Aglycemia induces apoptosis under hypoxic conditions in A549 cells.

Many of the cancer cells produce energy with accelerated glycolysis and perform lactic acid production even under normoxic conditions called the "Warburg effect". Metabolism can directly or indirectly regulate the apoptotic mechanism so that cancer cells take advantage of reprogrammed metabolism to avoid apoptosis. The aim of this study is to examine the mechanism of apoptosis by incubating human lung carcinoma cells (A549) under different metabolic conditions in hypoxia or normoxia environments. A549 cells were incubated in the normoxic or hypoxic condition that contained 5 mM glucose (Glc 5), 25 mM glucose (Glc 25), or 10 mM galactose (OXPHOS/aglycemic), and the mechanism of apoptosis was investigated. In the hypoxia condition, the rate of early apoptosis in aglycemic OXPHOS cells was increased (15.5% ±7.1). In addition, the activity of caspase-3 (6.1% ± 0.9), caspase-9 (30.4% ± 0.9), and cytochrome c expression level increased; however, the mitochondrial membrane potential (51.9% ± 0.4) was found to be decreased. Changing the amount of oxygen in glycolytic cells had no effect on apoptosis. However, it has been determined that apoptosis is stimulated under hypoxia conditions in aglycemic cells in which galactose is used instead of glucose. Considering that the majority of cancer cells are hypoxic, these data are important in determining targets in therapeutic intervention.

Mitochondrial oxidative phosphorylation became functional under aglycemic hypoxia conditions in A549 cells.

BACKGROUND: Normal cells produce energy (ATP) through mitochondrial oxidative phosphorylation in the presence of oxygen. However, many of the cancer cells produce energy with accelerated glycolysis and perform lactic acid production even under normoxic conditions called "The Warburg Effect". In this study, human lung carcinoma cells (A549) were incubated in either a normoxic or hypoxic environment containing 5 mM glucose (Glc 5), 25 mM glucose (Glc 25), or 10 mM galactose (OXPHOS/aglycemic), and then the bioenergetic pathway was anaylsed. METHODS AND RESULTS: HIF-1α stabilization of A549 cells with different metabolic conditions in normoxia and hypoxia (1% O2) was determined using the western blot method. After that, L-lactic acid analysis, p-PDH/PDH expression ratio, ATP analysis, and citrate synthase activity experiments were also performed. It was determined that HIF-1α stabilization reached the maximum level at the 4 h. It has been found that glycolytic cells produce approximately five times more lactate than OXPHOS cells under both normoxia and hypoxia conditions and also have a higher p-PDH/PDH ratio. It has been determined that citrate synthase activity in hypoxia of all metabolic conditions is lower than normoxia. It has been determined that Glc 5 and Glc 25 cells have more ATP production under normoxia than Glc 5 and Glc 25 cells in hypoxia. OXPHOS cells have showed more ATP production in hypoxia. CONCLUSION: It has been determined that oxidative phosphorylation became functional in a hypoxic aglycemic environment despite the metabolic programming regulated by HIF-1α. This data is important in determining targets for therapeutic intervention.

Resveratrol increases the sensitivity of breast cancer MDA-MB-231 cell line to cisplatin by regulating intrinsic apoptosis.

OBJECTIVES: Breast cancer is one of the most common types of cancer. Chemotherapeutic agents used during treatment induce cytotoxic effects also on normal cells in the tissues. Anti-oxidants used in combination with chemotherapeutic agents have been shown to reduce toxicity on normal cells to a minimum, and some anti-oxidant substances have chemotherapeutic effects. Cisplatin (CDDP) is a platinum class drug that is used clinically in the treatment of many cancers. Resveratrol (RSV) is a natural polyphenol with potent anti-oxidant and anticancer properties. In this study, we aimed to investigate apoptotic effects of using cisplatin and RSV alone or in combined treatment of MDA-MB-231 cells. MATERIALS AND METHODS: The cytotoxic effects of the drugs on MDA-MB-231 cells were determined by MTT method. Subsequently, the change in CDDP-induced apoptotic effect after RSV addition was examined using the AnnexinV FITC labeling, and TUNEL staining method. Activation of caspase-9, -3 in MDA-MB-231 cells was measured by flow cytometer. The mitochondrial membrane potential (MMP), the major factor on the intrinsic pathway, was measured using flowcytometry. RESULTS: The combined dose (23 µM CDDP + 72 µM RSV) produced more cytotoxicity than the agents used alone, leading to early apoptosis (8.2%), 31% depolarization, and 23% DNA fragmentation. Caspase-9 was found to be 30.5% in this combined group and caspase-3 was 26.3%. CONCLUSION: RSV, an effective anti-oxidant, and CDDP as an effective drug in cancer treatment, were found to increase apoptosis when given in the MDA-MB-231 cell.

Effects of integrin-linked kinase on protein kinase b, glycogen synthase kinase-3ß, and ß-catenin molecules in ovarian cancer cells.

Objectives: This study examines the impact of integrin-linked kinase (ILK), protein kinase B (AKT), glycogen synthase kinase-3ß (GSK-3ß), and ß-catenin signal molecules in SKOV-3 ovarian cancer cells adhered to fibronectin. Materials and Methods: Expression levels of α4, αv, ß1, and ß6 integrin subunits known as the fibronectin ligand were investigated with the flow cytometry technique. The effects of ILK, AKT, GSK-3ß, and ß-catenin on the binding of SKOV-3 cells to fibronectin were examined by using the Real-Time Cellular Analysis (RTCA) method. Additionally, the interaction of these proteins was investigated by using Western blot analysis. Results: The results show that the expression levels of integrin subunits were ranked as αv (67.8%), followed by α4 (48.55%), ß6 (32.05%), and ß1 (31%) on SKOV-3 cells. RTCA results showed that ILK (10 µM Cpd22), GSK-3ß (50 µM GSK-3ß inhibitor-XI), AKT (35 µM FPA 124), and ß-catenin (50 µM cardamonin) inhibitors decreased significantly (P<0.01) binding to fibronectin at 24 hr. Western studies in SKOV-3 cells adhered to fibronectin have shown that in inhibition of ILK, AKT expression was strongly inhibited, whereas, in the inhibition of AKT, ILK expression was strongly inhibited. Furthermore, the expression of ß-catenin is partially reduced in inhibition of these two molecules. In ß-catenin inhibition, AKT and ILK expressions are also strongly inhibited. Conclusion: ILK, AKT, GSK-3ß, and ß-catenin were found to be fundamental molecules in binding of SKOV-3 cells to fibronectin. ILK and AKT affect strongly the level of expression of each other, and both also affect the signal path of ß-catenin.

Apoptotic effects of ε-viniferin in combination with cis-platin in C6 cells.

Glioblastoma (GBM) is one of the most common and lethal forms of primary brain tumors in human adults. Treatment options are limited, and in most cases ineffective. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases like cancer. ε-viniferin is a resveratrol dimer and well known for having antiproliferative and apoptotic effects on cancer cells. Cisplatin is a platinum containing anti-cancer drug. In this study, we aimed to investigate antiproliferative and apoptotic effects of using cis-platin and ε-viniferin alone or in combined treatment of C6 cells. Cell proliferation was detected by WST-1. Mitochondrial membrane potential changes in the cells (ΔΨm) were evaluated using cationic dye JC1. Apoptotic index which is a hallmark of late apoptosis was detected by using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and apoptotic alterations were observed by transmission electron microscope (TEM). Activation of caspase-8, -9, -3 in C6 cells at various incubation periods was measured by flow cytometer. Apoptotic index increased at highest level in only combined treatment cells (91.6%) after 48 h incubation. These results were supported by TEM images. Caspase-8 activation in C6 cells increased to a maximum (12.5%) after 6 h by using combined cis-platin/ε-viniferin treatment (13.25/95 µM). Caspase-9 was activated at 44.5% after combined treatment for 24 h. This rate is higher than using cis-platin (14.2%) or ε-viniferin (43.3%) alone. The combined 13.25 µM/cisplatin and 95 µM ε-viniferin treatment caused maximum caspase-3 activation in C6 cells (15.5%) at the end of the 72 h incubation. In conclusion, it was observed that caspase-8, -9, -3 activation which was determined in vitro, trigerred apoptotic mechanism in C6 cells by using low concentrations of combined cis-platin and ε-viniferin.

Calcium Mobilization and Inhibition of Akt Reduced the Binding of PEO-1 Cells to Fibronectin.

OBJECTIVES: To investigate the effects of intracellular calcium (Ca2+) mobilization, ß-catenin and Akt signal pathways after the binding of metastatic ovarian cells to fibronectin. MATERIALS AND METHODS: The expression levels of α4ß1 and αvß6 integrin were determined using α4, ß1, αv, and ß6 antibodies using flow cytometry on PEO-1 cells. The effect of [Ca2+]i on cell adhesion capacity was investigated using RTCA after stimulating PEO-1 cells using thapsigargin and tunicamycin. The binding rate of PEO-1 cells to fibronectin was also investigated in the presence of either different concentrations of cardamonin, which inhibits the accumulation of ß-catenin, or different concentrations of FPA 124, which is a specific inhibitor for the PKB/Akt signal pathway, using RTCA. RESULTS: RTCA analysis results showed that increasing [Ca2+]i through leakage of the calcium pool was strongly effective on PEO-1 cell binding to fibronectin. Extracellular calcium influx also reduced the binding of PEO-1 cells. Cell binding to fibronectin was also inhibited with a ratio of 64% in the presence of 100 µM cardamonin compared with untreated control cells. Finally, it was found that PKB/Akt inhibition with 15 µM FPA 124 decreased the binding of PEO-1 cells to fibronectin with a ratio of 88% compared with untreated control cells. CONCLUSION: PEO-1 cell binding to fibronectin via integrins could be related to intracellular Ca2+ mobilization and Akt signaling.

Vincristine and ɛ-viniferine-loaded PLGA-b-PEG nanoparticles: pharmaceutical characteristics, cellular uptake and cytotoxicity.

The objective of this study was to prepare the ɛ-viniferine and vincristine-loaded PLGA-b-PEG nanoparticle and to investigate advantages of these formulations on the cytotoxicity of HepG2 cells. Prepared nanoparticle has shown a homogeneous distribution with 113 ± 0.43 nm particle size and 0.323 ± 0.01 polydispersity index. Zeta potential was determined as -35.03 ± 1.0 mV. The drug-loading percentages were 6.01 ± 0.23 and 2.01 ± 0.07 for ɛ-viniferine and vincristine, respectively. The cellular uptake efficiency of coumarin-6-loaded nanoparticles was increased up to 87.8% after 4 h. Nanoparticles loaded with high concentrations of both drugs showed a cytotoxic effect on HepG2 cells, having the percentage of cell viability of between 43.23% and 47.37%. Unfortunately, the percentage of apoptotic cells after treated with drugs-loaded nanaoparticles (10.93%) was similar to free forms of drugs (12.1%) that might be due to low ɛ-viniferine release in biological pH at 24 h.

Direct and protective effects of single or combined addition of vincristine and ε-viniferin on human HepG2 cellular oxidative stress markers in vitro.

The objective of this study is to examine the direct effects of low doses and high doses of ε-viniferin, a substance known to be an antioxidant, and vincristine sulphate, a chemotherapeutic agent, alone and in combination [ε-viniferin + vincristine] on HepG2 cell strain, as well as evaluate oxidative stress after incubation periods of 3, 6, and 24 h. Direct effect was determined right after the incubation period; however, for protective effect, antioxidant protection response was determined after the treatment for 1 h with 500 µM H2O2, which is an oxidative stressor. For this purpose, superoxide dismutase was determined for enzyme activity, and lipid hydroperoxide (LPO) and reduced glutathione concentrations were studied as indicators of oxidative stress. Results show that low [3.63 µM vincristine + 3.75 µM ε-viniferin] and high [11.25 µM vincristine + 15.8 µM ε-viniferin] doses of combination groups showed similar direct antioxidant effect on LPO levels as protective when compared to the H2O2 control group (p < 0.05). Superoxide dismutase enzyme showed a direct antioxidant effect in low and high dose combination groups. In addition, when the incubation period was increased to 24 h, a protective effect was observed in both dose groups (p < 0.05). Reduced glutathione activities showed a direct effect in the low dose combination group, and a protective effect in both the low and high doses in the 24 h. These results show that combined usage of drugs in HepG2 cell strain possesses a protective effect against exogenically produced oxidative stress conditions.

Synthesis and evaluation of new dithiocarbamic acid 6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]-imidazol-2-yl methyl esters.

A novel series of dithiocarbamic acid 6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]imidazol-2-yl methyl esters were synthesized and their cytotoxic and apoptotic activities were evaluated on HeLa cells. Some of these compounds showed potent cytotoxic activities and are able to induce the apoptosis mechanism in this cell line. Especially, 2c, 2d, and 2f had a high cytotoxic activity with an IC50 value of 8 or 10 µM at 24 h. These three compounds also induced HeLa cell apoptosis as compared to mitoxantrone. Particularly, 3 µM of 2f induced a high rate of early apoptotic cells (12.9%) at 6 h whereas mitoxantrone induced early apoptosis (5.5%) at 24 h. Compound 2c demonstrated a high ADP/ATP ratio (9.31) in HeLa cells at 12 h compared to mitoxantrone or other compounds, suggesting that 2c might induce HeLa cell apoptosis through the mitochondrial pathway. Caspase-3 activity started to increase after treatment with 6 µM of 2c for 6 h, and the maximal peak of activity was obtained at 12 h of incubation time. All three compounds were found to be potent apoptotic inducers compared to mitoxantrone.

Towards novel anti-tumor strategies for hepatic cancer: ɛ-viniferin in combination with vincristine displays pharmacodynamic synergy at lower doses in HepG2 cells.

Hepatocellular carcinoma is the fifth most common cancer and the third leading cause of cancer-related deaths worldwide. The efficacy of novel combination treatments are increasingly evaluated with use of integrative biology research and development (R&D) strategies and methodological triangulation. We investigated the anti-tumor effect of ɛ-viniferin alone, and the putative synergy of ɛ-viniferin with vincristine on the growth of HepG2 cells in vitro. Growth inhibition and apoptosis induction were determined by MTT assay and annexin V/propidium iodide (PI), respectively. Morphological changes and DNA fragmentation were investigated under electron microscopy and by agarose gel electrophoresis, respectively. The results collectively showed that treating cells with ɛ-viniferin and vincristine significantly inhibited cell viability at lower doses as compared to each agent applied alone. IC(50) values for ɛ-viniferin and vincristine were determined as 98.3 and 52.5 µM at 24 h, respectively. IC(50) value of ɛ-viniferin in combination with vincristine was 15.8+11.25 µM (mean/SD) at 24 h. The viability of cells treated with 17.9 µM vincristine alone for 24 h was 79.62%; it reduced to 26.53% when 25 µM ɛ-viniferin was added in combination with vincristine (p<0.05). We found that combination of drugs promoted the sensitivity of cells against to vincristine treatment. The effect of combined use was in support of a synergistic pharmacodynamic effect. Moreover, low doses of the combination regimen induced phosphatidyl re-localization, morphological changes, and DNA fragmentation, and therefore caused apoptotic death. This study thus suggests that low concentrations of ɛ-viniferin and vincristine can enhance the anti-tumor effects efficiently by inducing HepG2 cell apoptosis. Further studies in other model systems are warranted with a view to potential future applications in the clinic of such combination regimens and their putative mechanism of action in the observed synergy reported here.

Effects of fibronectin and type IV collagen on osteosarcoma cell apoptosis.

The aims of this study are the investigation of the effects of fibronectin and type IV collagen extracellular matrix proteins and the role of caspase-3 and -9 on cis-platin induced U2-OS apoptosis were studied. First the cytotoxic effects of cis-platin on cell system were investigated by colorimetric method and than morphological and ELISA analysis were used for determination of cell apoptosis when induced with cis-platin. In addition, after adhering the cells to fibronection or type IV collagen proteins, the apoptotic rate and the effects of caspase-3 and -9 were also investigated by ELISA in presence of specific inhibitors. U2-OS cells showed 20% cytotoxicity after treatment with 2.4 microM of cis-platin for 48 h. Morphological and the numerical data showed that cis-platin was able to induced apoptosis on cells as a dose-dependent manner. Caspase-3 and -9 inhibitors inhibited cis-platin-induced apoptosis in U2-OS cells, respectively. The binding of cells to 10 microg/mL of fibronectin but not type IV collagen enhanced the apoptosis about 2.5 fold that effects inhibited with caspase-3 inhibitor. The caspase-3 and -9 are involved in the apoptotic signals induced by cis-platin in U2-OS. The binding to fibronectin, but not type IV collagen enhanced the apoptotic response of U2-OS and fibronectin-dependent apoptosis was activated by caspase-3. These finding might be useful for patients to fight against osteosarcoma.

Anticancer activity of some bisbenzimidazoles.

The discovery of DNA topoisomerases has added a new dimension to the study of anticancer drugs. Bisbenzimidazole derivatives are important compounds known as DNA topoisomerase I inhibitors. In the present study, some symmetrical bisbenzimidazole derivatives were synthesized and investigated for their anticancer activity. Anticancer activity screening was applied on HT-29 (colon carcinoma) and MCF-7 (breast carcinoma) cell lines by investigation of cytotoxicity, analysis of DNA synthesis, and DNA fragmentation assays. One of the seven compounds tested showed significant cytotoxicity in both cell lines and caused DNA degradation in the HT-29 cell line.

Synthesis of 2-substituted-N-[4-(1-methyl-4,5-diphenyl-1H-imidazole-2-yl)phenyl]acetamide derivatives and evaluation of their anticancer activity.

In the present study 18 novel imidazole-(benz)azole and imidazole-piperazine derivatives were synthesized in order to investigate their probable anticancer activity. The structures of the compounds were confirmed by IR, (1)H NMR and EI-MS spectral data. Cytotoxicity (MTT), analysis of DNA synthesis and detection of apoptotic DNA assays were applied to determine anticancer activity of the compounds against colon (HT-29) and breast (MCF-7) carcinoma cell lines. Most of the compounds, showed greater activity against HT-29 cells than MCF-7 cells. Some of them indicated considerable cytotoxicity against both of the carcinogenic cell lines. However, their inhibitory activity on DNA synthesis was relatively poor. Anticancer activity screening results revealed that 11, 12 and 13 were the most active compounds in the series. They exhibited significant cytotoxicity against both of the carcinogenic cell lines and caused DNA fragmentation of the HT-29 cells.

Antiproliferative effects of some N-benzylideneanilines.

A series of Schiff bases including N-benzylideneaniline (NBA) nuclei were prepared. The chemical products obtained were characterized by mass spectometry (APCI), 1H NMR, and IR spectroscopy in order to seek their cytotoxic and proliferation effects on human small lung (A549) and cervical (HeLa) cancer cell lines with biochemical assays. All of the synthesized compounds showed antiproliferative effects to different extents.

Synthesis and cytotoxic and analgesic activities of some 1, 5-diaryl-3-ethoxycarbonylpyrrole derivatives.

Some 1,5-diaryl-3-ethoxycarbonyl-2-methylpyrrole derivatives were obtained by reacting 1-aryl-3-ethoxycarbonylpent-1,4-diones and a suitable aniline derivative or sulfanilamide under Paal-Knorr pyrrole synthesis conditions. The cytotoxicity of the compounds was tested and all compounds, except for compound 2 h, showed a time-dependent increase in cytotoxic activity. Analgesic activities of the compounds were determined by using the tail-flick and tail-immersion methods; some of the compounds showed potent analgesic activity.

Synthesis and effects on intracellular calcium of some 1,3-bis-(heteroaryl substituted)benzene derivatives.

A series of 1,3-bis-(heteroaryl substituted)benzene derivatives was designed as promising molecules which might increase intracellular Ca2+ level in F2408 fibroblast-like cells by affecting the Ca2+ channels on plasma membrane. Mentioned compounds were obtained by the treatment of isophtalaldehyde with benzil or 1,2-phenylenediamine derivatives. In this way, 13 compounds were synthesised and their structure elucidations were performed by IR, 1H NMR and mass spectroscopic data and elemental analysis results. Some of the compounds showed Ca2+ being released from the intact cells.