tissue specific surface proteins. And many surface proteins were expressed heterogeneously on mES cells. We also find that human ES cells express a wide variety of tissue specific surface proteins.

Conclusions/Significance

Our results indicate that global gene expression is not simply a result of leaky gene expression, which could be attributed to the loose chromatin structure of ES cells; it is also propagated to the functional level. ES cells may use diverse surface proteins to receive signals from the diverse extracellular stimuli that initiate differentiation. Moreover, the promiscuous expression of tissue specific surface proteins illuminate new insights into the strategies of cell surface marker screening.