The role of galanin in the progression and prognosis of colorectal cancer: the unfinished story.

The paper presents a summary of immunohistochemical (IHC) and biochemical investigations on the presence of galanin (Gal), one of the neuropeptides abundant in the enteric nervous systems, and three types of its receptors (GalR1-3) in colorectal cancer (CRC) tissue and non-involved colon wall and their associations with clinical-pathological data of the CRC patients. We were the first to morphologically demonstrate the presence of endogenous Gal in CRC sections and measure its content in homogenates of tumor tissue and dissected compartments of unchanged colon wall. The prominent atrophy of myenteric plexuses displaying Gal immunoreactivity (Gal-Ir) located close to the tumor invasion was found to be accompanied by higher Gal content in the tumor-adjacent muscularis externa than in tumor-distant tissue. In further studies for the first time, we demonstrated by the IHC technique the presence of the GalR1-3 receptors in the CRC tumors and the colon mucosa and found that higher GalR3-Ir in the tumor tissue correlated with longer overall survival of CRC patients. Furthermore, we discovered that lower GalR1 expression in submucosal plexuses located near the tumor correlated with a better prognosis in patients with CRC. These findings suggest that GalR1 could be considered as a novel therapeutic target in CRC. In conclusion, our morphological investigations provided novel data documenting the involvement of Gal and its receptors in the progression of CRC and showed the usefulness of the IHC technique for the prognosis of CRC patients.

High-Dose Fenofibrate Stimulates Multiple Cellular Stress Pathways in the Kidney of Old Rats.

We investigated the age-related effects of the lipid-lowering drug fenofibrate on renal stress-associated effectors. Young and old rats were fed standard chow with 0.1% or 0.5% fenofibrate. The kidney cortex tissue structure showed typical aging-related changes. In old rats, 0.1% fenofibrate reduced the thickening of basement membranes, but 0.5% fenofibrate exacerbated interstitial fibrosis. The PCR array for stress and toxicity-related targets showed that 0.1% fenofibrate mildly downregulated, whereas 0.5% upregulated multiple genes. In young rats, 0.1% fenofibrate increased some antioxidant genes' expression and decreased the immunoreactivity of oxidative stress marker 4-HNE. However, the activation of cellular antioxidant defenses was impaired in old rats. Fenofibrate modulated the expression of factors involved in hypoxia and osmotic stress signaling similarly in both age groups. Inflammatory response genes were variably modulated in the young rats, whereas old animals presented elevated expression of proinflammatory genes and TNFα immunoreactivity after 0.5% fenofibrate. In old rats, 0.1% fenofibrate more prominently than in young animals induced phospho-AMPK and PGC1α levels, and upregulated fatty acid oxidation genes. Our results show divergent effects of fenofibrate in young and old rat kidneys. The activation of multiple stress-associated effectors by high-dose fenofibrate in the aged kidney warrants caution when applying fenofibrate therapy to the elderly.

Impaired Expression of the Salvador Homolog-1 Gene Is Associated with the Development and Progression of Colorectal Cancer.

Salvador homolog-1 (SAV1) is a component of the Hippo pathway that regulates tissue growth and homeostasis by affecting diverse cell processes, including apoptosis, cell division, and differentiation. The aberrant expression of Hippo pathway components has been observed in various human cancers. This study aimed to examine the expression level of the SAV1 gene in colorectal cancer (CRC) and its prognostic value and associations with tumor progression. We obtained matched pairs of tumor tissue and non-cancerous mucosa of the large intestine from 94 CRC patients as well as 40 colon biopsies of healthy subjects collected during screening colonoscopy. The tissue samples and CRC cell lines were quantified for SAV1 mRNA levels using the quantitative polymerase chain reaction method, while SAV1 protein expression was estimated in the paired tissues of CRC patients using immunohistochemistry. The average level of SAV1 mRNA was decreased in 93.6% of the tumor tissues compared to the corresponding non-cancerous tissues and biopsies of healthy colon mucosa. A downregulated expression of SAV1 mRNA was also noted in the CRC cell lines. Although the average SAV1 immunoreactivity was increased in the CRC samples compared to the non-cancerous tissues, a decreased immunoreactivity of the SAV1 protein in the tumor specimens was associated with lymph node involvement and higher TNM disease stage and histological grade. The results of our study suggest that the impaired expression of SAV1 is involved in CRC progression.

The immunoreactivity of GLI1 and VEGFA is a potential prognostic factor in kidney renal clear cell carcinoma.

Kidney renal clear cell carcinoma (KIRC) is the most common type of kidney cancer and its pathogenesis is strongly associated with VHL-HIF-VEGF signaling. SHH ligand is the upstream SHH pathway regulator, while GLI1 is its major effector that stimulates as a transcription factor, i.a. expression of VEGFA gene. The aim of present study was to assess the prognostic significance of SHH, GLI1 and VEGFA immunoreactivity in KIRC tissues. The analysis included paired tumor and normal samples from 34 patients with KIRC. The immunoreactivity of SHH, GLI1 and VEGFA proteins was determined by immunohistochemical (IHC) renal tissues staining. The IHC staining results were assessed using the immunoreactive score (IRS) method which takes into account the number of cells showing a positive reaction and the intensity of the reaction. Increased GLI1 protein immunoreactivity was observed in KIRC tissues, especially in early-stage tumors, according to the TNM classification. Elevated expression of the VEGFA protein was noted primarily in high-grade KIRC samples according to the Fuhrman/WHO/ISUP scale. Moreover, a directly proportional correlation was observed between SHH and VEGFA immunoreactivity in TNM 3 + 4 and Fuhrman/ISUP/WHO 3 + 4 tumor tissues as well as in samples of patients with shorter survival. We also observed an association between shorter patient survival as well as increased and decreased immunoreactivity, of the VEGFA and GLI1, respectively. The aforementioned findings suggest that the expression pattern of SHH, GLI1 and VEGFA demonstrates prognostic potential in KIRC.

WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

BACKGROUND: The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). OBJECTIVE: The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. METHODS: WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). RESULTS: WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. CONCLUSIONS: There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

Cholinergic signaling influences the expression of immune checkpoint inhibitors, PD-L1 and PD-L2, and tumor hallmarks in human colorectal cancer tissues and cell lines.

BACKGROUND: Cancer cells express immunosuppressive molecules, such as programmed death ligands (PD-L)1 and PD-L2, enabling evasion from the host's immune system. Cancer cells synthesize and secrete acetylcholine (ACh), acting as an autocrine or paracrine hormone to promote their proliferation, differentiation, and migration. METHODS: We correlated the expression of PD-L1, PD-L2, cholinergic muscarinic receptor 3 (M3R), alpha 7 nicotinic receptor (α7nAChR), and choline acetyltransferase (ChAT) in colorectal cancer (CRC) tissues with the stage of disease, gender, age, risk, and patient survival. The effects of a muscarinic receptor blocker, atropine, and a selective M3R blocker, 4-DAMP, on the expression of immunosuppressive and cholinergic markers were evaluated in human CRC (LIM-2405, HT-29) cells. RESULTS: Increased expression of PD-L1, M3R, and ChAT at stages III-IV was associated with a high risk of CRC and poor survival outcomes independent of patients' gender and age. α7nAChR and PD-L2 were not changed at any CRC stages. Atropine and 4-DAMP suppressed the proliferation and migration of human CRC cells, induced apoptosis, and decreased PD-L1, PD-L2, and M3R expression in CRC cells via inhibition of EGFR and phosphorylation of ERK. CONCLUSIONS: The expression of immunosuppressive and cholinergic markers may increase the risk of recurrence of CRC. These markers might be used in determining prognosis and treatment regimens for CRC patients. Blocking cholinergic signaling may be a potential therapeutic for CRC through anti-proliferation and anti-migration via inhibition of EGFR and phosphorylation of ERK. These effects allow the immune system to recognize and eliminate cancer cells.

Pro-Inflammatory Cytokine Gene Expression in Penile Cancer: Preliminary Studies.

Background and Objectives: Penile cancer is a rare neoplasm in developed countries with an incidence of 0.8/100,000 per male inhabitant. Despite the development of personalized medicine and multimodal treatment, the outcome of penile cancer treatment is insufficient. Our study aimed to assess the levels of pro-inflammatory cytokines' mRNA such as interleukin 1-A (encoded by IL1A gene, alias IL-1A), interleukin 1-B (IL1B, IL-1B), interleukin 1 receptor antagonist (IL1RN, IL-1RN), interleukin 6 (IL6, IL-6), transforming growth factor ß1 (TGFB1, TGFß-1), and Interferon-gamma (INFG, INF-γ) in penile cancer tissue and associate them with tumor progression and patient survival. Material and Methods: Skin biopsies from patients suffering from penile cancer (n = 6) and unchanged foreskin from 13 healthy adult males undergoing circumcision due to a short frenulum were obtained. Pro-inflammatory cytokine mRNA levels were quantified through qPCR. Results: We observed higher expression of pro-inflammatory cytokine genes (IL-1A, IL-1B, IL-6, INF-γ, TGF-ß) in penile cancer tissue. The average follow-up period was 48 months (range: 38-54 months), during which only one penile tumor progression was observed However, this was without association with the nature of tumor (patient refused radical treatment). Conclusions: This is the first study to show increased expression of cytokines such as IL-1A, IL-1B, IL-6, INF-γ, and TGF-ß in penile cancer with positive correlation between TNM staging and INF-γ levels in tumor samples (rs = 0.672, p = 0.045), which may be associated with the immunosuppressive role of the tumor environment.

Anti-Inflammatory Klotho Protein Serum Concentration Correlates with Interferon Gamma Expression Related to the Cellular Activity of Both NKT-like and T Cells in the Process of Human Aging.

Klotho is a beta-glucuronidase that reveals both anti-inflammatory and anti-oxidative properties that have been associated with mechanisms of aging. The study aimed to analyze the relationships between the serum concentration of soluble α-Klotho and cellular activity of two populations of lymphocytes; T and NKT-like cells corresponding to the level of cytokine secretion; i.e., IFN-γ, TNF-α, and IL-6. The studied population comprised three age groups: young individuals ('young'), seniors aged under 85 ('old'), and seniors aged over 85 ('oldest'). Both NKT-like and T cells were either non-cultured or cultured for 48 h and stimulated appropriately with IL-2, LPS or PMA with ionomycin to compare with unstimulated control cells. In all studied age groups non-cultured or cultured NKT-like cells revealed higher expressions of TNF-α, IL-6, and IFN-γ than T cells. α-Klotho concentration in serum decreased significantly in the process of aging. Intriguingly, only IFN-γ expression revealed a positive correlation with α-Klotho protein serum concentration in both non-cultured and cultured T and NKT-like cells. Since IFN-γ is engaged in the maintenance of immune homeostasis, the observed relationships may indicate the involvement of α-Klotho and cellular IFN-γ expression in the network of adaptive mechanisms developed during the process of human aging.

Age-related effects of fenofibrate on the hepatic expression of sirtuin 1, sirtuin 3, and lipid metabolism-related genes.

BACKGROUND: Sirtuin 1 (Sirt1) and sirtuin 3 (Sirt3) participate in the regulation of lipid metabolism. Our aim was to investigate the effects of the hypolipemic drug fenofibrate (FN) on hepatic Sirt1 and Sirt3 expression, in relation to the expression of lipid metabolism-related genes and in the context of aging. METHODS AND RESULTS: Young and old male Wistar rats were fed standard chow or supplemented with 0.1% or 0.5% FN for 30 days (n=7-10 in each group). In young rats, 0.1% FN did not affect Sirt1 expression, however, 0.5% FN decreased Sirt1 and both doses reduced Sirt3 protein levels. In old rats, 0.5% FN decreased hepatic Sirt1 mRNA and both doses reduced Sirt1 protein levels, but not Sirt3 expression. Although hepatic Pparα protein levels did not change, FN treatment of young rats induced Cpt1b expression, whereas Lcad, Acox1, Pmp70, and Hmgcs2 expression increased only after 0.1% FN, and Fas2 expression decreased after 0.5% FN. In the liver of old rats, both doses increased Cpt1b and Lcad expression. Only 0.1% FN increased Pmp70 and Hmgcs2 expression, and only 0.5% FN increased Acox1 and Fas2 mRNA levels. CONCLUSIONS: Treatment with fenofibrate at low or high doses may downregulate the expression of Sirt1 and Sirt3 proteins in the rat liver. The dosage of FN affects molecular changes, and aging alters the response to 0.5% FN.

Galanin Receptors (GALR1, GALR2, and GALR3) Immunoexpression in Enteric Plexuses of Colorectal Cancer Patients: Correlation with the Clinico-Pathological Parameters.

Galanin (GAL) is an important neurotransmitter released by the enteric nervous system (ENS) neurons located in the muscularis externa and submucosa enteric plexuses that acts by binding to GAL receptors 1, 2 and 3 (GALR1, 2 and 3). In our previous studies, the GAL immunoexpression was compared in colorectal cancer (CRC) tissue and the adjacent parts of the large intestine wall including myenteric and submucosal plexuses. Recently we have also found that expression levels of GALR1 and GALR3 proteins are elevated in CRC tissue as compared with their expression in epithelial cells of unchanged mucosa. Moreover, higher GALR3 immunoreactivity in CRC cells correlated with better prognosis of CRC patients. To understand the distribution of GALRs in enteric plexuses distal and close to CRC invasion, in the present study we decided to evaluate GALRs expression within the myenteric and submucosal plexuses located proximally and distally to the cancer invasion and correlated the GALRs expression levels with the clinico-pathological data of CRC patients. The immunohistochemical and immunofluorescent methods showed only slightly decreased immunoexpression of GALR1 and GALR3 in myenteric plexuses close to cancer but did not reveal any correlation in the immunoexpression of all three GAL receptors in myenteric plexuses and tumour progression. No significant changes were found between the expression levels of GALRs in submucosal plexuses distal and close to the tumour. However, elevated GALR1 expression in submucosal plexuses in vicinity of CRC correlated with poor prognosis, higher tumour grading and shorter overall survival. When myenteric plexuses undergo morphological and functional alterations characteristic for atrophy, GALRs maintain or only slightly decrease their expression status. In contrast, the correlation between high expression of GALR1 in the submucosal plexuses and overall survival of CRC patients suggest that GAL and GALRs can act as a components of local neuro-paracrine pro-proliferative pathways accelerating the invasion and metastasis of cancer cell. The obtained results suggest an important role of GALR1 in submucosal plexuses function during the progression of CRC and imply that GALR1 expression in submucosal plexuses of ENS could be an important predictive factor for CRC progression.

Altered mRNA Expression of NFKB1 and NFKB2 Genes in Penile Lichen Sclerosus, Penile Cancer and Zoon Balanitis.

BACKGROUND: The nuclear factor-κB transcription factors 1 and 2 (NFKB1 and NFKB2) are key components of the NF-κB pathway, which responds to inflammatory signals. Since the NFKB1/2 factors are activated via different inflammatory molecules, we aimed to check their expression levels in penile cancer (PC), penile dermatoses: lichen sclerosus (PLS) and zoon balanitis (ZB). METHODS: Skin biopsies from altered and healthy looking foreskin were obtained from 59 (49 LS; early PLS: 13, moderate PLS: 32, severe PLS: 4; 6 PC; 4 ZB) and unchanged foreskin from 13 healthy control adult males undergoing circumcision. NFKB1/2 mRNA levels were quantified by qPCR. RESULTS: The highest levels of NFKB1 and NFKB2 were observed in PC, ca. 22 and 3.5 times higher than in control, respectively. NFKB1 expression was correlated with PLS progression (rs = 0.667) and was ca. 20 times higher in advanced PLS than in controls and early PLS. Occurrence of micro-incontinence was associated with elevated NFKB1 levels in PLS. CONCLUSION: This is the first study regarding gene profiles of NFKB1/2 in PC and penile dermatoses. New drugs targeting modulation of canonical-activated NF-κB pathway should be studied and introduced to the treatment of PLS and PC apart from other treatments.

Aging and short-term calorie restriction differently affect the cardiac and skeletal muscle expression of genes regulating energy substrate utilization in male rats.

Aging affects the energy metabolism differently in the cardiac and skeletal muscles. The study aim was to assess the effects of short-term calorie restriction (SCR) and refeeding on the expression of genes involved in the control of cardiac and skeletal muscle energy metabolism in old vs. young male rats. Young (4 mo) and old (24 mo) rats were subjected to 60% SCR for 30 days, and refed ad libitum for 2 or 4 days. In the cardiac (CM) and skeletal muscles (SM) we compared the gene expression (qPCR) of carnitine palmitoyltransferase-I (Cpt-I), peroxisome proliferator-activated receptor beta/delta (Ppar-ß/δ), glucose transporter 4 (Glut4), peroxisome proliferator-activated receptor-γ coactivator-1α (Pgc-1α), and sirtuin 3 (Sirt3). In CM, aging increased Cpt-I expression but did not affect the other genes. In SM, Cpt-I, Glut4, Pgc-1α, and Sirt3 mRNA levels were lower in old than young rats. In CM of only young rats SCR increased Cpt-I expression which remained elevated after refeeding. Upon SCR, the expression of Ppar-ß/δ, Glut4, Pgc-1α, and Sirt3 in CM increased in young but not old rats, and refeeding re-established control levels. In SM of young rats SCR increased Ppar-ß/δ and Pgc-1α, and decreased Sirt3 expression, whereas refeeding generally decreased these mRNA levels. In SM of old rats SCR decreased only Pgc-1α expression. The adaptive response to SCR and subsequent refeeding is muscle tissue-specific and differs in young and old male rats. SCR appears to increase the efficiency of glucose and fatty acid utilization in the cardiac muscle of young, but not old male rats.

Galanin Receptors (GalR1, GalR2, and GalR3) Expression in Colorectal Cancer Tissue and Correlations to the Overall Survival and Poor Prognosis of CRC Patients.

Colorectal cancer (CRC) is the second most common cause of cancer in women and the third in men. The postoperative pathomorphological evaluation of patients with CRC is extremely important for future therapeutic decisions. Although our previous studies demonstrated high galanin (GAL) presence within tumor tissue and an elevated concentration of GAL in the serum of CRC patients, to date, there is a lack of data regarding GAL receptor (GalR) protein expression in CRC cells. Therefore, the aim of this study was to evaluate the presence of all three types of GalRs (GalR1, GalR2 and GalR3) within epithelial cells of the human colon and CRC tissue with the use of the immunohistochemical method and to correlate the results with the clinical-pathological data. We found stronger immunoreactivity of GalR1 and GalR3 in CRC cells compared to epithelial cells of the unchanged mucosa of the large intestine. No differences in the GalR2 protein immunoreactivity between the studied tissues were noted. We also found that the increased immunoexpression of the GalR3 in CRC tissue correlated with the better prognosis and longer survival (p < 0.0079) of CRC patients (n = 55). The obtained results suggest that GalR3 may play the role of a prognostic factor for CRC patients. Based on data from the TCGA-COAD project deposited in the GDC Data Portal, we also found that GalR mRNA in cancer samples and the adjacent normal tissue did not correlate with immunoexpression of the GalR proteins in CRC cells and epithelial cells of the unchanged mucosa.

NKT-like (CD3 + CD56+) cells differ from T cells in expression level of cellular protective proteins and sensitivity to stimulation in the process of ageing.

BACKGROUND: NKT-like cells are T lymphocytes coexpressing several NK cell-associated receptors. They are effector lymphocytes of innate and adaptive immunity, and their number increases with age. The study aimed to analyze the expression of cellular protective proteins, i.e. sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in NKT-like and T cells of the young ('young', 31 subjects, age range 19-24 years), seniors aged under 85 ('old'; 30 subjects, age range 65-84 years) and seniors aged over 85 ('oldest', 24 subjects, age range 85-94 years). Both NKT-like and T cells were cultured for 48 h and stimulated with IL-2, LPS and PMA with ionomycin and compared with unstimulated control cells. RESULTS: The oldest seniors varied from the other age groups by significantly increased expression of SIRT1 and HSP70 in both NKT-like and T cells observed in both stimulated and nonstimulated cells. The analyzed lymphocyte populations of the oldest revealed not only the highest expression of these proteins but also insensitivity to all types of applied stimulation. When NKT-like cells were compared to T cells, higher expression of the studied protective proteins was observed in both stimulated and unstimulated NKT-like cells. Neither CD3 + CD56+ nor CD3+ cells revealed elevated expression of SOD2, and these cells responded to stimulation until very advanced age. T cells revealed higher sensitivity to stimulation with IL-2 regarding SIRT1 and HSP70 expression. NKT-like cells were more sensitive to stimulation with PMA and ionomycin concerning the expression of these proteins. IL-2 did not induce a significant increase in SOD2 expression in the studied age groups. CONCLUSIONS: The oldest seniors developed an adaptive stress response in both T and NKT-like cells regarding the expression of SIRT1 and HSP70, which was increased and insensitive to further stimulation in contrast to SOD2, which showed a more inducible pattern of expression. CD3 + CD56+ cells exhibited higher expression of cellular protective proteins than CD3+ cells in both stimulated and control, nonstimulated cells. NKT-like and T cells showed a distinct sensitivity to the applied stimulatory factors in the respective age groups.

Expression levels of sonic hedgehog pathway genes and their targets are upregulated in early clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU­SKI43, cyclopamine and GLI­antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786­O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription­quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of SHH, smoothened (SMO), glioma­associated zinc finger protein (GLI)1­3, BCL2 apoptosis regulator (BCL2), MYC proto­oncogene (MYC), vascular endothelial growth factor A (VEGFA) and cyclin D1 (CCND1) were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of VEGFA, as well as low CCND1 mRNA expression, were associated with short overall survival, and increased VEGFA expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786­O cells in the G2/M phase and decreased the expression levels of GLI1, BCL2, VEGFA and CCND1. RU­SKI43 inhibited cell migration and decreased the expression levels of BCL2, MYC and CCND1 in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of CCND1 and VEGFA may serve as prognostic factors of this disease. Cyclopamine and RU­SKI43 appear to be potential anti­renal cell carcinoma drugs; however, this hypothesis requires verification by further in vivo studies.

Sestrins' Expression in the Liver Is Not Altered by Short-Term Calorie Restriction in Young and Old Rats.

INTRODUCTION: Short-term calorie restriction (SCR) may have a positive impact on health. We hypothesized that sestrins, a family of stress-inducible proteins (Sesn1, Sesn2, Sesn3) are involved in the response to SCR in the liver. METHODS: Young-adult (4-month) and old (24-month) male Wistar rats were either fed ad libitum (control groups) or received 60% of food intake on a daily basis for 30 days (SCR groups). In blood sera, biochemical parameters and fibroblast growth factor 21 (FGF21) concentration were measured (ELISA). Liver samples were collected for analyses of genes' expression (real-time PCR) and protein levels (Western blotting). RESULTS: SCR caused improvements in blood glucose and lipids and parameters of liver function but did not affect the serum FGF21 concentration. SCR caused changes typically associated with calorie restriction in the gene expression of fatty acid synthase (fasn), ATP-citrate lyase (acly), and sirtuin 1 (sirt1). In the liver of young SCR rats, protein level of Sesn2 tended to increase, while Sesn3 tended to decrease, accompanied by reduced sesn3 expression. In old SCR rats, reduced sesn1 expression was reflected by decreasing trend for Sesn1 content. Activation of AMP-activated protein kinase (phospho-Thr172) and protein content of peroxisome proliferator-activated receptor gamma coactivator 1-alpha were not affected by SCR. CONCLUSION: Sestrins' hepatic expression is only minimally affected by SCR in young and old rats. We propose that sestrins may not be a major effector of mild SCR in the liver of young or old rats.

Serum WNT4 protein as an indicator of chronic glomerulonephritis but not a marker of inflammatory cell infiltration and fibrosis: A preliminary study.

BACKGROUND: The WNT signaling pathway contributes to renal fibrosis, which is a hallmark of chronic kidney disease (CKD). Serum concentration of WNT4 could be used to monitor the kidney disease; however, no data have yet been published on the subject. OBJECTIVES: This study measures WNT4 protein in serum of CKD patients depending on the stage, type of nephropathy, the non-nephrotic (NNP) or nephrotic proteinuria (NP), inflammatory cell infiltration in kidney parenchyma (IIKP), interstitial fibrosis in biopsy and serum creatinine. We also evaluated the usefulness of the serum WNT4 as a marker of fibrosis and IIKP. MATERIAL AND METHODS: The WNT4 protein level in serum of CKD patients and healthy individuals was measured using enzyme-linked immunoassay (ELISA). Patients' blood biochemical profiles and kidney biopsies were evaluated with common laboratory methods. RESULTS: The serum level of WNT4 protein was higher in CKD patients (i) regardless of the underlying etiology and at early stages of disease; (ii) with lupus nephritis and Immunoglobulin A (IgA) nephropathy; (iii) without or with a small area of IIKP; and (iv) with a small area covered with fibrosis. No difference was observed between NNP and NP patients. The utility of serum WNT4 as a marker of IIKP and fibrosis was not confirmed. Negative correlations with total and low-density lipoprotein (LDL)-cholesterol were found in CKD and IIKP patients. In patients with serum WNT4 above the median value, serum creatinine was higher. However, no correlation between serum WNT4 and creatinine level was found. CONCLUSIONS: The observed increase in serum WNT4 protein in the early stages of CKD and in patients diagnosed with immune-mediated glomerular disease may suggest that WNT4 may act as a mediator of inflammation. A certain association with the dysregulation of serum lipid metabolism can also be suspected. Serum WNT4 protein may be considered as the indicator of chronic glomerulonephritis, but not a diagnostic marker of IIKP and fibrosis.

Piceatannol, a Structural Analog of Resveratrol, Is an Apoptosis Inducer and a Multidrug Resistance Modulator in HL-60 Human Acute Myeloid Leukemia Cells.

Acute myeloid leukemia is characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells. Despite recent advances in the treatment of this disease, the prognosis and overall long-term survival for patients remain poor, which drives the search for new chemotherapeutics and treatment strategies. Piceatannol, a polyphenolic compound present in grapes and wine, appears to be a promising chemotherapeutic agent in the treatment of leukemia. The aim of the present study was to examine whether piceatannol induces autophagy and/or apoptosis in HL-60 human acute myeloid leukemia cells and whether HL-60 cells are able to acquire resistance to piceatannol toxicity. We found that piceatannol at the IC90 concentration of 14 µM did not induce autophagy in HL-60 cells. However, it induced caspase-dependent apoptosis characterized by phosphatidylserine externalization, disruption of the mitochondrial membrane potential, caspase-3 activation, internucleosomal DNA fragmentation, PARP1 cleavage, chromatin condensation, and fragmentation of cell nuclei. Our findings also imply that HL-60 cells are able to acquire resistance to piceatannol toxicity via mechanisms related to MRP1 activity. Our results suggest that the use of piceatannol as a potential chemotherapeutic agent may be associated with the risk of multidrug resistance, warranting its use in combination with other chemotherapeutic agents.

Short-term fenofibrate treatment improves ultrastructure of hepatocytes of old rats.

INTRODUCTION: Fenofibrate (FN) is a hypolipemic drug used for the treatment of mixed dyslipidemia. Since in our previous study FN administration to young and old rats adversely affected the serum activity of liver marker enzymes, we decided to examine the effects of FN on liver ultrastructure of young and old animals. MATERIAL AND METHODS: Young and old rats were fed standard rodent chow supplemented with 0.1% FN for 30 days. Liver samples obtained from animals under full anesthesia were processed by routine methods to obtain ultrathin and histological sections for the examination by light microscopy (LM) and transmission electron microscopy (TEM). Furthermore, liver lysates were analyzed by Western blotting for the expression of the autophagy-related proteins LC3A/B and beclin 1. RESULTS: The ultrastructure of hepatocytes in both age groups was well-preserved, with the presence of abundant mitochondria, numerous peroxisomes and lysosomes, glycogen stored in the form of rosettes, and occasionally autolysosomes. However, hepatocytes of old control rats contained less mitochondria and peroxisomes, and more lipid droplets than cells of young animals. The effects of FN on liver ultrastructure were age-depended. FN increased the relative number of mitochondria and peroxisomes in the hepatocytes of old, and did not affect their number in young rats. Moreover, FN decreased and increased the relative number of lipid droplets in the hepatocytes of old and young rats, respectively. At the LM level, Oil Red O staining revealed smaller and larger lipid droplets within hepatocytes and non-parenchymal liver cells. In the livers of young and old rats lipid droplets were distributed mainly in the periportal zones of hepatic lobules. Morphometric analysis confirmed that livers of control old rats contained more lipid-stainable areas than those of young ones; however, no effect of FN was observed either in young or old rats. Despite larger size of autolysosomes and autophagic vacuoles in hepatocytes of old rats, the expression of autophagy-related proteins did not differ in the livers of control and fenofibrate-treated young and old animals. CONCLUSIONS: The results of our study suggest that fenofibrate, apart from its hypolipemic action, may have beneficial effect on the energy metabolism in the liver of old individuals by increasing the number of mitochondria and peroxisomes in hepatocytes.