Top PDF PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

In the experiment presented in Figure 5A, the animals were treated with the drugs for a period of 22 days. In order to avoid overlooking transient effects on apoptosis, the tumors of the animals were harvested within 24 h after the last application of either drug. As shown in Figure 5A, monotherapy with either DOX or GDC-0941 over a period of 22 days significantly attenuated tumor growth (Figure 5A; Table S2), without causing any obvious side effects. Compared to the continuous low-dose application of DOX, GDC-0941 had a more pronounced effect on tumor growth inhibition. In fact, GDC-0941 completely stopped tumor growth after 7 days of treatment (Figure 5A; Table S2). However, the antitumor effects were not increased when tumors were additionally treated with DOX (Figure 5A, Table S2). A similar result was observed when a lower dose of GDC-0941 (i.e. 1/3 of the initial dose) was combined with DOX (Table S2). Moreover, tumor regrowth was not different between the treatment groups after withdrawal of the drugs (Table S3). Although immunohistochemical analysis of paraffin-embedded tumor sections did not reveal any significant difference in TUNEL positive cells (data not shown), the combination increased the percentage of cells positive for active caspase 3 when compared to application of either DOX or GDC-0941 alone (Figure 5B). These data demonstrated that the combination therapy consisting of DOX plus GDC-0941 elevates caspase 3 activity in vivo. However, this increase in caspase 3 activity caused by the combination treatment does not translate into a cooperative suppression of tumor growth.
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The role of nibrin in doxorubicin-induced apoptosis and cell senescence in Nijmegen Breakage Syndrome patients lymphocytes.

The role of nibrin in doxorubicin-induced apoptosis and cell senescence in Nijmegen Breakage Syndrome patients lymphocytes.

sitivity, genomic instability and cancer development. Since NBS1 deficient cells are characterized by genomic instability and NBS patients suffer from haematopoietic malignancies, we hypothesized that the molecular pathways leading to DNA damage-induced senescence might be impaired in patients affected with this disease. Most cell lines derived from NBS patients were established following transformation with viral oncogenes, which inhibit key regulatory genes such as the tumor suppressor gene proteins p53 and pRb, thus allowing the cell to bypass the senescence program and become immortal [8]. Accordingly, spontaneously immortal- ized T cell lines, S3R and S4, carrying the same mutation within the NBN gene, but with a seemingly functional p53/p21 response after gamma irradiation [9], are a very useful cellular model in studying the mechanisms of DNA damage-induced senescence. Therefore we used two cell lines derived from NBS patients (S3R and S4) and the control, L5 cell line (spontaneously immortalized spleenocytes obtained from a healthy donor) to examine if they are prone to DNA damage-induced senescence. To induce DNA damage and DDR activation we used doxorubicin, which is a DNA damaging agent acting through different mechanisms. It can lead to the formation of direct and indirect DNA damage through: intercalation into DNA, DNA binding and alkylation, DNA cross- linking, interference with DNA unwinding or DNA strand separation, helicase activity as well as inhibition of topoisomerase II and generation of free radicals [10].
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Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose.

Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose.

Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accu- mulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time- dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our find- ings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.
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Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

Nature is bountiful having provided us with a plethora of natural compounds with thera- peutic efficacy. They have attracted worldwide attention since they are contemplated to be safe and pose minimal risk to normal cells [13]. Andrographis paniculata (Acanthaceae) is com- monly used for the alleviation of a wide spectrum of ailments, which include meningitis, acute hepatitis and other acute inflammatory conditions and is very common for its ethnic usage in India and other Southeast Asian countries. Andrographolide, a diterpenoid lactone isolated from A.paniculata, has a broad range of pharmacological effects, such as anti-oxidant, anti- inflammatory, anti-HIV, immunomodulatory, hepatoprotective and anti-cancer activities [14– 17]. Recent studies have shown that Andrographolide-induced cytotoxicity is attributable to autophagy but not apoptosis in human liver cancer cells [18]. In another study, it has been shown that Andrographolide suppresses autophagy, and sensitizes human cancer cells in a p53-independent manner to cisplatin induced apoptosis [19]. Even though it is a remarkable bio-active molecule, Andrographolide is poorly water soluble which renders it difficult to pre- pare clinical formulations. In order to develop pharmacophores showing better anti-prolifer- ative efficacy than Andrographolide, we had selected Andrographolide for chemo-selective functionalizations at C14 hydroxyl group. As per our earlier study, we had been able to explain that Andrographolide transformed to an ester derivative (AG–4, previously abbreviated as 6a, S1 Fig) at C14 hydroxyl substantially improved its solubility and anti-proliferative efficacy [20]. Therefore, we intended to ascertain the molecular mechanism of anti-leukemic effect by AG–4 in-depth, particularly in the induction of various forms of PCD.
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TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment.

TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment.

Very recently, disruption of the normal bone marrow micro- environment through leukemia-driven inhibition of osteoblastic function and transient increase of osteoclasts has been demon- strated in a murine model of AML [43]. Bone is the largest reservoir of TGF-b, a key regulator of mechanical properties of the bone matrix [44], which can modulate the functions of MSCs in the bone microenvironment. Upon reaching the bone microen- vironmental niches, AML cells are exposed to a high level of active TGF-b, which in turn mediates a cascade of events that favor the tumor cell survival. We therefore further investigated the role of TGF-b under conditions mimicking BM microenvironment. Using in vitro co-culture assay system, we have demonstrated that 1D11 abrogated cell cycle arrest by the excess TGF-b1. These effects were seen in AML samples regardless of their FLT3 mutation status, indicating that microenvironmental regulation is broadly applicable to diverse AML subsets. Notably, 1D11 enhanced Ara- C–induced apoptosis in the MV4;11 cells not only under normoxic but also under hypoxic conditions prevalent in leukemic BM microenvironment. These findings suggest prominent role of TGF-b in AML cell survival under hypoxia. HIF-1a also functions as a transcriptional activator of CXCR4 [25], and we demon- strated that CXCL12/CXCR4 is an important component of the Figure 5. Combination of 1D11, Plerixafor, and Ara-C induces potent antitumor effects in vivo . Ba/F3-ITD-luciferase leukemia cells were injected into nude mice as described in Materials and Methods. (A) Serial bioluminescence images (i) and luciferase intensity (ii) of mice in the groups receiving 1D11, Ara-C, Plerixafor, one of the combinations indicated, or no treatment (control) were taken on days 13 and 17 after tumor cell injection. *P#0.05, **P,0.01. (B) Histologic sections of bone marrow taken from mice on day 17 were stained with H&E or anti-GFP antibody. doi:10.1371/journal.pone.0062785.g005
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2-Methyl 2-butanol suppresses human retinoblastoma cells through cell cycle arrest and autophagy

2-Methyl 2-butanol suppresses human retinoblastoma cells through cell cycle arrest and autophagy

2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time- dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.
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Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

Fuji xenograft studies were performed at Eisai Tsukuba. The protocols were approved by the IACUC of Eisai Tsukuba (Permit Number: 13-D-0307-002 for efficacy study and 14-D- 0047-001 for pharmacodynamics study). Fuji cells were harvested during mid-log phase growth, and resuspended in HBSS with 50% Matrigel. Balb/C-nu mice received 1 × 10 7 cells (0.1 mL cell suspension) subcutaneously in the right flank. Mice carrying tumors of approxi- mately 200 mm 3 for efficacy study (25 days after injection) or 260 mm 3 for pharmacodynamics study (26 days after injection) were sorted into treatment groups with similar mean tumor vol- umes. Tazemetostat, EPZ011989 or vehicle (0.5% methylcellulose + 0.1% Tween-80 in water) was administered at the indicated doses on twice a day schedules for either 7, 29 or 35 days by oral gavage. Due to reaching tumor volume endpoint on 2000 mm 3 some of the mice in the vehicle group needed to be euthanized on day 14. As reference compound, doxorubicin HCl in saline was dosed at 10 mg/kg. Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg), and adjusted for the last recorded weight of individual animals. Mice were monitored for overall health status daily during the dosing period or twice a week after the dosing period, and their tumor volumes were monitored twice a week using a digital caliper throughout the experiment. On day 7 of the pharmacodynamics study, mice were sampled in a prespecified fashion. Sampling included nonterminal bleeds (0.15 mL) from the retro-orbital venous plexus with anesthesia by isoflurane inhalation. Blood samples were processed for plasma, with Na- heparin as anticoagulant. The plasma samples were frozen at −80°C and stored before bioana- lysis of compound levels. Tumors were harvested from specified mice. Tumor tissue from each animal was frozen at −80°C or stored in RNAlater (Ambion) and kept at −20°C until total RNA purification was performed.
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Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells.

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells.

This study was designed to begin to address this important question and to select the optimal HDACIs which show the greatest enhancement on cytarabine sensitivities in pediatric AML cells. Such information is mechanistically important and has significant clinical implications, as well. To begin to identify which HDAC isoforms are involved in cytarabine sensitivity, we examined the expression profiles of class I, II, and IV HDACs in 4 pediatric AML cell lines. Our results suggested that HDACs 5 and 11 are unlikely involved in cytarabine sensitivities due to the lack or marginal expression of these enzymes. Using THP-1 cells which express high levels of both classes I and II HDACs, we then used equal doses of three different HDACIs (MS-275, VPA, and SAHA) with different substrate specificities to further narrow down the HDAC isoforms likely to be involved in augmenting cytarabine sensitivity. Results from these studies suggested that HDAC8 is unlikely to be involved in cytarabine-induced apoptosis in THP-1 cells since none of the HDACI treatments resulted in significant enzyme inhibition, although they all enhanced cytarabine-induced apoptosis.
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E3 ubiquitin ligase Cbl-b potentiates the apoptotic action of arsenic trioxide by inhibiting the PI3KAkt pathway Yingchun Li, Xiujuan Qu, Jinglei Qu, Ye Zhang, Jing Liu, Yuee Teng, Xuejun Hu, Kezuo Hou and Yunpeng Liu

E3 ubiquitin ligase Cbl-b potentiates the apoptotic action of arsenic trioxide by inhibiting the PI3KAkt pathway Yingchun Li, Xiujuan Qu, Jinglei Qu, Ye Zhang, Jing Liu, Yuee Teng, Xuejun Hu, Kezuo Hou and Yunpeng Liu

Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 ki- nase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apopto- sis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC 50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.
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Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis.

Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis.

Expression of cFLIP is regulated by NF-κB signaling, constitutive activation of which results in robust pro-proliferative and anti-apoptotic signaling in NHL cells [13]. We therefore investi- gated whether DZNep affects nuclear localization/activation of NF-κB subunits (p50 and p65) and thus reduces NF-κB-regulated gene expression. DZNep treatment did not significantly af- fect p50 or p65 translocation (data not shown) or the expression of cFLIP mRNA (Fig. 6A). Al- though cFLIP mRNA expression was not reduced by DZNep treatment, investigated cFLIP mRNA stability, using actinomycin D to block transcription, revealed impaired cFLIP mRNA stability in DZNep-treated cells compared to buffer treated controls (Fig. 6B). However DZNep treatment increased the transcription of another NF-kB target, A20, here used as a con- trol. Incubation of cells with the translation inhibitor cycloheximide (CHX) revealed a lower cFLIP protein half-life in DZNep-treated cells than in controls (Fig. 6C–D). These results sug- gest that DZNep targets cFLIP expression through mRNA destabilization and enhances cFLIP protein degradation independently of NF-kB activation.
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Characterization of TRIB2 following PI3K inhibition

Characterization of TRIB2 following PI3K inhibition

AKT can also promote cell survival by activating the cyclic AMP-response element binding protein (CREB). Phosphorylation of CREB induces the binding of accessory proteins that are needed for the transcription of anti-apoptotic genes, including Bcl-2 (B- cell lymphoma 2) and Mcl-1 (myeloid cell leukaemia 1) (19). Related to this function, AKT can affect apoptosis by the phosphorylation of BAD (Bcl-2-associated death promoter). Bad controls the release of cytochrome c from mitochondria to initiate the caspase cascade, a process that is critical for the induction of apoptosis (12). BAD can also bind to Bcl-2 or Bcl-XL (B-cell lymphoma-extra-large), therefore blocking their anti- apoptotic activities (19). When phosphorylated by AKT, the BAD-Bcl-2/Bcl-XL complexes are disrupted and BAD is bound instead to the 14-3-3 chaperon protein, being sequestered into the cytosol. In addition, AKT will also phosphorylate BAX (Bcl-2- associated X protein), blocking its translocation to the mitochondria (19). Another way that AKT directly affects the apoptotic response is the inactivation of pro-caspase9, impeding the initiation of caspase cascade, and the phosphorylation of MDM2 (murine double minute 2), leading to the inhibition of p53-mediated apoptosis (19). A critical AKT protein target are the members of the FOXO (Forkhead box O) family of transcription factors including FOXO1a, FOXO3a and FOXO4 (19). These transcription factors have an essential role in promoting apoptosis and inducing cell cycle arrest. These (FOXOs and p53) are discussed in depth in section 1.4 and 1.5 respectively.
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Follistatin is a novel biomarker for lung adenocarcinoma in humans.

Follistatin is a novel biomarker for lung adenocarcinoma in humans.

Methods and Results: The study population consisted of 80 patients with lung adenocarcinoma, 40 patients with ovarian adenocarcinoma and 80 healthy subjects. Serum FST levels in patients and healthy subjects were measured using ELISA. The results showed that the positive ratio of serum FST levels was 51.3% (41/80), which was comparable to the sensitivity of FST in 40 patients with ovarian adenocarcinoma (60%, 24/40) using the 95th confidence interval for the healthy subject group as the cut-off value. FST expressions in lung adenocarcinoma were examined by immunohistochemical staining, we found that lung adenocarcinoma could produce FST and there was positive correlation between the level of FST expression and the differential degree of lung adenocarcinoma. Furthermore, the results showed that primary cultured lung adenocarcinoma cells could secrete FST, while cells derived from non-tumor lung tissues almost did not produce FST. In addition, the results of CCK8 assay and flow cytometry showed that using anti-FST monoclonal antibody to neutralize endogenous FST significantly augmented activin A-induced lung adenocarcinoma cells apoptosis.
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Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

To determine the susceptibility of CARP to doxorubicin, ARVMs were incubated at different concentrations of doxorubicin for 24 h and CARP levels analyzed by immunoblot. Our data show that CARP is extremely sensitive to doxorubicin when compared to actin; as little as 0.5 m M doxorubicin was sufficient to suppress CARP protein expression and a time course of ARVMs treated with 1 m M doxorubicin showed a significant decrease in CARP levels as early as 3 h to less than 20% of control by 24 h (Figure 2A). The immunoblot data were corroborated by immunofluorescent images of ARVMs treated for 24 h with 1 m M doxorubicin, with immunostaining of sarcomeric and nuclear CARP often appearing diffuse and less intense compared to control cells (Figure 3). Since CARP resides in both the cytoplasm and nucleus of cardiomyocytes, we examined the relative susceptibility of CARP to doxorubicin in both compart- ments. Interestingly, there was a decrease in cytoplasmic CARP concomitant with a transient increase in nuclear CARP followed Figure 5. Doxorubicin inhibits CARP expression at the transcriptional level. A: ARVMs were pretreated with 10 mg/ml cycloheximide (Cyclo), a protein synthesis inhibitor, in the presence or absence of 1 mM doxorubicin (Doxo) and cell lysates analyzed by immunoblot for CARP and actin and corresponding densitometry analysis is shown below. B: Comparison of CARP mRNA decay (quantified by RT-PCR) in ARVMs pretreated with 5 mg/ml actinomycin D (act D) in the presence or absence of 1 mM doxorubicin. C: NRVMs were transfected with a CARP promoter luciferase reporter (CARP-pGL3) and treated with increasing concentrations of doxorubicin. Cell lysates were assayed for luciferase activity and values were normalized to a promoterless control (pGL3 basic). Shown are mean6SD from 4 independent experiments. The qRT-PCR and luciferase-reporter experiments were performed in triplicate. * P,0.05, ANOVA.
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Chemoprevention of skin cancer with 1,1-Bis (3'-indolyl)-1-(aromatic) methane analog through induction of the orphan nuclear receptor, NR4A2 (Nurr1).

Chemoprevention of skin cancer with 1,1-Bis (3'-indolyl)-1-(aromatic) methane analog through induction of the orphan nuclear receptor, NR4A2 (Nurr1).

3,39-Diindolylmethane (DIM) (Fig. S1) is a natural product derived from indole-3-carbinol (I3C) which is present in crucifer- ous vegetables such as brussels sprouts, broccoli and cauliflower. DIM has generated much interest in cancer research because of its low toxicity and cytotoxic effects on cancer cells in vitro and inhibition of tumor growth in vivo [24]. For example, DIM induced expression of cell cycle inhibitors such as p21 and p27 and downregulated-cyclin proteins including cyclin D1 and also decreased expression of survival and antiapoptotic proteins including survivin, bcl-2, bax and induced poly (ADP-Ribose) polymerase (PARP) cleavage, mitochondrial cytochrome c release and procaspase cleavage [25][26][27]. A series of novel synthetic 1,1-bis(3 9-indolyl)-1-(p-substituted phenyl) methane analogs (C- DIMs), are also potent anticancer agents [25][28][29] and their activities are structure-dependent. The p-t-butylphenyl and p- biphenyl derivatives activate peroxisome proliferator-activated receptor c (PPARc) whereas the unsubstituted p-phenyl and p- methoxyphenyl analogs activate the orphan receptor NR4A1 (Nurr77/TR3) [30]. Studies in our laboratory have reported a synergistic effect between 1,1-bis(3 9-indolyl)-1-(p-biphenyl) meth- ane (DIM-C-pPhC6H5) and Docetaxel in non-small cell lung cancer cells through enhanced induction of cleaved PARP, bax and N-cadherin and inhibition of phospho-Akt, cyclin D1, survivin, NF-kB, Mcl-1 and phospho JNK2 [29][31].
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O prurido mediado pelo receptor para o peptídeo liberador de gastrina (GRPR) é dependente da via de sinalização PI3KΎ/Akt

O prurido mediado pelo receptor para o peptídeo liberador de gastrina (GRPR) é dependente da via de sinalização PI3KΎ/Akt

experiments, mouse GRPR cDNA was cloned as a XhoI-HindIII frag- ment into the pcDNA3.1 ( ⫺)hygro plasmid vector. Human embryonic kidney cells (HEK293; ATCC CRL-1573) were transfected with the GRPR plasmid DNA. HEK293 cells transfected with pcDNA3.1 ( ⫺) vec- tor only were used as a control. Forty-eight hours after transfection, the cells were rinsed with PBS three times. Transfected HEK293 cells were incubated with either GRP (10 and 100 n M ) or PBS at 37°C for 10 and 30 min. Control HEK293 cells were treated with GRP (10 and 100 n M ) or PBS in a similar manner. A subset of transfected HEK293 cells remained untreated and served as a negative control. After incubation, the cells were harvested and lysed by protein immunoprecipitation lysis buffer (Thermo Fisher Scientific) with proteinase inhibitor and phosphatase inhibitor (Roche Diagnostics). Cell lysates were centrifuged at 4°C for 20 min. The supernatants were collected and protein concentrations were determined by BCA assay. Equal amounts of cell lysates were loaded onto NuPAGE Novex 4 –12% Bis-Tris mini gels, electrophoresed, and trans- ferred to PVDF using standard protocols. The blot was first probed with anti-GRPR (catalog #ab39883; Abcam), anti-Akt antibody (catalog #9272S; Cell Signaling Technology), anti-phospho-Akt (catalog #05- 1003; Millipore), and HRP-conjugated anti-GAPDH (catalog #3683; Cell Signaling Technology) antibody to identify the control GAPDH band at 37 kDa, 24 h at 4°C. Then, the blots were incubated with HRP-conjugated goat anti-rabbit antibody, followed by detection of the ECL substrate.
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Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells.

Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells.

Nrf2 is a highly unstable protein and its half-life is about 15 min in untreated cells [20]. A well established mechanism that controls Nrf2 activation is that oxidative stress or Nrf2 inducers can increase Nrf2 protein stability, resulting in its accumulation in the cells [28,29]. The Keap1-Nrf2 complex appears to play critical role in facilitating the degradation of Nrf2 [55–57]. It has been suggested that ubiquitination of the cytoplasmic Nrf2 involves the Keap1-Cul3-dependent E3. Keap1 functions as a BTB-containing substrate adaptor protein for Cul3 and brings Nrf2 into the Cul3- Rbx1 complex for ubiquitination [55–57]. However, the molec- ular mechanisms underlying the stabilization of Nrf2 protein by various exogenous Nrf2 inducers have not been clearly addressed. Although D3T has long been of interest as an effective inducer of Nrf2, the mechanisms underlying D3T mediated Nrf2 activation are not fully understood. Studies have suggested that the interaction between D3T and the sulfhydryl groups of Keap1 can cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation [58]. In addition, mitogen-activated protein kinases (MAPKs) have recently been shown to be involved in the activation of Nrf2 by D3T [59]. In this study, a significant increase in Nrf2 protein levels was observed in MG132-treated PC12 cells, suggesting that Nrf2 protein is degraded by the proteasome in the cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in PC12 cells treated with D3T alone or Figure 3. Flow cytometry of apoptotic PC12 cells with Annexin V-FITC showed that D3T treatment significantly prevents ethanol-induced apoptosis. Apoptosis was measured in PC12 cells cultured in control medium (Control), treated with 200 mM ethanol (EtOH), treated with 50 mM D3T alone (D3T), or treated with both ethanol and D3T (EtOH+D3T). Data are expressed as fold change over control and represent the Mean 6 SEM of three separate experiments. *p,0.05 vs. control. #p,0.05 vs. EtOH.
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Are iron oxide nanoparticles safe? Current knowledge and future perspectives

Are iron oxide nanoparticles safe? Current knowledge and future perspectives

was assessed by using the same tests and no significant positive response was obtained, although cell redox status was slightly dis- turbed [69]. Liu et al. [92] found no increase in the incidence of chromosome aberrations in Chinese hamster lung cells treated with ION (10 nm) with positively charged PEI surface or with neutral non-functional PEG-coated ION (10 and 30 nm). Besides, normal human fibroblasts exposed to meso-2,3-dimercaptosuccinic acid (DMSA)-coated maghemite nanoparticles showed also no increases in DNA damage attributed in part to the inhibition of potential toxicity by the DMSA coating, which acts as a barrier avoid- ing direct contact between fibroblasts and the nanoparticle core [93]. DNA damage was neither observed after treatment of L-929 fibroblasts with bare or TEOS-coated ION (magnetite) [36] or in lymphoblastoid TK6 cells and primary human leukocytes treated with uncoated nanomagnetite [58]. More recently, Couto et al. [27] also demonstrated absence of ION effects on genetic material of human T-lymphocytes reporting no chromosome aberrations in cells treated with PAA-coated and non-coated nanomagnetite for 48 h. In agreement with these studies, Paolini et al. [40] reported absence of genotoxic and carcinogenic effects of rhamnose-coated ION (magnetite) on mouse fibroblast Balb/c-3T3 cells. Further- more, a number of studies evaluating the potential mutagenicity induced by different ION by means of Ames test, with or with- out metabolic activation, were also reported with negative results [34,94,95]. However, Gomaa et al. [85] described both positive and negative results in the Ames test for ION (magnetite) depending on the administered dose and the presence of metabolic activa- tion. Supporting this observation, Liu et al. [92] concluded recently that ION mutagenicity could be dependent on nanoparticle size and surface coating after having found a positive mutagenic response (Ames test) in cells treated with PEG-coated ION (10 nm) but not in cells treated with PEI-coated ION (10 nm) or PEG-coated ION (30 nm).
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Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells.

Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells.

concentration, including chromatin condensation, formation of apoptotic bodies, extensive intra-cytoplasm vesicles, and multi- nucleated giant cells (Fig. 3A). These changes resembled changes in K562 cells treated with TPA, a drug known to induce K562 cells to differentiate towards the megakaryocytic lineage [16,20]. Co-treatment with the pancaspase inhibitor z-VAD-fmk partially blocked lapatinib-induced inhibition of viability and apoptosis induction, suggesting that lapatinib activates both caspase- dependent and caspase-independent cell death pathways (Figs. 3C and 3D). Interestingly, at conditions that reduced viability more than 95%, fewer than 40% of the K562 cells were positive for apoptosis (Fig. 2C), in contrast, 80% of HL-60 cells were positive for apoptosis after lapatinib treatment (right panel of Fig. 2C). The morphological features of lapatinib-treated HL-60 cells correlated with the high percentage of apoptotic cells. High levels of dead cells were detected at days 1–3, indicating that the reduction in K562 cell numbers after lapatinib treatment is not due to growth arrest or induction of apoptosis at later time points. This raises the possibility that other, non-apoptotic modes of cell death might be induced by lapatinib in K562 cells.
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PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells.

PI3K and ERK-induced Rac1 activation mediates hypoxia-induced HIF-1α expression in MCF-7 breast cancer cells.

system. Hypoxia has been known to promote ERK translocation to the nucleus, where ERK may exert part of its biological activity [49]. Previous report suggested that phosphorylation of p300 by the ERK increases HIF-1a transcriptional activity by increasing HIF-1/p300 complex formation [50]. However, our results showed that decreased activation of PI3K or ERK by inhibitor did not change the activated HIF-1a mRNA transcription level under hypoxia in MCF-7 human breast cancer cells. The role of other signaling molecules that function downstream of PI3K and ERK in controlling HIF-1a expression remains to be determined. ROS plays a central role in the key intracellular signal transduction pathway for regulation of angiogenesis [51,52]. An earlier report showed that thymosin beta-4-induced HIF-1a stabilization in human cervical tumor cells is ROS-dependent [53]. However, the mechanism whereby ROS influences HIF-1a expression has not been described. Some studies reported that overproduction of ROS induced HIF-1a accumulation via the PI3K/Akt-PKC-HDAC pathway [54]. In contrast, our results show that hypoxia increased the production of ROS in breast cancer cell line MCF-7. Furthermore, decreased activation of
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<i>In Silico</i>, <i>In Vitro</i> and <i>In Vivo</i> Assessment of Safety and Anti-inflammatory Activity of Curcumin

<i>In Silico</i>, <i>In Vitro</i> and <i>In Vivo</i> Assessment of Safety and Anti-inflammatory Activity of Curcumin

and generation of inflammatory granulation (Ghosh et al., 2002). β-hexosaminidase an acid exoglycosidase produced, stored in rat mast cells granules and is released from the mast cells in parallel with histamine upon stimulation (Lynch et al., 1978 Lagunoff et al., 1970; Lagunoff, 1972). It is evidenced that at least 86% of the mast cell content of β -hexosaminidase is available for immunologic release from the secretory granules along with histamine released from the activated mast cells (Schwartz et al., 1979). Therefore, the release of β-Hexosaminidase from the activated immune cells has been considered as a potent degranulation marker for histamine release (Schwartz and Austen, 1980; Ozawa et al., 1993). Thus β- Hexosaminidase and histamine are ideal markers to evaluate the inflammatory response in vitro and in vivo. Inflammation is a key etiological factor for several disease conditions such as hypersensitivity, asthma, Inflammatory Bowel Disease (IBD), rheumatoid arthritis and many others. Most of the currently marketed therapeutic drugs are associated with adverse side effects and are not suitable for chronic therapies and so some of them were withdrawn from the markets. For instance, Non-Steroidal Anti-Inflammatory (NSAID’s) drugs are reported to have adverse drug interactions and hence are not prescribed along with warfarin, antihypertensives and diuretics. Thus, treatment of these inflammatory disorders still remains a growing health concern and has become a major challenge to the health professionals.
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