Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance

Upregulation of the MDR1 (multidrug resistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a beta-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H(2)O(2), whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position -296 to -260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H(2)O(2) response element (HRE), is situated at position -561 to -520. The HRE is required for H(2)O(2)-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H(2)O(2). Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H(2)O(2)-dependent transcription of MDR1. A minimal BRE (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity

Early Expression of Yeast Genes Affected by Chemical Stress

The variety of environmental stresses is probably the major challenge imposed on transcription activators and the transcriptional machinery. To precisely describe the very early genomic response developed by yeast to accommodate a chemical stress, we performed time course analyses of the modifications of the yeast gene expression program which immediately follows the addition of the antimitotic drug benomyl. Similar analyses were conducted with different isogenic yeast strains in which genes coding for relevant transcription factors were deleted and coupled with efficient bioinformatics tools. Yap1 and Pdr1, two well-known key mediators of stress tolerance, appeared to be responsible for the very rapid establishment of a transient transcriptional response encompassing 119 genes. Yap1, which plays a predominant role in this response, binds, in vivo, promoters of genes which are not automatically up-regulated. We proposed that Yap1 nuclear localization and DNA binding are necessary but not sufficient to elicit the specificity of the chemical stress response

ALDH1A inhibition sensitizes colon cancer cells to chemotherapy

Abstract Background Recent evidence in cancer research, developed the notion that malignant tumors consist of different subpopulations of cells, one of them, known as cancer stem cells, being attributed many important properties such as enhanced tumorigenicity, proliferation potential and profound multidrug resistance to chemotherapy. Several key stem cells markers were identified in colon cancer. In our study we focused on the aldehyde dehydrogenase type 1 (ALDH1) expression in colon cancer-derived cell lines HT-29/eGFP, HCT-116/eGFP and LS-180/eGFP, and its role in the chemoresistance and tumorigenic potential. Methods The effect of pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde (DEAB) and also effect of molecular inhibition by specific siRNA was evaluated in vitro in cultures of human colorectal cell lines. The expression level of different isoenzymes of aldehyde dehydrogenase was determined using qPCR. Changes in cell biology were evaluated by expression analysis, western blot and apoptosis assay. The efficiency of cytotoxic treatment in the presence of different chemotherapeutic drugs was analyzed by fluorimetric assay. Tumorigenicity of cells with specific ALDH1A1 siRNA was tested in xenograft model in vivo. Results Treatment by DEAB partially sensitized the tested cell lines to chemotherapeutics. Subsequently the molecular inhibition of specific isoforms of ALDH by ALDH1A1 or ALDH1A3 siRNA led to sensitizing of cell lines HT-29/eGFP, HCT-116/eGFP to capecitabine and 5-FU. On the model of athymic mice we observed the effect of molecular inhibition of ALDH1A1 in HT-29/eGFP cells by siRNA. We observed inhibition of proliferation of subcutaneous xenografts in comparison to control cells. Conclusion This research, verifies the significance of the ALDH1A isoforms in multidrug resistance of human colorectal cancer cells and its potential as a cancer stem cell marker. This provides the basis for the development of new approaches regarding the treatment of patients with colorectal adenocarcinoma and potentially the treatment of other tumor malignancies

Napabucasin overcomes cisplatin resistance in ovarian germ cell tumor-derived cell line by inhibiting cancer stemness

Background: Cisplatin resistance of ovarian yolk sac tumors (oYST) is a clinical challenge due to dismal patient prognosis, even though the disease is extremely rare. We investigated potential association between cisplatin resistance and cancer stem cell (CSC) markers in chemoresistant oYST cells and targeting strategies to overcome resistance in oYST. Methods: Chemoresistant cells were derived from chemosensitive human oYST cells by cultivation in cisplatin in vitro. Derivative cells were characterized by chemoresistance, functional assays, flow cytometry, gene expression and protein arrays focused on CSC markers. RNAseq, methylation and microRNA profiling were performed. Quail chorioallantoic membranes (CAM) with implanted oYST cells were used to analyze the micro-tumor extent and interconnection with the CAM. Tumorigenicity in vivo was determined on immunodeficient mouse model. Chemoresistant cells were treated by inhibitors intefering with the CSC properties to examine the chemosensitization to cisplatin. Results: Long-term cisplatin exposure resulted in seven-fold higher IC50 value in resistant cells, cross-resistance to oxaliplatin and carboplatin, and increased migratory capacity, invasiveness and tumorigenicity, associated with hypomethylation of differentially methylated genes/promotors. Resistant cells exhibited increased expression of prominin-1 (CD133), ATP binding cassette subfamily G member 2 (ABCG2), aldehyde dehydrogenase 3 isoform A1 (ALDH3A1), correlating with reduced gene and promoter methylation, as well as increased expression of ALDH1A3 and higher overall ALDH enzymatic activity, rendering them cross-resistant to DEAB, disulfiram and napabucasin. Salinomycin and tunicamycin were significantly more toxic to resistant cells. Pretreatment with napabucasin resensitized the cells to cisplatin and reduced their tumorigenicity in vivo. Conclusions: The novel chemoresistant cells represent unique model of refractory oYST. CSC markers are associated with cisplatin resistance being possible targets in chemorefractory oYST