Hakan Akca

Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

BackgroundMultidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs.ResultsN-acetylcysteine increased the resistance of both cells against vincristine and BSO decreased NAC-enhanced MRP1-mediated vincristine resistance, indicating that induction of MRP1-mediated vincristine resistance depends on GSH. Vincristine decreased cellular GSH concentration and increased GPx activity. Glutathione S-Transferase activity was decreased by NAC.ConclusionOur results demonstrate that NAC and BSO have opposite effects in MRP1 mediated vincristine resistance and BSO seems a promising chemotherapy improving agent in MRP1 overexpressing tumor cells.

Invasiveness and anchorage independent growth ability augmented by PTEN inactivation through the PI3K/AKT/NFkB pathway in lung cancer cells.

PTEN is inactivated in a subset of lung cancer; therefore, we investigated the involvement of PTEN inactivation in invasiveness of lung cancer cells. AKT at Ser473 was phosphorylated in several lung cancer cell lines with loss of PTEN expression. Therefore, we created a tetracycline inducible expression system of wild-type PTEN (PTEN-WT) as well as catalytically (PTEN-G129R) and lipid phosphatase (PTEN-G129E) inactive PTEN mutants using the PC14, PC9 and PC3 lung adenocarcinoma cell lines, in which endogenous PTEN expression was not detected and AKT at Ser473 was phosphorylated by Western blot analysis. Induction of PTEN-WT reduced phosphorylation of AKT and inhibited the transcriptional activity of NFkB, whereas PTEN mutants did not, suggesting that PTEN inactivation results in the activation of the AKT/NFkB pathway in PC14, PC9 and PC3 cells. Furthermore, overexpression of PTEN-WT suppressed anchorage independent growth in soft agar and reduced invasiveness in a trans-well chamber assay of PC14 cells. Neither PTEN-G129R nor PTEN-G129E had suppressive effects on anchorage independent growth and invasiveness. Augmentation of invasiveness by constitutively active AKT was also shown in mouse NIH3T3 cells. Therefore, it was strongly indicated that activation of the PI3K/AKT/NFkB pathway by PTEN inactivation results in augmented invasiveness in lung cancer cells and lipid phosphatase activity of PTEN plays a key role in this process.

Zinc oxide nanoparticles: Genotoxicity, interactions with UV-light and cell-transforming potential

The in vitro genotoxic and the soft agar anchorage independent cell transformation ability of zinc oxide nanoparticles (NPs) and its bulky forms have been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells, either alone or in combination with UVB-light. The comet assay, with and without the use of FPG and Endo III enzymes, the micronucleus assay and the soft-agar colony assay were used. For the comet assay a statistically significant induction of DNA damage, with and without the enzymes, were observed up of 100μg/mL. ZnO NPs were able to increase significantly the frequency of micronuclei, and similar results were observed in the cell transformation assay where such NPs were able to induce cell-anchorage independent growth. These effects were observed at doses up 100μg/mL. Although UVB-light was able to induce genotoxic damage and cell-anchorage growth, a significant antagonist interaction effect was observed in combination with ZnO NPs. These in vitro results, obtained with the selected cell lines, contribute to increase our genotoxicity database on the ZnO NPs effects as well as to open the discussion about their risk in photo-protection sun screens.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.

N‐acetylcysteine enhances multidrug resistance‐associated protein 1 mediated doxorubicin resistance

Background  Resistance of cancer cells against anticancer agents is caused partly by multidrug resistance‐associated protein 1 (MRP1). The exact mechanism of MRP1‐involved multidrug resistance has not yet been clarified, although glutathione (GSH) is likely to have a role for the resistance to occur. N‐acetylcysteine (NAC) is a pro‐glutathione drug. DL‐buthionine (S,R)‐sulfoximine (BSO) inhibits GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1‐mediated doxorubicin resistance in human embryonic kidney (HEK293) and its MRP1‐transfected 293MRP cells.

Tumour suppressor PTEN enhanced enzyme activity of GPx, SOD and catalase by suppression of PI3K/AKT pathway in non-small cell lung cancer cell lines.

Phosphates and tensin homologue deleted on chromosome 10 (PTEN) is a tumour suppressor gene which dephosphorilates phosphoinositol 3,4,5 triphosphates. Therefore PTEN can regulate PI3K/AKT pathway in cells. Because of promoter methylation or gene deletion, PTEN expression is commonly decreased or lost in non-small cell lung cancer (NSCLC) cell lines. Therefore, we hypothesized that PTEN could regulate the activity of superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx) and catalase. We first recreated PTENwt, G129R and G129E expressions in lung cell lines, in which endogenous PTEN expression was not detected. Then, we showed that PTEN could suppress AKT activity by its lipid phosphatase domain. We then examined the effect of recreated PTEN expressions in NSCLC cells. While PTENwt expression caused enhanced activity of SOD, GPx and catalase in transfected cells lines, neither G129R nor G129E expression effected enzyme activities. These results suggest that PTEN can up-regulate SOD, GPx and catalase activity by inhibition of PI3K/AKT pathway in NSCLC cell lines.

Utility of serum DNA and pyrosequencing for the detection of EGFR mutations in non-small cell lung cancer.

Mutations in the EGFR gene are critical determinants of treatment with EGFR tyrosine kinase inhibitors (TKIs) for non-small cell lung cancer (NSCLC) patients. DNA isolation from tumor samples usually requires surgery; therefore, we wanted to isolate DNA from circulating tumor cells by using the serum of NSCLC patients. This protocol was recently published. DNA was isolated from the serum of 52 Turkish NSCLC patients and their EGFR mutation status was examined by pyrosequencing. EGFR mutations were detected in 25 of the 52 patients (48.1%): 17 patients with delE746-A750, 2 with delE747-A750insP, and 6 with L858R. All mutations detected by pyrosequencing were confirmed by dideoxy sequencing, and the presence of the same mutations in the tumors was verified by using paraffin embedded tissues of all the patients. Mutations were detected more frequently in adenocarcinomas (24 of 36, 66.7%) than in squamous cell carcinomas (1 of 16, 6.3%) (P<0.001). These results confirm the utility of serum DNA and pyrosequencing for the detection of EGFR mutations in patients with advanced NSCLC.

Convolvulus galaticus, Crocus antalyensis, and Lilium candidum Extracts Show Their Antitumor Activity Through Induction of p53-Mediated Apoptosis on Human Breast Cancer Cell Line MCF-7 Cells

Conventional and newly emerging treatment procedures such as chemotherapy, catalytic therapy, photodynamic therapy, and radiotherapy have not succeeded in reversing the outcome of cancer diseases to any drastic extent, which has led researchers to investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. It has been reported that several members of the Convolvulaceae, Iridaceae, and Liliaceae families have antitumor activity against some tumor cell lines. Here we first report that Convolvulus galaticus, Crocus antalyensis, and Lilium candidum species have cytotoxic activity on human breast cancer cell line MCF-7 cells. Plant samples were collected and identified, and their cytotoxic effects on the MCF-7 cell line were examined at different concentrations of methanol extracts. We found that all three plants have cytotoxic effects on MCF-7 cells but that C. galaticus has the strongest cytotoxic effect even in the lowest extract concentration tested (0.32 μg/mL). Our results indicate that these plant extracts have cytotoxic effects on human breast carcinoma cell line MCF-7 cells and that this cytotoxic effect comes from p53-mediated stimulation of apoptosis.

Downregulation of SATB2 is critical for induction of epithelial-to-mesenchymal transition and invasion of NSCLC cells

OBJECTIVES The epithelial-to-mesenchymal transition (EMT) is considered as a key step in invasion of cancer cells. There are several regulator proteins responsible for induction of EMT, but underlying mechanisms are still unclear. SATB2 is an epigenetic regulator involved in osteoblastic differentiation. The role of SATB2 in EMT and invasion of NSCLC cells is unknown. Therefore, we aimed to explain roles of SATB2 with underlying molecular mechanisms in EMT and invasion of NSCLC cells. MATERIALS AND METHODS We used A549 and NCI-H1650 cells as a model to evaluate the effects of SATB2 in EMT and invasion of NSCLC cells. Cell culture, western blot analysis, siRNA-mediated gene knockdown, and invasion assay were performed in this study. RESULTS AND CONCLUSION In this study, we investigated the regulatory role of SATB2 expression in TGF-β-induced EMT and invasion of NSCLC cells, and found that SATB2 is downregulated in A549 cells and TGF-β can induce EMT in these cells, however, TGF-β can not induce EMT in SATB2 expressing cells such as H1650, PC3, II-18, Hcc78 and Hcc193. Our results demonstrated that SATB2 knockdown is sufficient to induce generation of fibroblast-like morphology, EMT and invasion of NSCLC cells by upregulating the expressions of Slug, Twist and Zeb1. Moreover, SATB2 knockdown promotes TGF-β-induced EMT and invasion in NSCLC cells. These results strongly suggest that SATB2 prevents induction of EMT by suppressing expression of EMT-inducing transcription factors in NSCLC cells. Furthermore, SATB2 could inhibit tumour initiation by suppressing stemness marker genes such as CD44, Nanog, Oct-4A and Sox-2. Consequently, our results clearly indicate that SATB2 plays pivotal role in EMT, invasion and stemness of NSCLC cells.

The protective effect of docosahexaenoic acid on the bilirubin neurotoxicity

Usually, all newborns demonstrate high serum unconjugated bilirubin (UCB) level. UCB may induce adverse effects in the central nervous system. We aimed to evaluate the cytotoxic effects of UCB and the protective effects of docosahexaenoic acid (DHA) on astrocyte cell cultures. The viability of astrocyte cells decreased after UCB treatment in a dose-dependent manner. Pre-incubation of DHA prevents the cells from UCB-mediated neurotoxicity. Our results shown that UCB leads to inhibition of antioxidant enzymes superoxide dismutase (SOD), catalase and GPx activity and induction of apoptosis. But only 4-h pretreatment of DHA can suppress of UCB-mediated inhibition of antioxidant enzymes SOD, catalase and GPx activity and induction of apoptosis in astrocytes. Our results strongly indicated that DHA has a protective effect on UCB-mediated neurotoxicity through inhibition apoptosis and antioxidant enzymes activity of SOD, CAT and GPx in rat primer astrocyte cell line