Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.     

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2^-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.     

Down regulation of CIAPIN1 reverses multidrug resistance in human breast cancer cells by inhibiting MDR1

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1), initially named anamorsin, a newly indentified antiapoptotic molecule is a downstream effector of the receptor tyrosine kinase-Ras signaling pathway. Current study has revealed that CIAPIN1 may have wide and important functions, especially due to its close correlations with malignant tumors. However whether or not it is involved in the multi-drug resistance (MDR) process of breast cancer has not been elucidated. To explore the effect of CIAPIN1 on MDR, we examined the expression of P-gp and CIAPIN1 by immunohistochemistry and found there was positive correlation between them. Then we successfully interfered with RNA translation by the infection of siRNA of CIAPIN1 into MCF7/ADM breast cancer cell lines through a lentivirus, and the expression of the target gene was significantly inhibited. After RNAi the drug resistance was reduced significantly and the expression of MDR1mRNA and P-gp in MCF7/ADM cell lines showed a significant decrease. Also the expression of P53 protein increased in a statistically significant way (p ≤ 0.01) after RNAi exposure. In addition, flow cytometry analysis reveals that cell cycle and anti-apoptotic enhancing capability of cells changed after RNAi treatment. These results suggested CIAPIN1 may participate in breast cancer MDR by regulating MDR1 and P53 expression, changing cell cycle and enhancing the anti-apoptotic capability of cells.     

Association of the XRCC1 gene polymorphisms with cancer risk in Turkish breast cancer patients

The X-ray repair cross-complementing group 1 (XRCC1) gene is believed to play an important role in base excision repair and displays genetic polymorphisms. Data on the role of XRCC1 polymorphisms in cancer susceptibility is inconsistent. In the present study, we investigated the effect of two XRCC1 polymorphisms, Arg194Trp and Arg399Gln, on breast cancer risk in a case- control study involving Turkish breast cancer patients and healthy women. Both alleles exhibited a similar distribution among cases and controls leading to lack of any significant association between the XRCC1 polymorphisms and breast cancer risk, either in homozygotes and heterozygotes or combined. The allele frequency of the codon 194 variant was very low in cases and healthy individuals (5.3 and 3.9%, respectively) compared to that of the variant 399Gln allele (39.7 and 37.4%). Our results do not support evidence for a role of the XRCC1 polymorphism in developing breast cancer.     

Optimization of photodynamic therapy response by survivin gene knockdown in human metastatic breast cancer T47D cells

Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.     

Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists

The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up‐ and down‐regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.     

Determination of physiological thiols by electrochemical detection with piazselenole and its application in rat breast cancer cells 4T-1

Glutathione (GSH), the most abundant cellular thiol, has been shown to play an important role in maintaining cellular redox equilibrium that is pivotal for cell growth and function. In the present paper a novel electrochemical probe of piazselenole containing a Se-N bond was well developed for the determination of GSH. The cyclic voltammogram of piazselenole scanned at 100 mV/s displayed an irreversible reduction peak at -0.106 V (vs Ag/AgCl electrode) and a significant peak current decrease could be further provoked with the addition of GSH into piazselenole solution. On the basis of the peak current decrease of piazselenole recorded by differential pulse voltammetry with the increase of GSH concentration, a working curve was constructed for GSH determination in the range of 5.0 x 10(-10) approximately 2.2 x 10(-8) M with the linear regression equation as DeltaiP (10(-6)A) = 0.0952 + 0.4287 x CGSH (10(-8) M) and the detection limit (3sigma) as 83 pM. The proposed method was satisfactorily applied to the extracts of rat breast cancer cells 4T-1 for intracellular thiols detection.     

Glutathione peroxidase-1 regulates ASK1-dependent apoptosis via interaction with TRAF2 in RIPK3-negative cancer cells

Glutathione peroxidase (GPx) is a selenocysteine-containing peroxidase enzyme that defends mammalian cells against oxidative stress, but the role of GPx signaling is poorly characterized. Here, we show that GPx type 1 (GPx1) plays a key regulatory role in the apoptosis signaling pathway. The absence of GPx1 augmented TNF-α-induced apoptosis in various RIPK3-negative cancer cells by markedly elevating the level of cytosolic H₂O₂, which is derived from mitochondria. At the molecular level, the absence of GPx1 led to the strengthened sequential activation of sustained JNK and caspase-8 expression. Two signaling mechanisms are involved in the GPx1-dependent regulation of the apoptosis pathway: (1) GPx1 regulates the level of cytosolic H₂O₂ that oxidizes the redox protein thioredoxin 1, blocking ASK1 activation, and (2) GPx1 interacts with TRAF2 and interferes with the formation of the active ASK1 complex. Inducible knockdown of GPx1 expression impaired the tumorigenic growth of MDA-MB-231 cells (>70% reduction, P = 0.0034) implanted in mice by promoting apoptosis in vivo. Overall, this study reveals the apoptosis-related signaling function of a GPx family enzyme highly conserved in aerobic organisms.Subject terms: Apoptosis, Cancer prevention     

Exploring the interaction of calycosin with cyclin D1 protein as a regulator of cell cycle progression in lung cancer cells

Cyclin D1 has been shown to play a pivotal role in the proliferation of lung cancer cells through regulation of cell cycle progression. Therefore, targeting this protein can be used as a potential strategy in lung cancer treatment. Calycosin has been reported to show potential anticancer effects, however, its possible anticancer mechanisms remain unclear. Therefore, in this study we aimed to explore the interaction of cyclin D1 and calycosin to determine the binding properties and probable structural changes of cyclin D1. We carried out in-depth experimental and computational binding assays of calycosin with cyclin D1 under simulated physiological environment, using intrinsic, extrinsic, synchronous fluorescence, circular dichroism, and differential scanning calorimetry (DSC) analysis. The results showed a spontaneous static mechanism driven from hydrogen bonding and van der Waals forces between hydrophilic residues of cyclin D1 with hydroxyl groups of calycosin. We determined that calycosin led to secondary and tertiary structural changes of cyclin D1 through exposure of hydrophobic residues. Also, it was determined that calycosin resulted in an apparent decrease in the heat capacity changes (ΔCp) and midpoint of unfolding transition (Tm) values of cyclin D1. Cellular studies also indicated that calycosin caused the inhibition of lung cancer cell proliferation through cell cycle arrest at G1 phase, which may be due to denaturation of cyclin D1, although it needs further investigation in the future studies. In general, this study may provide useful preliminary data about the development of calycosin-based anticancer platforms.     

A comparison of breast cancer tumor cells with varying expression of the Her2/neu receptor by Raman microspectroscopic imaging

The Her2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast carcinomas. The roles of Her2/neu have been reported before in literature, showing different relations to intracellular lipid composition. Here, we use Raman microspectroscopic imaging to reveal the chemical composition of single live cells from breast carcinoma cell lines MDA-MB-231, MDA-MB-435s and SK-BR-3, which express Her2/neu receptor in different extent. Average Raman spectra of the different cell populations show prominent lipid presence in all cell lines. With high significance, Raman difference spectra reveal increased lipid contents, as well as a lower degree of fatty acid saturation in the MDA-MB cell lines with respect to the SK-BR-3 cells. These results are confirmed by hierarchical cluster analysis of single cells. High internal consistency of the chemical compositions in the cell lines is shown by hierarchical cluster analysis on a single matrix composed of the data of different cells from a single cell line. Although Her2/neu expression is highest for SK-BR-3 cells, their lipid contents are lower than that of the MDA-MB cell lines, which express less to no Her2/neu receptors. Rather than metabolic rate or senescence, the degree of metastaticity of the cells appears to be related to the polyunsaturated fatty acid contents of the cells.     

Down-regulation of Bcl-2 and Akt induced by combination of photoactivated hypericin and genistein in human breast cancer cells

Presented experiment considers combination of genistein and photodynamic therapy with hypericin with a view to achieve higher therapeutic outcome in human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, both identified in our conditions as photodynamic therapy resistant. Since genistein is known to suppress Bcl-2 expression, we predicted that photodynamic therapy with hypericin might benefit from mutual therapeutic combination. In line with our expectations, combined treatment led to down-regulation of Bcl-2 and up-regulation of Bax in both cell lines as well as to suppression of Akt and Erk1/2 phosphorylation induced by photoactivated hypericin in MCF-7 cells. Although Akt and Erk1/2 phosphorylation was not stimulated by photodynamic therapy with hypericin in MDA-MB-231 cells, it was effectively suppressed in combination. Variations in cell death signaling favoring apoptosis were indeed accompanied by cell cycle arrest in G₂/M-phase, activation of caspase-7, PARP cleavage and increased occurrence of cells with apoptotic morphology of nucleus. All these events corresponded with suppression of proliferation and significantly lowered clonogenic ability of treated cells. In conclusion, our results indicate that pre-treatment with tyrosine kinase inhibitor genistein may significantly improve the effectiveness of photodynamic therapy with hypericin in MCF-7 and MDA-MB-231 breast cancer cells.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

XANES determination of the platinum oxidation state distribution in cancer cells treated with platinum(IV) anticancer agents

Here we describe the use of X-ray absorption near edge spectroscopy (XANES) to provide information about the relative proportions of platinum(II) and platinum(IV) complexes by analyzing the XANES edge height. The intracellular reduction of platinum(IV) complexes in cancer cells has been observed directly, and the proportion of reduction after 2 h was found to correlate with the reduction potentials of the complexes.     

H2 formation in the reaction of O(1D) with CH4

The reaction of O(¹D) with CH₄ was studied to determine the efficiency of H₂ production in a direct process, and it was found to be 0.11 ± 0.02. Thus the two channels which account for all of the reaction between O(¹D) and CH₄ in the gas phase are      

Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.     

Antiproliferative effect of 2-Hydroxy-6-tridecylbenzoic acid from ginkgo biloba sarcotestas through the aryl hydrocarbon receptor pathway in triple-negative breast cancer cells

Abstract

This study aims to isolate the potential antiproliferative and cytotoxic compounds from ginkgo biloba sarcotestas (GBS) and investigates the underlying mechanism in human MDA-MB-231 and mouse 4T-1 triple-negative breast cancer cells. Our results showed that 2-Hydroxy-6-tridecylbenzoic acid was isolated by cytotoxicity-guided fractionation where different fractions were assessed using MTT assay against MDA-MB-231 and 4T-1 cells. Colony formation assay showed that 2-Hydroxy-6-tridecylbenzoic acid significantly inhibited cell proliferation. The inhibition was associated with the enhancement of cytochrome P450 (CYP) 1B1 expression in a dose- and time-dependent manner and no significant change of CYP1A1 expression by qPCR and Western blot assays in MDA-MB-231 and 4T-1 cells. The mechanism was further demonstrated by the activation of aryl hydrocarbon receptor (AhR) pathway with the upregulation of AhR, AhR nuclear translocator (ARNT) and AhR-dependent xenobiotic response elements (XRE) activity. These findings may have implications for development of anticancer agents containing 2-Hydroxy-6-tridecylbenzoic acid as functional additives.     

Determination of genomic N3‐methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser‐induced fluorescence

Methylating substances alter DNA by forming N3‐methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF), we synthesized the N3mT‐3’‐phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N’‐nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5‐hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5‐methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE‐LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.