Serum availability affects expression of common house-keeping genes in colon adenocarcinoma cell lines: implications for quantitative real-time PCR studies.

Careful selection of housekeeping genes (HKG) is prerequisite to yield sound qPCR results. HKG expression varies in response to hypoxia but the effect of manipulations of serum availability, a common experimental procedure, remains unknown. Also, no data on HKG expression stability across colon adenocarcinoma lines that would aid selection of normalizers suitable for studies involving several lines are available. Thus, we evaluated the effect of serum availability on the expression of commonly used HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in seven colon adenocarcinoma cell lines (Caco-2, DLD-1, HCT116, HT29, Lovo, SW480, and SW620). Sets of stably expressed line-specific and pan-line HKG were validated against absolutely quantified CDKN1A, TP53, and MDK transcripts. Both serum availability and line type affected HKG expression. UBC was fourfold down-regulated and HPRT1 1.75-fold up-regulated in re-fed HT29 cultures. Line-to-line variability in HKG expression was more pronounced than that caused by altering serum availability and could be found even between isogenic cell lines. PPIA, RPLP0, YWHAZ, and IPO8 were repeatedly highly ranked while ACTB, B2M, UBC, and PGK1 were ranked poorly. Normalization against PPIA/RPLP0/SDHA was found optimal for studies involving various colon adenocarcinoma cell lines subjected to manipulations of serum availability. We found HKG expression to vary, more pronouncedly by line type than growth conditions with significant differences also between isogenic cell lines. Although using line-specific normalizers remains optimal, a set of pan-line HKG that yields good estimation of relative expression of target genes was proposed.

Early induction of stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein following photodynamic therapy.

BACKGROUND: There are proteins, responsible for many basic cell functions (transmission of extracellular signals to cytoplasm or nucleus, cell growth, proliferation, migration, survival), which are activated and overexpressed in response to acute oxidative stress, especially tyrosine kinases. The oxidative stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein (Ossa/C9orf10) protects cancer cells from oxidative stress-induced apoptosis by Src family kinases activation. METHODS: In this study precursor of protoporphyrin IX, 5-aminolevulinic acid and its encapsulated form were used in treating MCF-7 human breast cancer cells. After light illumination, cells were collected at different time points and used for evaluation (immunocytochemistry, Western blot analysis) of expression of above proteins, c-Src and Ossa. RESULTS: Our results showed that 5-aminolevulinic acid-mediated photodynamic therapy caused decrease of c-Src expression at 7h after irradiation. The strongest expression was observed at 24h after treatment. Encapsulated form of 5-aminolevulinic acid in terms of PDT caused similar changes of expression of c-Src protein. Furthermore, we observed strong Ossa expression at 7h after treatment in comparison to very low expression at time points 0, 18 and 24h. CONCLUSION: We would like to emphasize that our results showed high expression of Ossa at early time interval after PDT, which was accompanied by a low expression of c-Src kinase, what could protect cancer cells from PDT through activation of c-Src in response to oxidative stress.

Immunocytochemical studies on the nuclear ubiquitous casein and cyclin-dependent kinases substrate following 5-aminolevulinicacid-mediated photodynamic therapy on MCF-7 cells.

BACKGROUND: Recent data indicates that nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) may play role in tumor growth. In present study authors examined whether photodynamic therapy with 5-aminolevulinic acid (5-ALA) induces NUCKS expression in breast cancer cell line, MCF-7. METHODS: In the experiment concentration of 5-ALA was 6.5mM. Excitation wavelength was 630 ± 20 nm, total light dose of light 5 or 10 J/cm(2) and irradiance 60 mW/cm(2) was used. Cells were collected at established time points and Western blot and immunocytochemical studies were performed using antibody against NUCKS. RESULTS: Studies proved strong cytotoxic effects in cells following PDT with 6.5mM of precursor and 10 J/cm(2). Western blot analysis revealed the strongest expression of NUCKS at 7h after PDT. At next time points, 18 and 24h, expression of NUCKS decreased and became similar to that of control group. Further immunocytochemical studies showed very strong expression of NUCKS following PDT with 5-ALA and light irradiation of 5 J/cm(2). Early, at 0 h, that expression was predominantly seen in nuclei, while at 7h expression of NUCKS was observed in disseminated manner within entire cells in both nuclei and cytoplasm, with prevalence of cytoplasmic staining. CONCLUSIONS: Authors suggest that NUCKS is involved in cellular responses following PDT, and since parallel induction of NUCKS and proapoptotic marker Bax and inhibition of anti-apoptotic Bcl-2 was observed, this protein might also be involved in induction of apoptosis following PDT.

Paraoxonase (PON)-1 activity in overweight and obese children and adolescents: association with obesity-related inflammation and oxidative stress.

BACKGROUND: Paraoxonase-1 (PON1) is a HDL-attached extracellular esterase which is believed to contribute to the anti-atherogenic and anti-inflammatory properties of HDL. A decrease in PON1 is a risk factor for cardiovascular disease and has recently been found to be associated with juvenile obesity. The issue of a possible association between enzyme activity and/or its phenotype distribution and obesity-related metabolic abnormalities, inflammation, and oxidative stress has not been addressed yet. OBJECTIVES: To evaluate PON1 activity and phenotype distribution with respect to obesity and obesity-related metabolic disorders, inflammation and oxidative stress in children and adolescents. MATERIAL AND METHODS: PON1 arylesterase activity was measured spectrophotometrically in 156 children and adolescents (47 lean, 27 overweight and 82 obese). Enzyme phenotype was determined using dual substrate (phenyl acetate/paraoxon) method. PON1 activity and phenotype distribution were related to the presence of obesity, metabolic syndrome, insulin resistance, hyperinsulinemia, hypertriglyceridemia, high blood pressure, low HDL level, impaired fasting glucose and/or glucose tolerance as well as inflammatory and oxidative stress indices. RESULTS: PON1 arylesterase activity decreased in general and central obesity, high blood pressure, and hyperinsulinemia conditions and correlated with BMI, CRP, adipocyte fatty acid-binding protein, superoxide dismutase, catalase, glutathione peroxidase, free thiols, and HOMA in a gender-dependent manner. PON1 decreases were independently associated with central obesity in girls, explaining 17% in PON1 variability, and with elevated CRP in boys, explaining 12% in its variability. PON1 phenotype was not associated with frequency of metabolic abnormalities. CONCLUSIONS: PON1 decreases in central obesity, exacerbating obesity-related inflammation and oxidative stress. The enzyme associations are gender-dependent: obesity and oxidative stress affects PON1 in girls whereas inflammation in boys.

Visfatin in juvenile obesity - the effect of obesity intervention and sex.

BACKGROUND: The association of visfatin, an adipocytokine relevant to the development of inflammation and metabolic disorders, with juvenile obesity needs to be re-established as previously used tests occurred to be nonspecific. OBJECTIVE: To evaluate visfatin association with a metabolic profile of 88 overweight/obese and 26 lean children/adolescents as well as changes in its levels following weight reduction programme (diet + enhanced physical activity ± metformin). DESIGN: A case-control and cohort study. RESULTS: Visfatin was higher in obese than lean and overweight individuals (2·07 vs. 1·53 and 1·47 ng mL(-1) , P = 0·034). Of metabolic syndrome components, central obesity combined with either insulin resistance (IR) or hyperinsulinemia (HI) was associated with increases in circulating visfatin. In girls, visfatin correlated with leptin (r = 0·40, P = 0·009) and thiols (r = -0·36, P = 0·009), which explained 24% in visfatin variability. In boys, visfatin correlated with waist circumference (r = 0·36, P = 0·036), BMI% (r = 0·38, P = 0·025), whole body insulin sensitivity index (r = -0·36, P = 0·036), IL-6 (r = 0·38, P = 0·024) and thiobarbituric acid reactive substances (TBARS) (r = 0·52, P = 0·001), of which IL-6 and TBARS were independent predictors of visfatin elevation, explaining 42% in data variability. Visfatin was significantly lower following weight reduction programme than at baseline (1·43 vs. 1·83 ng mL(-1) , P = 0·033). Visfatin reduction correlated neither with changes in metabolic parameters nor was it affected by metformin. ΔVisfatin correlated exclusively with baseline visfatin (r = 0·612, P < 0·0001), which explained 38% in data variability. CONCLUSIONS: Central obesity combined with HI/IR contributes to visfatin elevation. Visfatin association with metabolic/biochemical variables is gender dependent. Diet + enhanced physical activity are effective in visfatin reduction, the degree of which depends on baseline visfatin.

Circulating adipocyte fatty acid-binding protein, juvenile obesity, and metabolic syndrome.

Adipocyte fatty acid-binding protein (A-FABP) links obesity and metabolic syndrome (MetS) and might be targeted in future therapies. Its utility as a MetS biomarker has been suggested in adults but has not been examined in children/adolescents. Our objectives were to identify metabolic parameters associated with A-FABP elevation in children and adolescents and to evaluate the effect of obesity intervention and A-FABP diagnostic utility. A-FABP and anthropometric, metabolic, and inflammatory indices were measured in 31 lean and 114 overweight/obese children and adolescents and reassessed after obesity intervention (1 year; diet and enhanced physical activity, with or without metformin). A-FABP was significantly higher in overweight/ obese than lean individuals, where it correlated with insulin, waist circumference (WC), and 2-h glucose independent of body mass index (BMI), age, gender, and developmental stage. The pattern of A-FABP associations differed between sexes. As a MetS indicator, A-FABP had 68% accuracy. The weight reduction program was effective in reducing A-FABP, BMI%, WC, triglycerides, and cholesterol. In conclusion, elevation in A-FABP is associated with MetS components independent of BMI status and can be reduced by diet and enhanced physical activity. A-FABP as a single MetS biomarker has a moderate accuracy.

In vitro and in vivo matrix metalloproteinase expression after photodynamic therapy with a liposomal formulation of aminolevulinic acid and its methyl ester.

Photodynamic therapy (PDT) is a well-known method for the treatment of malignant tumors, and its principles have been well established over the past 30 years. This therapy involves the application of a chemical called a photosensitizer and its subsequent excitation with light at the appropriate wavelength and energy. Topical photodynamic therapy with aminolevulinic acid (5-ALA) is an alternative therapy for many malignant processes, including nonmelanoma skin cancers such as basal-cell carcinoma (BCC). Our novel approach for this study was to use a liposomal formulation of 5-ALA and its methyl ester (commercially available as metvix) both in vitro and in vivo, and to check whether the liposome-entrapped precursors of photosensitizers can induce the expression of metalloproteinases (MMPs) in animal tumor cells and in other tissues from tumor-bearing rats and in selected cell lines in vitro. We also checked whether the application of tissue inhibitors of matrix metalloproteinases (TIMPs) has any effect on MMPs in the above-mentioned experimental models, and if they can cause complete inhibition of MMP expression. Immunohistochemical studies revealed that after the PDT, the intensity of expression of MMPs in healthy animals was very low and seen in single cells only. After the PDT in tumor-bearing rats, MMP-3 was expressed in the tumor cells with the highest intensity of staining in the tissues directly adjacent to the tumors, while MMP-2 and -9 were not found. In the control groups, there was no observed expression of MMPs. In vitro studies showed that MMP-3 was expressed in MCF-7 cells after PDT, but MMP-9 was not observed and MMP-2 was only seen in single cases. Our studies confirmed that the application of an MMP-3 inhibitor may block an induction of MMP-3 expression which had previously been initiated by PDT. The preliminary data obtained from cancer patients revealed that new precursors are effective in terms of PDT, and that using MMP inhibitors should be considered as a potential enhancing factor in clinical PDT.

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme.

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.