Background/Aims: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab’s therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. Methods: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab’s effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab’s administration and the analysis was determined by qRT-PCR. Results: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients’ blood before and 2 hours after the anti-TNF drug administration. Conclusion: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study.
Background: Autophagocytosis is a biological process involving the controlled distribution of cell fragments and organelles in order to obtain an additional source of energy. LAMP3 (lysosome-associated membrane protein 3) is a heavily glycosylated integral membrane protein located mainly in the lysosomal membrane. Recent studies have shown that it participates in tumor metastasis and drug resistance. Its main role is contribution to tumor cells proliferation, migration and invasion. The aim of this study was to determine changes in transcriptional activity of LAMP3 at various stages of colorectal cancer. In addition, an attempt was made to select miRNAs potentially regulating LAMP3 expression using bioinformatic databases. Methods: The study was conducted on healthy colon samples and colon cancer samples in four stages. Molecular analysis included the extraction of total RNA, purification of the obtained extracts, expression profile analysis using oligonucleotide microarray technique and in silico determination of miRNAs potentially regulating the LAMP3 expression. Results: The level of LAMP3 expression is higher in colorectal adenocarcinoma cells than in non-cancerous cells and depends on the stage of the disease. Conclusions: LAMP3 may promote cancer progression, metastasis and cause the resistance to treatment.
I N T R O D U C T I O N :The main limitation of the use of amphotericin B (AmB)effective in the treatment of systemic fungal infectionsis its high toxicity to human cells. The mechanism of AmB toxicity is not clear. Caspase-related and BCL-2 proteins participate in the regulation of apoptosis. Thus, they may be involved in drug toxicity. In this study we evaluated the influence of AmB on the transcriptional activity of genes related to caspases and the BCL-2 family. We also tested the influence of modified forms of AmB: AmB-Cu 2+ (the complex with copper(II) ions) and the AmB-ox (oxidized form). M A T E R I A L A N D M E T H O D S :Human RPTECs (Renal Proximal Tubule Epithelial Cells) were treated with AmB, AmB-Cu 2+ and AmB-ox. Total RNA was extracted using the phenol-chloroform method. The expression profiles of genes related to caspase activity and BCL-2 were determined using oligonucleotide microarrays (HG-U133A 2.0, Affymetrix). Analysis included 67 ID related to caspases and 32 ID associated with BCL-2, according to the Affymetrix database. R E S U L T S : The analysis revealed upregulation of the BCL-2 and BCL2L1genes in the cells treated with AmB-Cu 2+ , in comparison to the control. In both the AmB and AmB-Cu 2+ -treated cells, differentiating genes were associated with inflammation and mitophagy activated by intrinsic signals. In the cells treated with AmB-ox, the BCL-2 genes were downregulated. C O N C L U S I O N S :The results suggest that AmB and AmB-Cu 2+ activate genes involved in the regulation of inflammation and autophagy induced by intrinsic signals, but overexpression of BCL-2 and BCL2L1 may protect AmB-Cu 2+ --treated cells from death. In the cells treated with AmB-ox extrinsic signals prevail, indicating the distinct molecular mechanism of its cytotoxicity.
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