Małgorzata Kapral

Molecular targets of metformin antitumor action

Epidemiological studies have shown that metformin, a first line therapeutic agent for diabetes mellitus, reduced the risk of developing various malignancies. Several preclinical studies established some possible mechanisms of its anticancer effects. The primary effect of metformin action is a decrease in cell energy status, which activates AMP-activated kinase (AMPK), a cellular metabolic sensor. This event is followed by a decrease in serum concentrations of insulin and insulin growth factor I (IGF-I), the potent mitogens for cancer cells. In addition to the indirect mode of action, metformin may exhibit direct inhibitory effect on cancer cells by targeting mammalian target of rapamycin (mTOR) signaling and anabolic processes. This review gathers information on mechanisms of metformin antitumor activity, with special attention given to the impact of this antidiabetic drug on insulin/PI3K/mTOR and AMPK signaling. Furthermore, the factors required for this novel activity of metformin are discussed.

A microarray study of gene expression profiles in nasal polyps

OBJECTIVE Nasal polyposis (NP) is a multifactorial disease manifesting in chronic inflammation of upper respiratory tract of unknown etiology. We studied mRNA gene expression profiles in NP compared with normal mucosa as well as pointed at genes characteristic of different expression in examined tissues. MATERIAL AND METHODS Fifty-three patients with NP (36 eosinophilic and 17 neutrophilic NP) were included into the study. Transcriptional activity of genes was analyzed using oligonucleotide microarray in 17 NP and 8 cases of normal nasal mucosa. A study of mRNA expression of selected genes was performed using QRT-PCR. RESULTS We identified 556 genes, which were differentially expressed between the studied and the control group. Among them 217 showed significantly higher expression, whereas 339 lower expression in NP than in controls. The microarray and QRT-PCR results were compatible for 7 of 8 evaluated genes. In NP strongly significant higher transcriptional activity of MMP10, NOS2A, ALOX15 and IL-8 genes was observed. In the control group, significantly higher expression of DMBT1, ALOX12 and LTF genes was detected. CONCLUSION The analysis of gene expression in inflammatory changed nasal polyp tissues may become a supplementary method in diagnostics and treatment. Molecular alterations may indicate changes during the clinical course of the disease.

Transforming growth factor β isoforms (TGF-β1, TGF-β2, TGF-β3) messenger RNA expression in laryngeal cancer

PURPOSE Cancerogenesis is a multistage process controlled by many cytokines, including growth factors. The aim of the study was the comparison of transcriptional activity of transforming growth factor beta (TGF-beta) genes in laryngeal squamous cell carcinomas and adjacent nonneoplastic tissues. MATERIALS AND METHODS Tissues samples were obtained from 32 patients with laryngeal squamous cell carcinoma in histologic grades G1 to G3 who underwent surgical treatment at the ENT Clinics of Medical University of Silesia in Katowice, Poland. Quantification of gene expression was performed by real-time quantitative reverse transcriptase polymerase chain reaction technique. RESULTS In tumor cells, expression of TGF-beta1 and TGF-beta2 isoforms (P < .001) was higher than in normal tissues. There was a positive correlation between the expression of TGF-beta1 and TGF-beta2 genes in tumors (R = 0.78, P = .0000) and adjacent normal tissues (R = 0.77, P = .0000). CONCLUSIONS The results suggest that TGF-beta1 and TGF-beta2 messenger RNAs may be useful as molecular markers in distinguishing cancer from nonneoplastic tissues in laryngeal area.

Zmiany potencjału proliferacyjnego fibroblastów pochodzących z polipów nosowych, w hodowlach in vitro, pod wpływem pochodnych wit. D

INTRODUCTION Recurrent polyposis in the same patient resulting in the necessity of repeated surgeries forced to search for new pharmacological therapeutic methods. At present, locally acting glycocorticosteroids have the greatest value in the treatment of nasal polyposis. Polyps grow is connected with inflammation process and proliferation of fibroblasts. OBJECTIVE An evaluation of calcitriol and tacalcitol influence on proliferation of fibroblasts extracted from nasal polyps. MATERIAL consisted of 9 tissue samples coming from nasal polyps sampled during polypectomies. The testing was performed on the polyps fibroblasts after the sixth passage after the primary culture was established. Three days after the culture was started the cells were poured with nutrient medium without serum added and after further 24 hours was replaced by nutrient medium with takalcitol and calcitriol in the defined concentrations. The expression of the genes coding histone H3 was evaluated with the use of RT-PCR technique. RESULTS Tacalcitiol and calcitriol in vitro decrease proliferation of fibroblasts sampled from nasal polyps. Inhibition is most effective for the concentration of 10-4M. Tacalcitiol and calcitriol also inhibit level of histone H3 gene expression. CONCLUSION Experimental data suggest tacalcitiol to be more effective in the same concentration. Present studies may indicate the direction of further investigation in the potential pharmacological treatment on nasal polyps.Summary Introduction Recurrent polyposis in the same patient resulting in the necessity of repeated surgeries forced to search for new pharmacological therapeutic methods. At present, locally acting glycocorticosteroids have the greatest value in the treatment of nasal polyposis. Polyps grow is connected with inflammation process and proliferation of fibroblasts. Objective An evaluation of calcitriol and tacalcitol influence on proliferation of fibroblasts extracted from nasal polyps. Material consisted of 9 tissue samples coming from nasal polyps sampled during polypectomies. The testing was performed on the polyps fibroblasts after the sixth passage after the primary culture was established. Three days after the culture was started the cells were poured with nutrient medium without serum added and after further 24 hours was replaced by nutrient medium with takalcitol and calcitriol in the defined concentrations. The expression of the genes coding histone H3 was evaluated with the use of RT-PCR technique. Results Tacalcitiol and calcitriol in vitro decrease proliferation of fibroblasts sampled from nasal polyps. Inhibition is most effective for the concentration of 10-4M. Tacalcitiol and calcitriol also inhibit level of histone H3 gene expression. Conclusion Experimental data suggest tacalcitiol to be more effective in the same concentration. Present studies may indicate the direction of further investigation in the potential pharmacological treatment on nasal polyps.

Modulating effect of inositol hexaphosphate on arachidonic acid-dependent pathways in colon cancer cells

Cyclooxygenase (COX) and lipoxygenase (LOX) are key enzymes of arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in the pathogenesis of inflammatory bowel diseases and colorectal cancer. The aim of the study was to examine the influence of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the expression of genes encoding COX and LOX isoforms and synthesis of their products (PGE2 and LTB4) in colon cancer cell line Caco-2 stimulated with pro-inflammatory agents (IL-1β/TNFα). Real-time RT-qPCR was used to validate mRNAs level of examined genes. The concentrations of COX-2 and 5-LOX proteins as well as PGE2 and LTB4 were determined by the ELISA method. Based on these studies it may be concluded that IP6 may limit inflammatory events in the colonic epithelium and prevent colon carcinomas by modulating the expression of genes encoding COX and LOX isoforms at both mRNA and protein levels as well as by affecting the synthesis and secretion of prostaglandins and leukotrienes.

Inositol Hexaphosphate Inhibits Proliferation and Induces Apoptosis of Colon Cancer Cells by Suppressing the AKT/mTOR Signaling Pathway

AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR) plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6), a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM). Cellular proliferative activity was monitored by 5-bromo-2′-deoxyuridine (BrdU) incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in colon cancer cells.

Zróżnicowanie polipów nosa w badaniach techniką mikromacierzy oligonukleotydowych

UNLABELLED Nasal polyps, according to many authors, generate as a result of chronic inflammation process with activation of cytokines, immunological reaction mediators that regulate proliferation, differentiation and cell apoptosis. Clarifying molecular mechanisms present in those disturbances may have diagnostic and prognostic value in evaluation of recurrence, dynamics and differentiation of nasal polyps as well as in their therapy. AIM The aim of the work was an analysis of nasal polyps on the basis of molecular, histopathological and clinical picture as well as comparing differentiated genes transcription in nasal polyps and proper nasal mucosa. MATERIAL AND METHOD Oligonucleotide array with HGU 133A - Affymetrix were used to analyze the expression of 22,283 genes in nasal polyp tissues from 17 patients. The control group consisted of 8 tissue samples from patients after nasal septoplasty surgery. RESULTS All the samples could be classified to nasal polyps group or proper mucosa group, it reflected significant differences in genes profile expression in both groups. The evaluation of 22,283 genes transcriptions showed that in most cases nasal polyps tissue reflect classification connected with dominant inflammation cells infiltration. The data obtained let distinguish subgroups connected with clinical condition of the patients. The subgroup with massive nasal and sinus polyposis, eosinophilia and differentiated lower respiratory airways hyperactivity and the subgroup without eosinophilia infiltration may be distinguished. The data obtained suggest that molecular mechanisms may influence on the promotion and kind of inflammation process as well as the clinical course of nasal polyps.Summary Nasal polyps, according to many authors, generate as a result of chronic inflammation process with activation of cytokines, immunological reaction mediators that regulate proliferation, differentiation and cell apoptosis. Clarifying molecular mechanisms present in those disturbances may have diagnostic and prognostic value in evaluation of recurrence, dynamics and differentiation of nasal polyps as well as in their therapy. Aim The aim of the work was an analysis of nasal polyps on the basis of molecular, histopathological and clinical picture as well as comparing differentiated genes transcription in nasal polyps and proper nasal mucosa. Material and method Oligonucleotide array with HGU 133A – Affymetrix were used to analyze the expression of 22 283 genes in nasal polyp tissues from 17 patients. The control group consisted of 8 tissue samples from patients after nasal septoplasty surgery. Results All the samples could be classified to nasal polyps group or proper mucosa group, it reflected significant differences in genes profile expression in both groups. The evaluation of 22 283 genes transcriptions showed that in most cases nasal polyps tissue reflect classification connected with dominant inflammation cells infiltration. The data obtained let distinguish subgroups connected with clinical condition of the patients. The subgroup with massive nasal and sinus polyposis, eosinophilia and differentiated lower respiratory airways hyperactivity and the subgroup without eosinophilia infiltration may be distinguished. The data obtained suggest that molecular mechanisms may influence on the promotion and kind of inflammation process as well as the clinical course of nasal polyps.

Differential Influence of Inositol Hexaphosphate on the Expression of Genes Encoding TGF-β Isoforms and Their Receptors in Intestinal Epithelial Cells Stimulated with Proinflammatory Agents

Transforming growth factor β (TGF-β) is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the mRNA expression of TGF-β1, TGF-β2, TGF-β3 and TβRI, TβRII, and TβRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1β in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-βs and their receptors in a time-dependent manner. IL-1β upregulated mRNA levels of all TGF-βs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF-β1 at 12 h. IP6 counteracted the stimulatory effect of IL-1β on TGF-β1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1β-stimulated mRNA expression of TGF-β2 and -β3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-βs and their receptors at transcriptional level.

Profil ekspresji genów kodujących TGF-β1 i jego receptory TGFβRI, TGFβRII i TGFβRIII w polipach nosa☆☆☆◊◊◊

Summary Background Transforming growth factor β (TGF-β) plays an important role in cells proliferation and differentiation as well as in local immunological response. Objectives An evaluation of genes expression profile for TGF-β1 and its receptors TGF-βRI, TGF-β RII and TGF-β βRIII as well as their potential role in the pathogenesis of nasal polyps in eosynophilic and neutrophilic polyps and in normal nasal mucosa. Material Material consisted of 22 patients. Nasal polyps were removed during standard polypectomy or FESS. In the histopathological evaluation there were 16 eosynophilic polyps and 5 neutrophilic ones. The control group consisted of 8 healthy patients from whom healthy nasal mucosa was taken during nasal septoplasty. Methods The expression of the genes coding TGF-β and its receptors was evaluated with the use of RT-PCR technique. Results TGF-β1 mRNA was present in 10 eosynophilic polyps out of 16. In neutrophilic polyps group (n = 6) mRNA TGFβ-1 was present in 3 samples. TGFβ-1 isoform was present in all the tissues of the control group. It was significantly larger expression of TGFβ-1 gene in normal mucosa in comparison with eosinophilic and neutrophilic polyps (p Conclusions Considering regulative function of TGFβ1 in inflammation processes, its low concentration in nasal polyps tissue may influence on migration and survival of inflammation cells. The high expression of genes coding TGFβRI, TGF-βRII and TGF-βRIII receptors in all the polyps and healthy tissues, show readiness to transduction of TGFβ. It may suggest that, less intensive TGFβ1 expression in nasal polyps may be connected with the presence of other than first TGFβ isoforms. This problem needs further investigations to set precise role of individual TGFβ isoforms and other growth factors in the pathogenesis of NSP as their interactions with local cytokines. It may help to work out more effective and specific therapeutic methods in nasal polyps therapy.