New Approach for Studying of Isoforms and High-Homology Proteins in Mammalian Cells.

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.

Antiproliferative effects of 13α/ß-steroids on triple-negative MDA-MB-231 breast cancer cells: unraveling intracellular signaling without ERα

Abstract This study aimed to investigate the activities of novel 20(R)-3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes (kuz7 and kuz8b) of natural 13ß- and epimeric 13α-series against triple-negative MDA-MB-231 breast cancer cells. High antiproliferative activity of synthesized compounds kuz8b and kuz7 against MDA-MB-231 triple-negative cancer cells was revealed. The steroid kuz7 of natural 13ß-configuration was more active against MDA-MB-231 cells than the 13α-steroid kuz8b. Cell cycle analysis revealed common patterns for the action of both tested compounds. The number of cells in the subG1 phase increased in a dose-dependent manner, indicating induction of apoptosis, which was also verified by PARP cleavage. In contrast, the number of cells in the G0/G1 phase decreases with increasing compound concentration. Steroid kuz7 at micromolar concentrations reduced the expression of GLUT1, a glucose transporter. High efficacy of the combination of kuz7 with biguanide metformin was shown, and synergistic effects on MDA-MB-231 cell growth and expression of the anti-apoptotic protein Bcl-2 were revealed. According to the obtained results, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and the decrease in GLUT1 expression, 13ß-steroid kuz7 is of interest for further preclinical studies both alone and in combination with the metabolic drug metformin

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes targeting HSP90-HER2 axis in breast cancer cells.

Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes were designed as potential HSP90 inhibitors. A series of the compounds was synthesized by oximation of (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-ones followed by O-acylation with acylamidobenzoic acids. The obtained compounds showed an antiproliferative effect on three breast cancer cell lines (MCF7, MDA-MB-231 and HCC1954). Compound 16s exhibited high antiproliferative potency against HCC1954 breast cancer cells with the IC50 value of 6 µM was selected for in-depth evaluation. Compound 16s did not inhibit the growth of normal epithelial cells. We have demonstrated that the compound 16s can induce apoptosis in cancer cells via inhibition of HSP90 "client" proteins including a key oncogenic receptor, HER2/neu. Described here compounds can be considered for further basic and preclinical investigation as a part of HSP90/HER2-targeted therapies.

Effective synthesis of novel dihydrobenzisoxazoles bearing the 2-aminothiazole moiety and evaluation of the antiproliferative activity of their acylated derivatives.

An effective method for the synthesis of 8-aryl-4,5-dihydrothiazolo[4',5':3,4]benzo[1,2-c]isoxazol-2-amines was developed. This method includes the α-keto bromination of 3-aryl-6,7-dihydrobenzo[c]isoxazol-4(5H)-ones followed by the condensation of the obtained bromo derivatives with thiourea in acetonitrile. Using virtual screening, a series of acylated derivatives of the obtained compounds were selected as potential HSP90 inhibitors. These compounds were prepared and evaluated as antiproliferative agents against three cancer cell lines (A431, 22Rv1, and MCF-7). Compounds 8b, 8c and 8q exhibiting high antiproliferative potency against MCF-7 breast cancer cells with IC50 values ranging from 2.3 to 9.5 µM were chosen for in-depth evaluation. The selected compounds had remarkable effects on HSP90 client proteins, including steroid hormone receptors and the anti-apoptotic factor BCL2. The obtained compounds are of interest for anticancer drug development.

Gene Transcription as a Therapeutic Target in Leukemia.

Blood malignancies often arise from undifferentiated hematopoietic stem cells or partially differentiated stem-like cells. A tight balance of multipotency and differentiation, cell division, and quiescence underlying normal hematopoiesis requires a special program governed by the transcriptional machinery. Acquisition of drug resistance by tumor cells also involves reprogramming of their transcriptional landscape. Limiting tumor cell plasticity by disabling reprogramming of the gene transcription is a promising strategy for improvement of treatment outcomes. Herein, we review the molecular mechanisms of action of transcription-targeted drugs in hematological malignancies (largely in leukemia) with particular respect to the results of clinical trials.

Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

Neural transplantation is a promising modality for treatment of neurodegenerative diseases, traumatic brain injury and stroke. Biocompatible scaffolds with optimized properties improve the survival of transplanted neural cells and differentiation of progenitor cells into the desired types of neurons. Silk fibroin is a biocompatible material for tissue engineering. Here, we describe thin-film scaffolds based on photocrosslinked methacrylated silk fibroin (FBMA). These scaffolds exhibit an increased mechanical stiffness and improved water stability. Photocrosslinking of fibroin increased its rigidity from 25 to 480 kPa and the contact angle from 59.7 to 70.8, the properties important for differentiation of neural cells. Differentiation of SH-SY5Y neuroblastoma cells on FBMA increased the length of neurites as well as the levels of neural differentiation markers MAP2 and ßIII-tubulin. Growth of SH-SY5Y cells on the unmodified fibroin and FBMA substrates led to a spontaneous phosphorylation of Src and Akt protein kinases critical for neuronal differentiation; this effect was paralleled by neural cell adhesion molecule elevation. Thus, FBMA is an easily manufactured, cytocompatible material with improved and sustainable properties applicable for neural tissue engineering.

PF­114, a novel selective inhibitor of BCR­ABL tyrosine kinase, is a potent inducer of apoptosis in chronic myelogenous leukemia cells.

A t(9;22) chromosomal translocation which forms the chimeric tyrosine kinase breakpoint cluster region (BCR)­Abelson murine leukemia viral oncogene homolog 1 (ABL) is a key mechanism underlying the pathogenesis of chronic myelogenous leukemia (CML). Pharmacological inhibition of BCR­ABL with imatinib (Gleevec) has been reported as an effective targeted therapy; however, mutations (including the kinase domain of ABL) suppress the efficacy of inhibitors. PF­114, a derivative of the third generation BCR­ABL inhibitor ponatinib, demonstrated a high inhibitory activity against wild-type and mutant BCR­ABL forms, such as the clinically important T315I. Furthermore, PF­114 exhibited preferential kinase selectivity, safety, notable pharmacokinetic properties and therapeutic efficacy in a murine model. Investigation into the mechanisms of CML cell death revealed an exceptional potency of PF­114 (at low nanomolar concentrations) for the CML­derived K562 cell line, whereas leukemia cell lines that lack the chimeric tyrosine kinase were markedly more refractory. The molecular ordering of events mechanistically associated with K562 cell death included the dephosphorylation of CrkL adaptor protein followed by inhibition of ERK1/2 and Akt, G1 arrest, a decrease of phosphorylated Bcl­2­associated death promoter, Bcl­2­like protein 11, BH3 interacting­domain death agonist, Bcl­extra large and Bcl­2 family apoptosis regulator, and reduced mitochondrial transmembrane potential. Increased Annexin V reactivity, activation of caspases and poly(ADP­ribose)polymerase cleavage were proposed to lead to internucleosomal DNA fragmentation. Thus, PF­114 may be a potent inducer of apoptosis in CML cells. Nevertheless, activation of STAT3 phosphorylation in response to PF­114 may permit cell rescue; thus, a combination of BCR­ABL and STAT3 inhibitors should be considered for improved therapeutic outcome. Collectively, the targeted killing of BCR­ABL­positive cells, along with other beneficial properties, such as in vivo characteristics, suggests PF­114 as a potential candidate for analysis in clinical trials with CML patients.

Comparison of the mRNA expression profile of B-cell receptor components in normal CD5-high B-lymphocytes and chronic lymphocytic leukemia: a key role of ZAP70.

The B-cell receptor (BCR) signaling pathway is of great importance for B-cell survival and proliferation. The BCR expressed on malignant B-CLL cells contributes to the disease pathogenesis, and its signaling pathway is currently the target of several therapeutic strategies. Although various BCR alterations have been described in B-CLL at the protein level, the mRNA expression levels of tyrosine kinases in B-CLL compared to that in normal CD5-high and CD5-low B-lymphocytes remain unknown. In the current study, we measured the mRNA expression levels of CD79A, CD79B, LYN, SYK, SHP1, and ZAP70 in purified populations of CD5-high B-CLL cells, CD5-low B-cells from the peripheral blood of healthy donors, and CD5-high B-cells from human tonsils. Here, we report a clear separation in the B-CLL dataset between the ZAP70-high and ZAP70-low subgroups. Each subgroup has a unique expression profile of BCR signaling components that might reflect the functional status of the BCR signaling pathway. Moreover, the ZAP70-low subgroup does not resemble either CD5-high B-lymphocytes from the tonsils or CD5-low lymphocytes from PBMC (P < 0.05). We also show that ZAP70 is the only gene that is differentially expressed in CD5-high and CD5-low normal B-lymphocytes, confirming the key role of Zap-70 tyrosine kinase in BCR signaling alterations in B-CLL.

Steroidal Pyrimidines and Dihydrotriazines as Novel Classes of Anticancer Agents against Hormone-Dependent Breast Cancer Cells.

Most breast and prostate tumors are hormone-dependent, making it possible to use hormone therapy in patients with these tumors. The design of effective endocrine drugs that block the growth of tumors and have no severe side effects is a challenge. Thereupon, synthetic steroids are promising therapeutic drugs for the treatment of diseases such as hormone-dependent breast and prostate cancers. Here, we describe novel series of steroidal pyrimidines and dihydrotriazines with anticancer activities. A flexible approach to unknown pyrimidine and dihydrotriazine derivatives of steroids with selective control of the heterocyclization pattern is disclosed. A number of 18-nor-5α-androsta-2,13-diene[3,2-d]pyrimidine, androsta-2-ene[3,2-d]pyrimidine, Δ1, 3, 5(10)-estratrieno[16,17-d]pyrimidine, and 17-chloro-16-dihydrotriazine steroids were synthesized by condensations of amidines with ß-chlorovinyl aldehydes derived from natural hormones. The synthesized compounds were screened for cytotoxicity against breast cancer cells and showed IC50 values of 7.4 µM and higher. Compounds were tested against prostate cancer cells and exhibited antiproliferative activity with IC50 values of 9.4 µM and higher comparable to that of cisplatin. Lead compound 4a displayed selectivity in ERα-positive breast cancer cells. At 10 µM concentration, this heterosteroid inhibited 50% of the E2-mediated ERα activity and led to partial ERα down-regulation. The ERα reporter assay and immunoblotting were supported by the docking study, which showed the probable binding mode of compound 4a to the estrogen receptor pocket. Thus, heterosteroid 4a proved to be a selective ERα modulator with the highest antiproliferative activity against hormone-dependent breast cancer and can be considered as a candidate for further anticancer drug development. In total, the synthesized heterosteroids may be considered as new promising classes of active anticancer agents.