Cell Death ELISA Assay Research Articles

What if amoxicillin/clavulanic acid reduces the cisplatin anticancer impact on oral cancer treatment?

Antibiotics-chemotherapeutics combination have become on the table for many cancer treatments. For this reason, we thought that further progress and development of studies to support chemotherapeutic approaches with the use of antibiotics may be beneficial in the clinical field.Cell lines (SCC-15, HTB-41, and MRC-5) were treated with 5–100 μM/ml concentrations of cisplatin (cisp) and amoxicillin/clavulanic acid (amx/cla) with combination (amx/cla-cisp) and alone in three different incubation periods. The all-cells viability was examined with WST-1 and apoptotic activity of the drugs were investigated via cell death ELISA assay kit.The cytotoxic impact of the 100 μM amx/cla-cisp combination was found to be reduced by up to 21.8%, which was significant given that the cytotoxic effect of only cisplatin therapy was 86.1%. Because our findings demonstrated that solo amx/cla therapy have almost no impact on proliferation or death, we focused on the amx/cla-cisp combination effect. It was found that the amx/cla-cisp combination has reduced the apoptotic fragment when comparing with the solely cisp-treated cells.Due to amx/cla-cisp combination on both cells but significantly on SCC-15 recovered the sole cisplatin effect, we believe that there might be a second thought when prescribing antibiotics while treating cancer patients. Not only the antibiotic's type but also the cancer type might interact to lessen the chemotherapeutic agent's impact which is clinically a dilemma to focus on.

Downregulation of MDM2 by small interference RNA induces apoptosis and sensitizes MCF-7 breast cells to resveratrol.

Recent studies have demonstrated the mouse double minute gene (MDM2), a main oncogene, as a novel and interesting therapeutic target for cancer therapy. The aim of this study was to investigate the involvement of MDM2 in antiproliferative and antimetastatic effects of resveratrol in breast cancer cells. MCF-7 cells were transfected with siRNA against MDM2 and resveratrol. Proliferation and apoptosis were evaluated by MTT assay and cell death ELISA assay, respectively. MDM2, p53, Bax, Bcl-2, caspase-3, MMP-2, and MMP9 expressions were determined by qRT-PCR and Western blotting. Transfection with si-MDM2 significantly suppressed the expression of MDM2 expression, resulting in MCF-7 cell growth inhibition and spontaneous apoptosis. Pretreatment with Si-MDM2 synergically increased antiproliferation and antimetatstatic effects of resveratrol. No significant anticancer effects were detected with negative control siRNA treatment. Our findings suggest that silencing of MDM2 by specific siRNA effectively induce apoptosis and also enhanced anticancer effects of resveratrol. Therefore, siMDM2 may be a potent combination in breast therapy.

Nettle Tea Inhibits Growth of Acute Myeloid Leukemia Cells In Vitro by Promoting Apoptosis.

Urtica dioica (UD), commonly known as “stinging nettle”, is a herbaceous flowering plant that is a widely used agent in traditional medicine worldwide. Several formulations of UD leaf extract have been reported to exhibit anti-inflammatory and antioxidant properties, with anticancer potential. The current study investigated the possible anticancer properties of nettle tea, prepared from Urtica dioica leaves, on acute myeloid leukemia (AML) cell lines, and deciphered the underlying molecular mechanisms. Treatment of AML cell lines (U-937 and KG-1) with UD aqueous leaf extract resulted in a dose- and time-dependent inhibition of proliferation, an increase in apoptotic hallmarks such as phosphatidylserine flipping to the outer membrane leaflet, and DNA fragmentation as revealed by cell-death ELISA and cell-cycle analysis assays. Apoptosis induction in U937 cells involves alterations in the expression of Bax and Bcl-2 upon exposure to nettle tea. Furthermore, the chemical composition of UD aqueous extract indicated the presence of multiple chemical agents, such as flavonoids and phenolics, mainly patuletin, m/p-hydroxybenzoic acid, and caffeic acid, among others, to which the pro-apoptotic and anti-tumor effects may be attributed.

Induction of AMPK activation by N,N'-diarylurea FND-4b decreases growth and increases apoptosis in triple negative and estrogen-receptor positive breast cancers.

PurposeTriple negative breast cancer (TNBC) is the most lethal and aggressive subtype of breast cancer. AMP-activated protein kinase (AMPK) is a major energy regulator that suppresses tumor growth, and 1-(3-chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) is a novel AMPK activator that inhibits growth and induces apoptosis in colon cancer. The purpose of this project was to test the effects of FND-4b on AMPK activation, proliferation, and apoptosis in breast cancer with a particular emphasis on TNBC.Materials and methods(i) Estrogen-receptor positive breast cancer (ER+BC; MCF-7, and T-47D), TNBC (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND-4b for 24h. Immunoblot analysis assessed AMPK, acetyl-CoA carboxylase (ACC), ribosomal protein S6, cyclin D1, and cleaved PARP. (ii) Sulforhodamine B growth assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h. Proliferation was also assessed by counting cells after 72h of FND-4b treatment. (iii) Cell death ELISA assays were performed after treating ER+BC and TNBC cells with FND-4b for 72h.Results(i) FND-4b increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in all subtypes. (ii) FND-4b decreased proliferation in all cells, while dose-dependent growth decreases were found in ER+BC and TNBC. (iii) Increases in apoptosis were observed in ER+BC and the MDA-MB-231 cell line with FND-4b treatment.ConclusionsOur findings indicate that FND-4b decreases proliferation for a variety of breast cancers by activating AMPK and has notable effects on TNBC. The growth reductions were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+BC cells were more susceptible to FND-4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose treatment. Further development of FND compounds could result in a novel therapeutic for TNBC.

Abstract P5-05-05: Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer

Abstract The ability of cancer cells to produce large amounts of lactate through aerobic glycosis (Warburg effect) is coupled to high rates of glucose uptake. Enhanced glucose uptake and glycolysis are closely correlated to increased breast tumor aggressiveness and poor prognosis. However, despite the importance of glucose uptake in supplying energy and preventing apoptosis of cancer cells, the majority of current efforts in searching for therapeutic agents targeting glucose metabolism have been aimed at modulating activities of different metabolic enzymes that are involved in glycolysis. Very limited studies have been done in developing novel therapeutic agents against glucose uptake in breast cancer cells. Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa protein kinase. Although ATM is traditionally considered to be a nuclear protein that functions as a signal transducer in the cellular response to DNA damage, it is now known that ATM is also present in the cytoplasm and has important cytoplasmic functions. We previously discovered that ATM activates Akt, a main regulator of glucose uptake, by stimulating its phosphorylation at Ser473 following insulin treatment. We also found that ATM participates in insulin-mediated glucose uptake in muscle cells, and KU-55933, a specific inhibitor of ATM, strongly inhibits this process. Recently, we found that KU-55933 inhibits cell proliferation by inducing apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. We have also found that KU-55933 inhibits migration of MDA-MB-231 by a cell invasion assay. Furthermore, we found that these cancer cells exhibit enhanced glucose uptake in response to insulin and the addition of KU-55933 leads to a dramatic reduction of insulin-mediated glucose uptake in these cells. To further test whether KU-55933's ability to induce apoptosis is linked to its inhibition of glucose uptake, we performed a cell death ELISA assay in MDA-MB-231 cells treated with KU-55933 and different concentrations of glucose. Our results show that KU-55933 induces apoptosis of MDA-MB-231 cells, resulting in a similar degree of cell death as glucose starvation, while cells treated with glucose in conjunction with KU-55933 have decreased apoptosis. Moreover, we performed a cell migration assay and found that KU-55933 strongly inhibits the migration of MDA-MB-231 cells (similar to that caused by glucose starvation), which is almost fully rescued by the extra glucose supplemented in the cell culture medium. We have also established a positional isotope labeling-based targeted metabolomics method that can directly measure the conversion from glucose to lactate through glycolysis in cancer cells. Our results show strong production of lactate from glucose in MDA-MB-231 cells even under normal aerobic growth conditions, and KU-55933 strongly inhibits this process. Our findings may lead to the development of KU-55933 and its analogs as a new generation of therapeutic agents against aggressive breast cancer. Citation Format: Yang D-Q, Harris B, Jiang S, Li Y, Freund D, Hegeman A, Cleary M. Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-05.

Abstract 15702: Role of the Drp-1 Receptors Mid49 and Mid51 in Regulating Fission and Cell Proliferation in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction that is partially due to hyperproliferation of apoptosis-resistant pulmonary arterial smooth muscle cells (PASMCs). This “neoplastic phenotype” of PAH PASMCs is due, in part, to excessive mitochondrial fission caused by activation of dynamin related protein-1 (Drp-1). A pool of Drp-1 exists in the cytosol. Upon activation it is recruited to the mitochondrial outer membrane by Drp-1 receptor proteins, such as MiD49 and MiD51. The biological functions of these newly discovered Drp-1 receptors in diseases, including PAH, is poorly understood. We hypothesize that in PAH increased expression of MiD49 and MiD51 contribute to increased mitochondrial fission and excessive cell proliferation. Methods: Immunoblots quantified MiD49 and MiD51 expression in human control (n=3) and PAH PASMCs (n=6). The effects of silencing expression of MiD49 and MiD51, using small interfering RNA (siRNA), on cell proliferation, cell cycle progression, and rates of apoptosis were assessed using flow cytometry, and a cell death ELISA assay, respectively. Mitochondrial network structure was assessed using confocal imaging of cells stained with tetramethylrhodamine (TMRM). Results: MiD49 and MiD51 expression was increased in human PAH PASMCs vs control PASMCs (P<0.05). There was marked inter-individual variation in expression profile with some patients expressing excess MiD49 and others excess MiD51. PAH PASMCs had fragmented mitochondrial networks and both siMiD49 and siMiD51 increased mitochondrial fusion. Either siMiD49 or siMiD51 completely blocked the proliferation of PAH PASMCs by arresting the cells in the G1/G0 phase. In contrast, only siMiD51 induced apoptosis in PAH PASMCs. Conclusion: MiD49 and MiD51 are critically involved in mitochondrial fission and cell proliferation in human PAH PASMCs. Inhibition of MiD49 or MiD51 enhances mitochondrial fusion and reduces cell proliferation, while silencing MiD51 also induces apoptosis. These newly discovered Drp-1 binding partners are promising therapeutic targets in PAH.

Effects of everolimus on macrophage-derived foam cell behavior.

The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10(-5)-10(-11) M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA(PLUS) assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p≤0.05). Calcein AM/DAPI staining showed that FC exposed to everolimus (10(-5) M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10(-5)M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10(-5)M) significantly increased MAP1LC3, caused an increased trend in clusterin (p=0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein expression was significantly decreased by everolimus (10(-5) M) in FC. Percentage of RAM-11 positive area exhibited a reduction trend within sections stented with EES compared to unstented proximal sections at 60 days (p=0.09). Everolimus, a potent anti-proliferative agent used in drug-eluting stents and bioresorbable vascular scaffolds, may inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of FC, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion. We explored the effects of everolimus on the behavior of human THP1 macrophage-derived foam cells in culture, including cell viability, mRNA levels, and pro-inflammatory cytokine production. We conclude that everolimus, a potent anti-proliferative agent used in drug-eluting stents/bioresorbable vascular scaffolds, may potentially inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of foam cells, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

Inhibition of ornithine decarboxylase alters the roscovitine-induced mitochondrial-mediated apoptosis in MCF-7 breast cancer cells

Polyamines (PAs) are small aliphatic amines that play a major role in multicellular functions. The PA levels are controlled by ornithine decarboxylase (ODC), the rate limiting enzyme of PA biosynthesis. α-difluoromethylornithine (DFMO) is the ODC inhibitor, which has been shown to act as an antiproliferative agent in human cancer cells by irreversibly inhibiting ODC, which is overexpressed in breast cancer cells. Roscovitine (ROSC; CYC202), a selective cyclin-dependent kinase inhibitor, induces cell cycle arrest and concomitantly apoptosis in tumor cells. In this study, we aimed to investigate the possible role of PAs in ROSC-induced apoptosis in estrogen-dependent MCF-7 breast cancer cells. Cell viability was assessed following the exposure of MCF-7 cells to DFMO and/or ROSC by MTT cell viability assay. To evaluate the drug-induced apoptotic events, DNA fragmentation by Cell Death ELISA assay and 4,6-diamidino-2-phenylindole staining, were utilized. The disruption of mitochondrial membrane potential, caspase-9 and PARP cleavage was also determined in order to investigate the role of mitochondrial-mediated apoptosis. The modulation of Bcl-2 family members was also evaluated using the immunoblotting technique. Drug-induced reactive oxygen species was determined by a fluorometer following 2',7'-dichlorofluorescein diacetate staining. We found that ROSC induced apoptosis in a dose- and caspase-dependent manner. The ODC specific inhibitor, DFMO, altered the apoptotic effects of ROSC by increasing the generation of reactive oxygen species, decreasing the PA intracellular pool and modulating pro-apoptotic and anti-apoptotic Bcl-2 family members. All these findings suggest that ODC may be a critical target for evaluating the PA metabolic pathway as a therapeutic target in ROSC-induced mitochondrial-mediated apoptosis in estrogen-dependent MCF-7 breast cancer cells.

Expression and Functional Roles of Caspase-5 in Inflammatory Responses of Human Retinal Pigment Epithelial Cells

To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells. Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis. Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1-specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α-induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate. Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses.

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin‐induced apoptosis

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX-PP) released from the secreted precursor protein (Pro-LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her-2/neu-driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX-PP on MIA PaCa-2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa-2 cells in culture, LOX-PP attenuated the ERK and AKT activities and decreased the levels of the NF-κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX-PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF-κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX-PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX-PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX-PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities.

Abstract 5416: Delta-tocotrienol potentiates antitumor activity of gemcitabine in pancreatic cancer through inhibition of nuclear factor-κB

Abstract Background: Gemcitabine is currently the standard therapeutic agent for advanced pancreatic cancer, but has limited efficacy due to chemoresistance. Chemoresistance to gemcitabine seems to be due to constitutive activation of nuclear factor-kappaB (NF-κB) and induction of its regulated gene products. We have already shown that δ-tocotrienol is the most active natural vitamin E in depleting the NF-κB activity in pancreatic cancer. Therefore in this study we used δ-tocotrienol to enhance the antitumor activity of gemcitabine in pancreatic cancer in vitro and in vivo. Methods: The antiproliferative (MTT assay) and proapoptotic (Cell Death ELISA assay) activities of gemcitabine and δ-tocotrienol alone and in combination were evaluated in human pancreatic cancer cell lines MiaPaCa-2 and AsPc-1. Additive/synergistic effects of the combination was determined using combination index and isobologram plot. The effect of the two drugs and combination on malignant transformation in MiaPaca-2 cells was determined by colony formation assay. The in vivo activity of the two drugs and combination was evaluated in SCID nude mice xenografted with AsPc-1 cells. Because the NF-κB has a central role in gemcitabine chemoresistance, we investigated the effect of the two drugs alone and in combination on the NF-κB activity and its regulated gene products in vitro and in vivo. Results: The combination of gemcitabine and δ-Tocotrienol inhibited the cell proliferation of pancreatic cancer cells (AsPc-1 and Mia PaCa-2) more than either drug alone. Gemcitabine and δ-tocotrienol combination at 1:2 ratio resulted in additive to synergistic effects on inhibition of cell proliferation of Mia PaCa-2 cells. The inhibition of malignant transformation and apoptosis induction (histone associated DNA fragments, caspase-3 activity, PARP-1 cleavage, and BAX expression) in pancreatic cancer cells was greater with δ-tocotrienol compared with gemcitabine. The combination of the two drugs resulted in additive/synergistic effects on apoptosis induction. Gemcitabine and δ-tocotrienol combination was more effective in inhibiting AsPc-1 tumor growth and apoptosis induction in mice than either drug alone. Gemcitabine was less effective in inhibiting the nuclear DNA binding of NF-κB/p65 activity, p65/p50 subunits, and antiapototic proteins Bcl-XL, XIAP and survivin expression compared to δ-tocotrienol. However the combination of gemcitabine and δ-Tocotrienol showed additive/synergistic effects on depletion of NF-κB activity and its regulated gene products in vitro and in vivo. Conclusion: This combination chemotherapy could be a potential novel strategy for advanced pancreatic cancer. This study supports the selection of δ-tocotrienol as a potential therapeutic adjuvant to potentiate gemcitabine antitumor activity in human pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5416.

TabAnti-HER2 (erbB-2) oncogene effects of phenolic compounds directly isolated from commercial Extra-Virgin Olive Oil (EVOO)

BackgroundThe effects of the olive oil-rich Mediterranean diet on breast cancer risk might be underestimated when HER2 (ERBB2) oncogene-positive and HER2-negative breast carcinomas are considered together. We here investigated the anti-HER2 effects of phenolic fractions directly extracted from Extra Virgin Olive Oil (EVOO) in cultured human breast cancer cell lines.MethodsSolid phase extraction followed by semi-preparative high-performance liquid chromatography (HPLC) was used to isolate phenolic fractions from commercial EVOO. Analytical capillary electrophoresis coupled to mass spectrometry was performed to check for the composition and to confirm the identity of the isolated fractions. EVOO polyphenolic fractions were tested on their tumoricidal ability against HER2-negative and HER2-positive breast cancer in vitro models using MTT, crystal violet staining, and Cell Death ELISA assays. The effects of EVOO polyphenolic fractions on the expression and activation status of HER2 oncoprotein were evaluated using HER2-specific ELISAs and immunoblotting procedures, respectively.ResultsAmong the fractions mainly containing the single phenols hydroxytyrosol and tyrosol, the polyphenol acid elenolic acid, the lignans (+)-pinoresinol and 1-(+)-acetoxypinoresinol, and the secoiridoids deacetoxy oleuropein aglycone, ligstroside aglycone, and oleuropein aglycone, all the major EVOO polyphenols (i.e. secoiridoids and lignans) were found to induce strong tumoricidal effects within a micromolar range by selectively triggering high levels of apoptotic cell death in HER2-overexpressors. Small interfering RNA-induced depletion of HER2 protein and lapatinib-induced blockade of HER2 tyrosine kinase activity both significantly prevented EVOO polyphenols-induced cytotoxicity. EVOO polyphenols drastically depleted HER2 protein and reduced HER2 tyrosine autophosphorylation in a dose- and time-dependent manner. EVOO polyphenols-induced HER2 downregulation occurred regardless the molecular mechanism contributing to HER2 overexpression (i.e. naturally by gene amplification and ectopically driven by a viral promoter). Pre-treatment with the proteasome inhibitor MG132 prevented EVOO polyphenols-induced HER2 depletion.ConclusionThe ability of EVOO-derived polyphenols to inhibit HER2 activity by promoting the proteasomal degradation of the HER2 protein itself, together with the fact that humans have safely been ingesting secoiridoids and lignans as long as they have been consuming olives and OO, support the notion that the stereochemistry of these phytochemicals might provide an excellent and safe platform for the design of new HER2-targeting agents.

Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal muscles of rats

The purpose of this study was to investigate the occurrence and possible mechanisms of apoptosis in skeletal muscles after burn injury. After a 40% body surface area burn to rats, TA muscles were examined for apoptosis at varying times by TEM, TUNEL and cell death ELISA assay. Thermal injury was found to induce apoptosis in skeletal muscle on the first day and maximal apoptosis appeared 4 days post-injury. Apoptotic ligands in serum assessed by ELISA revealed rapidly increase of TNF-alpha and subsequent increase of sFasL to sFas ratio after burn injury. It implied TNF-alpha induced apoptosis in early stage and FasL induced apoptosis in later stage after burn injury. Apoptosis-related genes/proteins in skeletal muscles examined by real-time PCR array and Western blotting showed pro-apoptotic genes/proteins, including Tnfrsf1a, Tnfrsf1b and Tnfsf6 in TNF ligand and receptor family, Bax and Bid in Bcl-2 family, caspase-3 and caspase-6 in caspase family, Dapk1, FADD and Cidea in death and CIDE domain family, Apaf-1 in CARD family, and Gadd45a were up-regulated, while anti-apoptotic gene Bnip1 was down-regulated compared with that of time-matched controls. In addition, increment of caspase-3, caspase-8 and caspase-9 activity provided further evidence for their role in apoptosis in skeletal muscle. Significant increase in expression in pro-apoptotic genes/proteins and activity of caspases suggested that death receptor-mediated signaling pathways and other apoptotic related pathways participated in apoptosis in skeletal muscle after burn injury. However, it was found that some anti-apoptotic genes such as Bcl2l1, Mcl-1, Nol-3, Il-10 and Prok2 were also up-regulated, which might imply the co-existence of protective response of the body after burns. In conclusion, the data suggest that apoptosis and pro-apoptotic signaling are enhanced in muscles of burned rats. To further elucidate the underlying apoptotic mechanisms mediating the atrophic response is important in establishing potential therapeutic interventions that could prevent and/or reduce skeletal muscle wasting and preserve its physiological function.

The mechanism of methylselenocysteine and docetaxel synergistic activity in prostate cancer cells

The study was designed to evaluate the combination treatment of methylselenocysteine (MSeC) and docetaxel and to delineate the underlying mechanism associated with observed in vitro synergy between MSeC and docetaxel in prostate cancer cells. Cells were treated with different concentrations and schedules (concurrent or sequential) of MSeC and docetaxel alone or in combination. Cell growth/death was assessed with sulforhodamine B assay, trypan blue assay, and time-lapse video. Loewe synergism/antagonism model was used to determine whether the combination effect was additive, synergistic, or antagonistic. Apoptosis and caspase-3 activity were evaluated with cell death ELISA assay and caspase activity assay, respectively. Synergy between MSeC and docetaxel was further assessed in the presence and absence of z-VAD-fmk, a pan-caspase inhibitor. Effect of MSeC and docetaxel alone or in combination on the cellular expression of the antiapoptotic protein survivin was measured with Western blot analyses. Pretreatment with MSeC was crucial to enhance docetaxel antitumor activity. The enhanced antitumor activity of the sequential combination treatment of MSeC and docetaxel (MSeC/docetaxel) was highly synergistic. Apoptosis increased after MSeC/docetaxel, compared with each drug alone or concurrent treatment. Pretreatment with z-VAD-fmk converted the synergy into antagonism, suggesting that the synergy is caspase-dependent apoptosis. The survivin level was down-regulated following MSeC/docetaxel treatment when compared with each drug alone. In conclusion, pretreatment with MSeC was essential to markedly sensitize cells to docetaxel. The synergy between MSeC and docetaxel in C2G prostate cancer cells is associated with increased level of caspase-dependent apoptosis and decreased level of survivin.

Cytotoxicity of a non-cyclooxygenase-2 inhibitory derivative of celecoxib in non-small-cell lung cancer A549 cells

Lung cancer is one of the most common causes of cancer death worldwide. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, overall survival remains poor. Therefore, new therapeutic targets are needed. Phosphoinositide-dependent kinase-1 (PDK1) is one potential target. The aim of the present studies was to investigate the potential of a celecoxib-derived PDK1 inhibitor (OSU03013), that does not inhibit cyclooxygenase-2, to kill lung cancer cells in vitro. Using human non-small-cell lung cancer A549 cells, OSU03013 dose-dependently induced apoptosis. After 6 h of treatment with 7.5 microM OSU03013, 26% of the cells were apoptotic, compared to 4% of the control cells as determined by measuring the sub-G1 peak of propidium iodide stained cells with flow cytometry. A similar increase in apoptosis was evident using the Cell Death ELISA assay. OSU03013-induced apoptosis was accompanied by a reduction in the mitochondrial membrane potential, the release of cytochrome c and the cleavage of caspase-3. Surprisingly, the phosphorylation of Akt at serine 473 was increased in A549 cells treated with 7.5 microM OSU03013. However, the toxicity of OSU03013 was reduced in A549 cells expressing a constitutively active form of Akt. These data demonstrate that OSU03013 induces apoptosis in A549 cells via the mitochondrial pathway. Inhibition of the Akt pathway appears uninvolved in this toxicity, although Akt can provide protection. These results also suggest the potential of celecoxib-derived agents to treat some forms of lung cancer.

Raphanus sativus and its isothiocyanates inhibit vascular smooth muscle cells proliferation and induce G 1 cell cycle arrest

Mu ( Raphanus sativus, Korean White Radish) crude extract (Mu-CE) has been studied for its anti-proliferative activity on mouse aortic smooth muscle cells. The abnormal growth of vascular smooth muscle cells (VSMC) is a prominent feature of vascular disease, including atherosclerosis, restenosis after angioplasty. We examined the mechanisms of the action of Mu-CE on VSMC proliferation. The viability of VSMC decreased to 35% at 24 h of treatment with Mu-CE. Treatment of Mu-CE showed potent inhibitory effects on the DNA synthesis of cultured VSMC. In addition, Mu-CE induced apoptosis using cell death ELISA assay. These inhibitory effects were associated with G 1 cell cycle arrest. Treatment of Mu-CE, which induced a cell-cycle arrest in G 1-phase, induced down-regulation of cyclins and CDKs and up-regulation of the CDK inhibitor p21 expression, whereas up-regulation of p27 by Mu-CE was not observed. Then, total isothiocyanates (ITC) including four different 4-(Methylthio)-3-butenyl isothiocyanate (MTBITC), allyl isothiocayanate (AITC), benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) was isolated from n-hexane extracts of Mu. When the VSMC were treated with ITC, the cell viability was significantly decreased. These findings indicate the efficacy of Mu-CE in inhibiting cell proliferation, G 1- to S-phase cell-cycle progress on VSMC.

Effect of dietary flavonols on oestrogen receptor transactivation and cell death induction.

Consumption of fruits and vegetables has been associated with cancer prevention; flavonoids are widely distributed in plant foods and considered to be the active ingredients. Quercetin and kaempferol are two of the most commonly found dietary flavonols, and have been reported to prevent cancer. We have previously reported that the isoflavone genistein and the flavone baicalein exert differential actions on the oestrogen receptor (OR) alpha in HepG2 cells. Because of the structural resemblance to both isoflavone and flavone, we examined the effects of these dietary flavonols on ORalpha- and ORbeta-specific transactivations and their subsequent involvement in inducing MCF-7 cell death. In the present study, both quercetin and kaempferol were able to compete for OR binding in a cell-free system and were agonistic to ORalpha and -beta expressed in HepG2 cells, while some additive effect was observed in the oestrogen response element (ORE)-driven transcription when 17beta-oestradiol was co-administered. Since the bcl-2 promoter contained two ORE, and ORE-driven transcriptional activity and Bcl-2 mRNA expression were increased by treatment with 10 microm-quercetin or kaempferol, it is possible that quercetin and kaempferol might up-regulate Bcl-2 expression through OR transactivation in MCF-7 cells. Cell death ELISA assay performed on MCF-7 cells indicated that an increase of apoptosis occurred at 25 microm-, but not 10 microm-, quercetin or kaempferol. Indirectly the results suggest that OR activation is not sufficient to induce apoptosis and that apoptosis is induced despite an increase in Bcl-2 expression.

Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway.

Elevated expression of interleukin-6 (IL-6) is implicated in the progression of hormone refractory prostate cancer. Previous studies demonstrated that IL-6 promotes androgen-independent growth of prostate cancer cells. In this study, the effect of IL-6 on apoptosis induced by androgen deprivation was investigated. The effect of IL-6 on apoptosis induced by androgen deprivation in LNCaP cells was examined by cell death ELISA and Western blot using cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9, as well as Bcl-xL and phosphorylated Bad. The Stat3 in IL-6-mediated anti-apoptosis in prostate cancer cells was examined using either dominant-negative or constitutively activated Stat3 mutants. Overexpression of IL-6 renders androgen sensitive LNCaP human prostate cancer cells more resistant to apoptosis induced by androgen deprivation. LNCaP cells undergo apoptosis after 72 hr of androgen deprivation, an outcome is largely absent in clones overexpressing IL-6 as measured by cell death ELISA and chromatin degradation assays. IL-6 over-expressing cells resulted in a significant decrease in the expression of cleaved PARP and cleaved caspase-9 as well as an increase in the expression of Bcl-xL and phosphorylated Bad. Addition of IL-6 antibody completely abolished the anti-apoptotic activity of IL-6. This protective effect of IL-6 was reversed by the expression of a dominant-negative Stat3 mutant, Stat3F. Furthermore, ectopic expression of a constitutively active Stat3 antagonized androgen deprivation-induced cell death of LNCaP cells. These results indicate that IL-6 protects androgen sensitive LNCaP cells from apoptosis induced by androgen deprivation, and Stat3 activation play an important role in IL-6-mediated anti-apoptosis in prostate cancer cells.

Inhibition of Apoptosis by Z-VAD-fmk in SMN-depleted S2 Cells

Spinal muscular atrophy is an autosomal recessive motor neuron degenerative disorder, caused by the loss of telomeric copy of the survival motor neuron gene (SMN1). To better understand how motor neurons are targeted in Spinal muscular atrophy patients, it is important to study the role of SMN protein in cell death. In this report, we employed RNA interference (RNAi) to study the loss-of-function of SMN in Drosophila S2 cells. A 601-base pair double-stranded RNA (dsRNA) of Drosophila SMN (dSMN) was used for silencing the dSMN. Our data indicate that dSMN RNAi resulted in more than 90% reduction of both RNA and protein. Further analysis of S2 cells by cell death ELISA and flow cytometry assays revealed that reduction of dSMN expression significantly increased apoptosis. The cell death mediated by SMN depletion is caspase-dependent and specifically due to the activation of the endogenous caspases, DRONC and DRICE. Significantly, the effect of dSMN RNAi was reversed by a peptide caspase inhibitor, Z-VAD-fmk. These results suggest that dSMN is involved in signal pathways of apoptotic cell death in Drosophila. Hence, the model system of reduced SMN expression by RNAi in Drosophila could be exploited for identification of therapeutic targets.