Ewa Langner

Apoptosis induction in human glioblastoma multiforme T98G cells upon temozolomide and quercetin treatment

Glioblastoma multiforme is the most aggressive primary brain tumour. At the cellular and molecular levels, several mechanisms responsible for apoptosis or autophagy induction are blocked. Identification of molecular targets stimulating cells to initiate programmed cell death should be performed for therapeutic purposes. A promising solution is the combination of temozolomide and quercetin. The aim of our study was to evaluate the effect of both drugs, applied alone and in combinations, on apoptosis and autophagy induction in human glioblastoma multiforme T98G cells. Our results clearly indicate that quercetin and temozolomide induce apoptosis very significantly, having no effect on autophagy induction. At the molecular level, it was correlated with caspase 3 and 9 activation, cytochrome c release from the mitochondrium and a decrease in the mitochondrial membrane potential. Both drugs are also potent Hsp27 and Hsp72 inhibitors. This suggests that the apoptotic signal goes through an internal pathway. Increased expression of caspase 12 and the presence of several granules in the cytoplasm after temozolomide treatment with or without quercetin preceding appearance of apoptosis may suggest that apoptosis is initiated by ER stress. Additionally, it was accompanied by changes in the nuclear morphology from circular to ‘croissant like’.

Temozolomide, quercetin and cell death in the MOGGCCM astrocytoma cell line.

The aim of the present study was to investigate the effect of Temozolomide (an alkylating chemotherapeutic agent) and quercetin (natural flavonoid) on cell death in the human astrocytoma cell line MOGGCCM (WHO grade III). Our results indicate that Temozolomide induces autophagy, while quercetin promotes severe necrosis in the cell line in a manner dependent on the drug concentration. We demonstrated for the first time that combinations of both drugs were much more effective in programmed cell death induction in glioma cells. At a low (5muM) drug concentration, quercetin potentiated a pro-autophagic effect of Temozolomide, while after treatment with a higher drug concentration (30muM), autophagy switched to apoptosis. Temozolomide attenuated the toxic effect of quercetin. Apoptosis was mediated by the mitochondrial pathway and the activation of caspase 3 and cytochrome C release, but no changes in caspase 8 expression was observed. It was accompanied by decreased mitochondrial membrane potential and inhibition of Hsp27 and Hsp72 expression. Autophagy was correlated with an increased level of LC3II. Temozolomide and quercetin also inhibited migratory phenotype of MOGGCCM cells and changed the nuclei morphology from a circular to an irregular shape. Our results indicate that quercetin acts in synergy with Temozolomide and when used in combination rather than in separate pharmacological application, both drugs are more effective in programmed cell death induction. Temozolomide administered with quercetin seems to be a potent and promising combination which might be useful in glioma therapy.

Kynurenic acid synthesis and kynurenine aminotransferases expression in colon derived normal and cancer cells

Abstract Background. Kynurenic acid (KYNA), a tryptophan metabolite, was found in human saliva, gastric juice, bile, pancreatic juice and mucus of rat small intestine. Methods. KYNA content in mucus aspirated from human caecum or colon ascendens and KYNA production in colon epithelial and cancer cells were determined using HPLC. Moreover, biological properties of KYNA and kynurenine aminotransferases (KATs) expression in colon epithelial and colon cancer cells were studied. Results. Considerably higher KYNA concentration was detected in samples from patients diagnosed with colon carcinoma (269.40 ± 107.00 pmol/ml, N = 4), Adenoma tubulovillosum (200.50 ± 36.72, N = 10) or Adenoma tubulare (243.50 ± 38.09, N = 9) than in control group (82.22 ± 7.61 pmol/ml, N = 30). Moreover, colon epithelium CCD 841 CoTr cells actively synthesized KYNA in a concentration- and time-dependent manner. This process was decreased by aminooxyacetic acid and L-glutamate in opposite to 4-aminopyridine treatment. Interestingly, KYNA production in colon cancer cells (HT-29 1.39 ± 0.27, LS-180 1.18 ± 0.15 and Caco-2 4.21 ± 0.30 pmol/1 × 105 cells/2 h) was considerably higher in comparison to normal colon epithelial cells (0.70 ± 0.07 pmol/1 × 105 cells/2 h). However, KATs I and II were expressed at similar level in both colon epithelium and cancer cells. Furthermore, KYNA exerted an antiproliferative effect at higher micro- and milimolar concentrations against colon cancer cells with the IC50 of 0.9, 0.2 and 1.2 mM for HT-29, LS-180 and Caco-2 cells, respectively. Conclusion. Summarizing, this is the first report presenting KYNA synthesis and KAT expression in colon derived normal and cancer cells.

Biological Properties of Melanoidins: A Review

Melanoidins are brown, high molecular weight products of Maillard reaction, which takes place during thermal processing of food. They are formed in a multistage reaction between reducing sugars and compounds possessing free amino groups and found in roasted coffee, bakery products, cooked meat, beer, honey, sweet wine, processed tomatoes, and fiber. Nowadays, melanoidins have attracted a lot of attention, not only as a functional food ingredient, but also as a potential pro-healthy dietary supplement. In this field, their antioxidant, antimicrobial, anticancer, and detoxifying activity have been described. Based on recent research developments, an overview of the biological properties of melanoidins with implications for human health is presented.

Melanoidins isolated from heated potato fiber (Potex) affect human colon cancer cells growth via modulation of cell cycle and proliferation regulatory proteins

Melanoidins are brown, nitrogen containing, high molecular weight end products of Maillard reaction with poorly established activity towards tumor cells. The goal of present study was to verify whether both heated potato fiber Potex extract (180°C for 2h) and melanoidins isolated from the extract exerts growth-inhibiting activity in human colon cancer cells in vitro. The cells of LS180 colon cancer cell line were tested upon treatment with roasted potato fiber extract (AM4) as well as with high (HMW) and low (LMW) molecular weight fractions isolated from the extract, since both may be regarded as/or contain melanoidins. The tested compounds at concentration of 1000 μg/ml reduced cell growth down to 45%, 69% and 54%, respectively. Furthermore, deregulated ERK1/2 signaling was revealed upon treatment. Moreover, multiple alternations in cell cycle regulators activity were found (i.e. cyclinD1, cyclin-dependent kinase 4 and 6, p21, p27, p53, pRb) leading to cell cycle cessation in G0 phase. Importantly, LMW compounds revealed markedly stronger potential to alter specific molecular targets comparing to HMW compounds. Summarizing, the results emphasize that both high and low molecular weight melanoidins contribute to antiproliferative activity of heated potato fiber in LS180 colon cancer cells in vitro.

Kinetic studies of the effects of Temodal and quercetin on astrocytoma cells

The aim of the present study was to investigate the kinetics of the effects exerted by Temodal and quercetin on the survival of the human astrocytoma MOGGCCM cell line. Our results indicate that quercetin was toxic and induced necrosis, whereas Temodal induced autophagy-mediated cell death most effectively. The amount of cell death directly correlated with drug concentration and length of exposure. During combined administration of both drugs, Temodal attenuated the cytotoxic effects of quercetin. Combinations of both drugs were effective in inducing programmed cell death, but the type of cell death was concentration-dependent. Co-administration of Temodal (100 μM) with a low quercetin concentration (5 μM) resulted in a very significant induction of autophagy; however, after treatment with quercetin at a higher concentration (30 μM), apoptosis became the primary mechanism of cell death. The sequence of drug administration was also important. The highest number of dead cells was observed after simultaneous administration of both drugs or after pre-incubation with Temodal followed by treatment with quercetin. Apoptosis was identified through activation of the mitochondrial pathway including cleavage of caspase-3 and release of cytochrome c. Autophagy was identified through increased levels of LC3II. Our results indicate that Temodal and quercetin are synergistic inducers of programmed cell death, better together than applied separately. This drug combination appears to be a potent and promising therapeutic relevant to the treatment of gliomas.

Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas

The aim of the present study was to investigate the effect of sorafenib and quercetin on the induction of apoptosis and autophagy in human anaplastic astrocytoma (MOGGCCM) and glioblastoma multiforme (T98G) cell lines. In MOGGCCM cells, sorafenib initiated mainly apoptosis, mediated by the mitochondrial pathway with mitochondrial membrane permeabilization, cytochrome c release to the cytoplasm, and activation of caspase 9 and 3. Additional incubation with quercetin potentiated the pro-apoptotic properties of sorafenib. In T98G cells, autophagy was observed most frequently after the sorafenib treatment. It was accompanied by increased beclin 1 and LC3II expression. Administration of quercetin after the sorafenib treatment resulted in an increased number of autophagic cells. After simultaneous drug application, the level of autophagy was lower in favour of apoptosis. Inhibition of heat shock proteins expression by specific small interfering RNA significantly increased the sensitivity of both the cell lines to induction of apoptosis, but not autophagy. We demonstrated for the first time that sorafenib and quercetin are very effective programmed cell death inducers in T98G and MOGGCCM cells, especially in cells with blocked expression of heat shock proteins.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions

Abstract Background and objective. Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. Results. Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. Conclusion. In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

The effect of quercetin and imperatorin on programmed cell death induction in T98G cells in vitro

BACKGROUND High expression of HSP27 and HSP72 in glioma cells has been closely associated with chemoresistance and decreased sensitivity to programmed cell death induction. Therefore, it is important to devise therapies that effectively target invasive cancer cells by inducing cell death. The aim of our study was to assess the effect of quercetin and imperatorin applied separately and in combinations on the apoptosis and autophagy induction in human T98G cells cultured in vitro. METHODS Cell death induction was analyzed by the staining method. The Western blotting technique and fluorimetric measurements of activity were used to assess the expression of marker proteins of apoptosis and autophagy. The specific siRNA transfected method was used for blocking of the expression of HSP27 and HSP72 genes. RESULTS The experiments revealed the highest percentage of apoptotic cells after using a 50?M concentration of both compounds. Simultaneous quercetin and imperatorin administration induced apoptosis more effectively than incubation with single drugs. These results were accompanied with decreased HSP27 and HSP72 expression, and a high level of caspase-3 and caspase-9 activity. Autophagy was not observed. Additional experiments were performed on a cell line with blocked Hsp27 and Hsp72 expression and significant increase the sensitivity to apoptosis induction upon quercetin and imperatorin treatment was noticed. CONCLUSIONS The present study indicates that quercetin and imperatorin are potent apoptosis inducers, especially when they act synergistically, which may be a promising combination useful in glioma therapy. Our results also demonstrated that blocking the HSP27 and HSP72 gene expression might serve as a therapeutic target for the human brain cancer.

Antiproliferative activity of melanoidins isolated from heated potato fiber (potex) in glioma cell culture model.

Potex constitutes a potato fiber preparation widely used as an ingredient to meat and bakery products which thermal treatment results in creation of new compounds. Melanoidins are high molecular weight brown end products of Maillard reaction, and few data presenting tumor cell growth inhibiting activity of melanoidins have been reported. Thus, in present study we utilized water extract of Potex roasted (180 °C for 2 h), whose chemical characterization revealed the presence of melanoidin complexes. Heated Potex extract inhibited C6 glioma cell proliferation in a dose-dependent manner measured by MTT method. High molecular weight components present in initial extract were responsible for stronger antiproliferative effect compared with low molecular weight fraction. Impaired MAPK (mitogen-activated protein kinase) and Akt signaling was found in cells treated with the extract. Moreover, flow cytometry analyses revealed the extract to induce G1/S arrest in glioma cells. Simultaneously, Western blot analysis showed elevated levels of p21 protein with concomitant decrease of cyclin D1. In conclusion, observed antiproliferative activity of melanoidins present in heated Potex was linked to disregulated MAPK and Akt signaling pathways, as well as to cell cycle cessation. These results suggest potential application of Potex preparation as a functional food ingredient and chemopreventive agent.