Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines

Context: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen.

Objective: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines.

Materials and methods: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco’s modified Eagle’s medium for 5 days, and next cultured in Hypoxic Chamber in 1% O₂, 10% O₂, 21% O₂. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software.

Results: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line.

Conclusion: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.     

Teriflunomide – The common drug with underestimated oxygen - Dependent anticancer potential

Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect.The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21%oxygen level occurred in routine cell culture environment.The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels.We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced.Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.     

The in vitro effect of boar semen on the contractility of rat uteri

Contradictory reports in the literature provoked the investigation of a possible oxytocic effect of boar semen on in vitro rat uterus preparations. When fresh boar seminal plasma (SP), dialyzed seminal plasma (DSP) or a filtrate of acetone-extracted seminal plasma (AE) were added to uterine preparations, both the frequency and force of spontaneous contractile activity tended to be depressed. When the uterine preparations were primed with oxytocin (1.6 USP units/100 ml bathing solution), treatment with SP and AE resulted in an increased frequency of contraction but no increase in force. DSP increased neither the frequency nor force of contractions. A solution of salts and organic compounds that approximated the small molecular or dialyzable components of boar semen ("synthetic boar seminal plasma" or BS) affected contractility in a manner similar to SP and AE. When the rat uterus was bathed in a low calcium physiological salt solution, none of the seminal treatments (SP, DSP, AE, or BS) were capable of initiating a contraction. Further, the force of carbachol-induced contractions in these uterine preparations was markedly depressed by these seminal treatments. In contrast, treatment with oxytocin nearly doubled the contractile force. The depressant effect of SP, DSP and AE on both spontaneous and carbachol-induced contractions appeared to be irreversible. Mineral analysis of SP, DSP, AE, BS and the low calcium salt solution showed the SP, AE and BS contained amounts of Ca(2+) and Mg(2+) which could have altered the uterine contractility. In addition, a non-dial-yzable factor in SP appeared to have a similar effect.     

Optimized Effective Potential for Atoms and Molecules

We describe the optimized effective potential method of density functional theory and the semi-analytical approximation due to Krieger, Li and Iafrate. Results for atomic and molecular systems including correlation contributions are presented and compared with conventional Kohn–Sham methods. The combination of the exact exchange energy functional with the correlation energy functional of Colle and Salvetti works extremely well for atomic systems, while further improvement is required for molecular systems.     

Arginase in bull testis

Bull testis arginase was about 1,000 times purified. The molecular weight was estimated to 120,000 (+/- 250). The enzyme was an anionic protein. Km was determined to be 3.4 mM. According to electrophoretic mobility and affinity to DEAE-cellulose as well as to the immunological properties, the arginase isolated from bull testis may be identified as arginase A1.     

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L‐Ser and/or L‐Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1‐mRNA and AST2‐mRNA by RT‐PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L‐Asp. AST1‐mRNA expression was independent on oxygen and cell line, but AST2‐mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L‐Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.     

Alkaline phosphatase from adult rat femur

Four alkaline phosphatase forms from adult rat femur were distinguished on polyacrylamide gel electrophoresis: two soluble forms of Mr 165,000 and 110,000 in the water extract, and three membrane-bound forms of Mr 130,000, 110,000 and 100,000 extractable with deoxycholate. Alkaline phosphatase after SDS-treatment disintegrated into three kinds of monomers: of Mr 80,000, 65,000 and 50,000. The soluble fraction (extract I) contained subunits of Mr 80,000 and 55,000--whereas the pellet fraction (extract II), subunits of Mr 65,000 and 50,000. Since for native forms only three types of subunits were found it seems that, apart from homodimers, there are also some heterodimers composed of the Mr 65,000 and 50,000 subunits forming the native enzyme of Mr 110,000-115,000. Two denatured monomers: of Mr 80,000 and 50,000 may form two native homodimeric forms of Mr 165,000 and 100,000 while in the pellet two monomers: of Mr 65,000 and 50,000 may correspond to three native alkaline phosphatase forms: of Mr 130,000, 110,000-115,000 and 100,000. Probably the Mr 110,000-115,000 form is a heterodimer composed of subunits of Mr 65,000 and 50,000.     

Differential effect of ethanol on formation of phosphorylated intermediates of the jejunal brush border alkaline phosphatase Mr 90,000 and 65,000 subunits

The influence of ethanol on formation of phosphorylated intermediates of different forms of alkaline phosphates in brush border of adult rat was studied. Ethanol enhanced phosphorylation of the Mr 65,000 subunit of jejunal enzyme but had no effect on the Mr 90,000 subunit. Commercial highly purified alkaline phosphate in the presence of ethanol also displayed a two times higher phosphorylation ability.     

Early Changes in the Nucleotide Pools of Germinating Rust Uredospores

Puccinia graminis uredospores were induced to germinate on a water surface and the nucleotide/nucleoside pools were determined by high performance liquid chromatography. Distinct phases of germination could be discriminated by correlating the changes of individual nucleotide pools with characteristic morphological changes. During the first 15 min no visible changes of the cell wall were observed whereas the adenine nucleotide pools increased strongly indicating an activation of the energy metabolism. The stage preceding hyphal growth revealed large increases in the precursor pool for cell wall synthesis (UDP-N-acetylglucosamine). The early phases of germination are obviously controlled by distinct metabolic transitions. Furthermore, correlations of pool contents and changes during germination with the germination efficiency of the three uredospore batches investigated are discussed.