Immunogenicity of recombinant analog of antitumor protein lactaptin

Therapeutic monoclonal antibodies and recombinant proteins including cytokines are commonly used in the treatment of cancer and inflammatory diseases. In most cases, these protein-based drugs exhibit a high therapeutic efficacy, which is unfortunately frequently associated with a variety of side effects. We have investigated the in vitro and in vivo immunogenicity of recombinant antitumor protein lactaptin (RL2). Based on the qRT-PCR analysis, we have shown that, in MDA-MB-231 human breast adenocarcinoma cells, RL2 suppresses the NF-kB signaling cascade that regulates the reactions of innate immunity. RL2 inhibits the expression of the CXCL1 protein and apoptosis inhibitor A20 and enhances expression of IkB, NF-kB repressor. The ELISA method has been used to evaluate the antibody titer in the blood of mice, which received single and triple intravenous or intraperitoneal injections of RL2. The multiplex immunoassay of 23 cytokines in the mice blood has shown that the RL2 injections lead to a slight increase in the levels of systemic pro-inflammatory cytokine interleukin-5 (IL-5) and keratinocyte chemoattractant (KC), a homologue of human macrophage inflammatory protein-1 (MIP-1). These observations indicate the low immunogenicity of the recombinant lactaptin analog, which can be considered to be a potential molecular drug candidate for further clinical development.     

iASPPsv antagonizes apoptosis induced by chemotherapeutic agents in MCF-7 cells and mouse thymocytes

iASPP was an inhibitory member of ASPP family and could specifically inhibit the apoptotic function of p53. iASPPsv was identified by our lab as the short isoform of iASPP, which encoded a 407aa protein and highly matched the carboxyl terminus of iASPP. In this study, iASPPsv was stably transfected into the breast cancer cell line MCF-7 by means of lentivirus to explore the effects of iASPPsv on biological functions of MCF-7. Thymocytes from iASPP/iASPPsv transgenic mice were also used to explore the effects of iASPP/iASPPsv on cell biological function. The results demonstrated that iASPPsv antagonized the growth inhibition induced by etoposide (VP-16) in MCF-7 cells. iASPPsv also down-regulated proapoptotic genes (Bax, Puma and Noxa) expression to inhibit apoptosis caused by VP-16. Moreover, iASPP and iASPPsv could both help the thymocytes of transgenic mice to resist the growth inhibition and apoptosis caused by dexamethasone (Dex) or VP-16. At the same time, DNA double strand break damage accumulated in either iASPPsv MCF-7 cells or iASPP/iASPPsv thymocytes. These findings showed that iAPSS/iASPPsv reduced the growth inhibition and apoptosis induced by Dex or VP-16, with DNA damage accumulating which might promote the pathogenesis and/or progression of cancer.     

The mechanism of neogambogic acid-induced apoptosis in human MCF-7 cells

Neogambogic acid (NGA), an active ingredient in garcinia, can inhibit the growth of some solid tumors and result in an anticancer effect. We hypothesize that NGA may be responsible for the inhibition of proliferation of human breast cancer cell line MCF-7 cells. To investigate its anticancer mechanism in vitro, MCF-7 cells were treated with various concentrations of NGA. Results of MTT (methyl thiazolyl tetrazolum) assay showed that treatment with NGA significantly reduced the proliferation of MCF-7 cells in a dose-dependent manner. NGA could increase the expression of the apoptosis-related proteins FasL, caspase-3, caspase-8, caspase-9, and Bax and decrease the expression of anti-apoptotic protein Bcl-2 accompanied by the mitochondrial transmembrane damage. The antiproliferative effect of NGA on MCF-7 cells is due to the G(0)/G(1) arrest, increased apoptosis and activation of Fas/FasL and cytochrome C pathway. These results provide an important insight into the cellular and molecular mechanisms through which NGA impairs the proliferation of breast cancer cells.     

Novel thiosemicarbazides induced apoptosis in human MCF-7 breast cancer cells via JNK signaling

Abstract

In this study, novel thiosemicarbazides and 1,3,4-oxadiazoles were synthesized and evaluated for their anticancer effects on human MCF-7 breast cancer cell lines. Among the synthesized derivatives studied, compound 2-(3-(4-chlorophenyl)-3-hydroxybutanoyl)-N-phenylhydrazinecarbothioamide 4c showed the highest cytotoxicity against MCF-7 breast cancer cells as it reduced cell viability to approximately 15% compared to approximately 25% in normal breast epithelial cells. Therefore, we focused on 4c for further investigations. Our data showed that 4c induced apoptosis in MCF-7 cells which was further confirmed by TUNEL assay. Western blotting analysis showed that compound 4c up-regulated the pro-survival proteins Bax, Bad and ERK1/2, while it down-regulated anti-apoptotic proteins Bcl-2, Akt and STAT-3. Additionally, 4c induced phosphorylation of SAPK/JNK in MCF-7 cells. Pretreatment of MCF-7 cells with 10?µM of JNK inhibitor significantly reduced 4c-induced apoptosis. Molecular docking results suggested that compound 4c showed a binding pattern close to the pattern observed in the structure of the lead fragment bound to JNK1. Collectively, the data of current study suggested that the thiosemicarbazide 4c might trigger apoptosis in human MCF-7 cells by targeting JNK signaling.     

Apoptosis induction by dohevanil, a DHA substitutive analog of capsaicin, in MCF-7 cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in a variety of red peppers of the genus Capsicum, is a type of vanilloid. It has been shown to induce apoptosis in many cell types. The effects of vanilloids on apoptosis induction are thought to be correlated with the length and degree of the unsaturation of the fatty acyl chains. In this study, we compared the effect of capsaicin and its docosahexaenoic acid (DHA, C22:6) analog (we named as dohevanil) on human breast cancer MCF-7 cells, which do not express caspase-3. Dohevanil, which was synthesized from DHA and vanillylamine, has longer and highly unsaturated fatty acyl chain than capsaicin. We showed that both vanilloids exhibit effects of growth inhibition and DNA fragmentation induction in MCF-7 cells. These effects of dohevanil were more potent than capsaicin. Because these effects were inhibited by z-VAD-fmk, a broad-spectrum caspase inhibitor, the vanilloids induced the apoptosis via caspase-dependent pathway not involving caspase-3. In conclusion, dohevanil has a more potent effect on apoptosis induction in MCF-7 cells than capsaicin.     

Role of mitochondrial oxidative stress in the apoptosis induced by diospyrin diethylether in human breast carcinoma (MCF-7) cells

Mitochondria and associated oxidative stress have been shown to play critical roles in apoptotic death induced by various stress agents. Previously, we reported the antitumor property of diospyrin (D1), a plant-derived bisnaphthoquinonoid, and its diethylether derivative (D7), which was found to cause apoptotic death in human cancer cell lines. The present study aims to explore the relevant mechanism of apoptosis involving generation of cellular reactive oxygen species (ROS) by D7 in human breast carcinoma (MCF-7) cells. It was found that while D7 inhibited the proliferation of tumor cells, the associated apoptosis induced by D7 was prevented by treating the cells with N-acetyl-L: -cysteine (NAC), an antioxidant, and cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition (MPT). Experiments using suitable inhibitors also demonstrated that D7 could alter the electron flow in mitochondrial electron transport chain by affecting target(s) between complex I and complex III, and indicated the probable site of D7-induced generation of ROS. These results were further supported by confocal microscopic observation on changes in mitochondrial organization and shape in cells treated with D7. Taken together, the results of our study clearly suggested that the apoptosis induced by D7 would involve alteration of MPT, cardiolipin peroxidation, migration of Bax from cytosol to mitochondria, decreased expression of Bcl-2, and release of cytochrome c, indicating oxidative mechanism at the mitochondrial level in the tumor cells.     

Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells

Selenium nanoparticles (Se NPs) have been recognized as promising materials for biomedical applications. To prepare Se NPs which contained cancer targeting methods and to clarify the cellular localization and cytotoxicity mechanisms of these Se NPs against cancer cells, folic acid protected/modified selenium nanoparticles (FA–Se NPs) were first prepared by a one-step method. Some morphologic and spectroscopic methods were obtained to prove the successfully formation of FA–Se NPs while free folate competitive inhibition assay, microscope, and several biological methods were used to determine the in vitro uptake, subcellular localization, and cytotoxicity mechanism of FA–Se NPs in MCF-7 cells. The results indicated that the 70-nm FA–Se NPs were internalized by MCF-7 cells through folate receptor-mediated endocytosis and targeted to mitochondria located regions through endocytic vesicles transporting. Then, the FA–Se NPs entered into mitochondria; triggered the mitochondria-dependent apoptosis of MCF-7 cells which involved oxidative stress, Ca²+ stress changes, and mitochondrial dysfunction; and finally caused the damage of mitochondria. FA–Se NPs released from broken mitochondria were transported into nucleus and further into nucleolus which then induced MCF-7 cell cycle arrest. In addition, FA–Se NPs could induce cytoskeleton disorganization and induce MCF-7 cell membrane morphology alterations. These results collectively suggested that FA–Se NPs could be served as potential therapeutic agents and organelle-targeted drug carriers in cancer therapy.     

Transient suppression of X-ray-induced apoptosis by exposure to power frequency magnetic fields in MCF-7 cells

Epidemiological studies suggest that exposure to power frequency magnetic fields may be a risk factor for breast cancer in humans. To study the relationship between exposure to 60-Hz magnetic fields (MFs) and breast cancer, cell cycle distribution, apoptosis, and the expression of related proteins (p21, Bax, and Bcl-2) were determined in MCF-7 cells following exposure to magnetic fields (60 Hz, 5 mT) alone or in combination with X rays. It was found that exposure of MCF-7 cells to 60-Hz MFs for 4, 8, and 24 h had no effect on cell cycle distribution. Furthermore, 60-Hz MFs failed to affect cell growth arrest and p21 expression induced by X rays (4 Gy). Similarly, 60-Hz MFs did not induce apoptosis or the expression of Bax and Bcl-2, two proteins related to apoptosis. However, exposure of cells to 60-Hz MFs for 24 h after irradiation by X rays (12 Gy) significantly decreased apoptosis and Bax expression but increased Bcl-2 expression. The effects of exposure to 60-Hz MFs on X-ray-induced apoptosis and Bax and Bcl-2 expressions were not observed at 72 h. These data suggest that exposure to 60-Hz MFs has no effects on the growth of MCF-7 cells, but it might transiently suppress X-ray-induced apoptosis through increasing the Bcl-2/Bax ratio.     

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3β (GSK3β), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3β. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.     

Nuclear thioredoxin-1 is required to suppress cisplatin-mediated apoptosis of MCF-7 cells

Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.     

The apoptosis of HEL cells induced by hydroxyures

Hydroxyurea has been used to synchronize cultured cells to S-phase and used to treat patients with sicklecell anemia.Recently,we found that hydroxyurea can induce the apoptosis of HEL(human erythroleukemia) cells.The induced HEL cells showed ultrastructurally chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies.However,the cells of K562,another human erythroleukemia cell line,did not show such morphological changes.Under fluoroscope,the HEL cells after induction of ten displayed a clear reduction in nuclear diameter and nuclear chromatin cleavage and condensation and the presence of nuclear ring and apoptotic bodies.Analysis with flow cytometry showed that the percentage of apoptotic cells is about 30-40% after HEL cells were induced by hydroxyurea for 3 days.DNA ladder can be observed by electrophoretic analysis.     

Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late stage hallmark of the apoptotic process. In mammalian apoptotic cells fragmentation is catalyzed by DFF40/ CAD DNase. DFF40/CAD primary activated through site-specific proteolytic cleavage by caspase 3. The absence of caspase 3 in MCF-7 leads to lack of oligonucleosomal DNA fragmentation under numerous apoptotic stimuli. In this study it was shown that palmitate induces apoptotic changes of nuclei and oligonucleosomal DNA fragmentation in casp3 deficient MCF-7. Activation and accumulation of 40-50 kDa DFF40 like DNases in nuclei and cytoplasm of palmitate-treated MCF-7 were detected by SDS-DNA-PAGE assay. Microsomes of apoptotic MCF-7 activate 40-50 kDa nucleases when incubated with human placental chromatin and induce oligonucleosomal fragmentation of chromatin in cell free system. Both DNases activation and chromatin fragmentation are suppressed in presence of caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome associated caspase 7 is suggested to play the principal role in induction of oligonucleosomal DNA fragmentation of casp3 defitient MCF-7.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

trans-Resveratrol induces apoptosis in human breast cancer cells MCF-7 by the activation of MAP kinases pathways

Polyphenols represent a large class of plant-derived molecules with a general chemical structure that act as potent free radical scavengers. They have long been recognized to possess several therapeutic activities ranging from anti-thrombotic to antioxidant. Moreover, the capability of polyphenols to act as reducing or oxidizing molecules depends on the presence of environmental metals and on the concentrations used. In this work we demonstrated that the stilbene trans-resveratrol was able to commit human breast cancer MCF-7 cells to apoptosis. Mainly, we evidenced a pivotal role of the mitochondria in this phenomenon as cytochrome c release into the cytosol was found after the treatment. We further showed that trans-resveratrol was able to affect cellular redox state. In particular, it induced an early production of ROS and lipid oxidation, and only later compromised the GSH/GSSG ratio. This mode of action was mirrored by a temporally different activation of JNK and p38(MAPK), with the former rapidly induced and the latter weakly activated at long intervals. The results obtained demonstrate a pro-apoptotic activity for trans-resveratrol, and suggest a preferential activation of different classes of MAP kinases in response to different oxidative stimuli (ROS versus GSH/GSSG alteration).     

Regulation of calnexin sub-cellular localization modulates endoplasmic reticulum stress-induced apoptosis in MCF-7 cells

The endoplasmic reticulum (ER) is the cellular compartment where proteins enter the secretory pathway, undergo post-translational modifications and acquire a correct conformation. If these functions are chronically altered, specific ER stress signals are triggered to promote cell death through the intrinsic apoptotic pathway. Here, we show that tunicamycin causes significant alteration of calnexin sub-cellular distribution in MCF-7 cells. Interestingly, this correlates with the absence of both tunicamycin-induced calnexin phosphorylation as well as tunicamycin-induced cell death. Under these conditions, calnexin-associated Bap31, an ER integral membrane protein, is subjected to a caspase-8 cleavage pattern within a specific sub-compartment of the ER. These results suggest that MCF-7 resistance to ER stress-induced apoptosis is partially mediated by the expression level of calnexin which in turn controls its sub-cellular localization, and its association with Bap31. These data may delineate a resistance mechanism to the ER stress-induced intrinsic apoptotic pathway.     

Effect of spider venom on cell apoptosis and necrosis rates in MCF-7 cells

The purpose of this study was to examine the effects of antitumor activity of the venom from the spider Macrothele raven (Araneae, Hexathelidae) on the human breast carcinoma cell line, MCF-7. The spider venom affected cell viability in a dose- and time-dependent manner as observed by [(3)H]-methyl thymidine incorporation assay. Cytotoxicity changes in MCF-7 cells caused by the spider venom at concentrations of 10, 20, and 40 mug/mL were determined by lactate dehydrogenase release assay. Flow cytometry showed that the spider venom induced apoptosis and necrosis of MCF-7 cells at these concentrations. MCF-7 cells treated with spider venom were accumulated on the G(2)/M and G(0)/G(1) phases. In addition, Western blotting analysis indicated that one of the pharmacological mechanisms of spider venom was to activate the expression of p21. In vivo examination of the inhibition of tumor growth in nude mice by the spider venom (at concentrations of 1.6, 1.8, and 2.0 mug/g mice) revealed that tumor size significantly decreased compared to controls by 21 days of treatment and at all points of analysis thereafter for 7 weeks (p < 0.01). We thus propose that the in vivo and in vitro effects of the spider venom can be possibly estimated.     

Adenine nucleotide (ADP/ATP) translocase 3 participates in the tumor necrosis factor induced apoptosis of MCF-7 cells

Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.