Protein kinaz d2’nin ilaç dirençli meme kanseri hücre hatlarındaki işlevleri.

Even though chemotherapy keeps its position as the most preferred and potent strategy of cancer treatment, resistance of tumor to the anti-neoplastic drug poses an obstacle for chemotherapy success. Multidrug resistance (MDR) is a phenomenon that is defined as the intrinsic or acquired resistance against structurally and functionally unrelated drugs. Acquisition of multidrug resistance can be through several distinct mechanisms such as increased drug efflux by ABC transporters, increased drug detoxification through phase I and II enzymes, altered death pathways and increased damage repair, making MDR a multifaceted problem that remodel many regulatory or metabolic circuits. MDR phenotype has also been linked to increased aggressiveness marked by mobility and invasiveness or vice versa. Protein kinase D2 (PKD2) is one of the isoforms in three-membered serine/threonine kinase family, PKD. PKD family members can possess redundant as well as specific roles on proliferation, survival, angiogenesis and motility, the events that are relevant to cancer. In glioblastoma, leukemia, colorectal, pancreas and breast cancer, tumor promoting and suppressing roles of PKD members have been shown. In particular, breast cancer, the most common cancer type in women, PKD2 and PKD3 appear to have oncogenic roles while PKD1 possesses tumor-suppressive functions. Specifically, PKD2 seems to be ubiquitous in many breast cancer types, while PKD1 and PKD3 are not. To this end, we aimed to characterize the ubiquitous member, PKD2, in a panel of breast cancer cell lines. We found that the expression of PKD2 does not differ between cell lines, whereas its basal level activity is higher in chemoresistant MCF7 derivatives compared to parental MCF7 cell line, implying that PKD2 may have role in drug resistance and associated phenotypes. Cell proliferation assay showed that PKD2 downregulation does not affect the drug resistance in MCF7/DOX cells. PKD2 knockdown also does not significantly change the expression of potential PKD targets that are implicated in MDR and apoptosis. MCF7/DOX cells are phenotypically different from parental cell line such that they have higher expression of epithelial to mesenchymal transition markers, higher mobility and invasive characteristics. Since PKD2 is also implicated in motility we checked whether PKD2 downregulation influences the migration of MCF7/DOX cells towards a chemoattractant. The migration assay showed that PKD2 downregulation suppresses the migration of MCF7/DOX cells. The data implied that under this experimental setup PKD2 did not alter the drug resistance whereas it changes the migration potential of doxorubicin resistant MCF7 cell line. Further research is needed to uncover the roles of other isoforms PKD1 and PKD3 as well as upstream regulators of PKD members in chemoresistance.M.S. - Master of Scienc

Characterization of Novel SWI/SNF Chromatin Remodeling Complex (GBAF) in Health and Disease

In eukaryotic systems, the genetic material of the cell –DNA– is packed into a protein-dense structure called chromatin. Chromatin structure is critical for preservation of the genetic material as well as coordination of vital processes such as DNA replication, transcription and DNA damage repair. The fundamental repeating unit of chromatin is nucleosome which is composed of an octamer of small alkaline proteins called histones and the DNA wrapped around this octamer. The nucleosomes are then packed into higher-order structures leading to formation of 3D chromatin architecture. The chromatin is a dynamic structure; the spacing between nucleosomes, or the folding of the larger chromatin segments is subjected to alterations during embryonic development, tissue specifications or simply during any event that require gene expression changes. Failure in proper regulation of chromatin structure has been associated with embryonic defects and disease such as cancer. This work has focused on a class of ATP-dependent chromatin remodeling complexes known as switch/sucrose-non-fermentable (SWI/SNF) or BRG-associated factors (BAF) complex. This family of complexes act on chromatin and alter its physical structure by mobilizing histones or nucleosome particles through the activity of its ATPase –BRG1 or BRM, enabling more accessible DNA for the other factors such as transcription factors to localize and recruit transcription machinery. In particular, we discovered and biochemically defined a novel version of this family of chromatin complexes that we named as GLTSCR1/1L-BAF (GBAF). GLTSCR1 and GLTSCR1L are two uncharacterized paralogous proteins that have been identified as BRG1-interacting proteins. Biochemically surveying the essence of this interaction, we realized that these proteins incorporates into a previously unknown SWI/SNF family complex that lacks well-characterized SWI/SNF subunits such as ARID1/2, BAF170, BAF47; instead, uniquely comprise GLTSCR1/1L and bromodomain-containing protein BRD9. Focusing on the GLTSCR1 subunit, we observed that its absence is well-tolerated by many different cell types except slight growth retardation by prostate cancer cells. Expanding the cohort of prostate cancer cells, we realized that not the paralogous subunits GLTSCR1 or GLTSCR1L but unique and non-redundant subunit BRD9 is the major GBAF-dependence in prostate cancer cells. We observed that especially the androgen-receptor positive cell lines have severe growth defects upon BRD9 knockdown or inhibition. In vivo, we showed that xenografts with BRD9 knockdown prostate cancer cells (LNCaP) have smaller tumor size. We demonstrated that BRD9 inhibition can block the expression of androgen-receptor targets. Similarly, BRD9knockdown and treatment with antiandrogen drug (enzalutamide) has overlapping transcriptional effects. Mechanistically, we showed that BRD9 interacts with AR and it colocalizes with AR in subset of AR -binding sites. Surprisingly, we realized that BRD9 depletion has similar transcriptional and phenotypic effects as BET protein inhibitors. BET protein family contains 4 bromodomain containing proteins (BRD2, BRD3, BRD4, BRDT). These proteins were previously shown to be critical for AR-dependent gene expression. We detected interaction between BRD9 and BRD2/4. We demonstrated that BRD4 and BRD9 had shared binding sites on genome, a fraction of which are co-bound by AR. At particular target sites we showed that BRD9 localization is dependent on BET proteins, but not the other way around. Taking together, we provided some evidences that GBAF targeting through BRD9 can be a novel therapeutic approach for prostate cancer. Growing body of reports suggested that current therapy options targeting the androgen receptor is failing due to acquired resistance. Therefore, targeting the AR pathways via its coregulators such as BET proteins or SWI/SNF complexes can serve as potent alternative approaches. Further research is needed to elucidate the roles of GBAF and BET proteins in androgen receptor independent prostate cancer cells, which are still responsive to GBAF or BET manipulations although to a lesser extent

Protein kinase D2 silencing reduced motility of doxorubicin-resistant MCF7 cells

Success of chemotherapy is generally impaired by multidrug resistance, intrinsic resistance, or acquired resistance to functionally and structurally irrelevant drugs. Multidrug resistance emerges via distinct mechanisms: increased drug export, decreased drug internalization, dysfunctional apoptotic machinery, increased DNA damage repair, altered cell cycle regulation, and increased drug detoxification. Several reports demonstrated that multidrug resistance is a multifaceted problem such that multidrug resistance correlates with increased aggressiveness and metastatic potential. Here, we tested the involvement of protein kinase D2, a serine/threonine kinase that was previously implicated in proliferation, drug resistance, and motility in doxorubicin-resistant MCF7 (MCF7/DOX) cell line, which served as an in vitro model for drug resistance and invasiveness. We showed that basal level activity of protein kinase D2 (PKD2) was higher in MCF7/DOX cells than parental MCF7 cells. To elucidate the roles of PKD2 MCF7/DOX, PKD2 expression was reduced via small interfering RNA (siRNA)-mediated knockdown. Results showed that acquired resistance of MCF7/DOX to doxorubicin was not affected by PKD2 silencing, while motility of MCF7/DOX cells was reduced. The results implied that PKD2 silencing might inhibit migration of MCF7/DOX cells without affecting chemoresistance significantly

ÇOKLU İLAÇ DİRENÇLİ MCF-7 HÜCRE HATLARINDA PROTEİN KİNAZ D2 İFADESİNİN İLAÇ DİRENÇLİLİĞİNE ETKİSİ

ÇOKLU İLAÇ DİRENÇLİ MCF-7 HÜCRE HATLARINDA PROTEİN KİNAZ D2 İFADESİNİN İLAÇ DİRENÇLİLİĞİNE ETKİS

Etoposide resistance in MCF-7 breast cancer cell line is marked by multiple mechanisms

Purpose: Acquired or intrinsic drug resistance is one of the major handicaps in the success of chemotherapy. Etoposide is a topoisomerase II poison widely used in chemotherapy. Similar to other topoisomerase inhibitors and DNA damaging agents, resistance to etoposide may arise as a result of alterations in target expression and activity, increased drug efflux and alterations in DNA damage response mechanisms. Here, we tested the involvement of such mechanisms in etoposide-resistant MCF-7 breast cancer cells

At the Crossroad of Gene Regulation and Genome Organization: Potential Roles for ATP-Dependent Chromatin Remodelers in the Regulation of CTCF-Mediated 3D Architecture

In higher order organisms, the genome is assembled into a protein-dense structure called chromatin. Chromatin is spatially organized in the nucleus through hierarchical folding, which is tightly regulated both in cycling cells and quiescent cells. Assembly and folding are not one-time events in a cell’s lifetime; rather, they are subject to dynamic shifts to allow changes in transcription, DNA replication, or DNA damage repair. Chromatin is regulated at many levels, and recent tools have permitted the elucidation of specific factors involved in the maintenance and regulation of the three-dimensional (3D) genome organization. In this review/perspective, we aim to cover the potential, but relatively unelucidated, crosstalk between 3D genome architecture and the ATP-dependent chromatin remodelers with a specific focus on how the architectural proteins CTCF and cohesin are regulated by chromatin remodeling

Poly (I:C)- and doxorubicin-loaded magnetic dendrimeric nanoparticles affect the apoptosis-related gene expressions in MCF-7 cells

Use of nanoparticles as drug carrier vectors has great potential to circumvent the limitations associated with chemotherapy, including drug resistance and destructive side effects. For this purpose, magnetic generation 4 dendrimeric nanoparticles were prepared to carry chemotherapeutic agent doxorubicin (G 4-DOX) and immune modulator polyinosinic:polycytidylic acid [Poly(I:C)]. As previously reported, DOX and Poly(I:C) was loaded onto G 4 nanoparticles (PIC-G 4-DOX). Cellular internalization study using confocal microscopy demonstrated high levels of cellular internalization of PIC-G 4-DOX nanoparticles by MCF-7 cells. This resulted in higher efficacy of PIC-G 4-DOX nanoparticles in killing MCF-7 breast cancer cells. Alteration in the expression levels of selected genes was determined by RT-qPCR analyses. Proapoptotic NOXA, PUMA, and BAX genes were upregulated, and SURVIVIN, APOLLON, and BCL-2 genes were downregulated, indicating the cell-killing effectiveness of PIC-G 4-DOX nanoparticles. Gene expression analysis provided some insights into the possible molecular mechanisms on cytotoxicity of DOX and Poly(I:C) delivered through G 4 magnetic nanoparticles. The results demonstrated that PIC-G 4-DOX can be useful for targeted delivery affecting apoptotic pathways, resulting in an advanced degree of cancer-cell-killing. They are promising for targeting cancer-cells because of their stability, biocompatibility, higher internalization, and toxicity.Publisher's Versio

Polycomb Paralog Chromodomain Inhibitors Active Against Both CBX6 and CBX8

Methyllysine reader proteins bind to methylated lysine residues and alter gene transcription by changing the compaction state of chromatin or by the recruitment of other multiprotein complexes. The polycomb paralog family of methyllysine readers bind to trimethylated lysine on the tail of histone 3 via a highly conserved aromatic cage located in their chromodomains. Each of the polycomb paralogs are implicated in several disease states. CBX6 and CBX8 are members of the polycomb paralog family with two structurally similar chromodomains. By exploring the structure-activity relationships of a previously reported CBX6 inhibitor we have discovered more potent and cell permeable analogs. Our current report includes potent, dual-selective inhibitors of CBX6 and CBX8. We have shown that the –2 position in our scaffold is an important residue for selectivity amongst the polycomb paralogs. Preliminary cell-based studies show that the new inhibitors impact cell proliferation in a rhabdoid tumor cell line. This report includes data on inhibitor design, inhibitor synthesis, compound characterization by LCMS, compound activity by fluorescence polarization, analysis of structure-activity relationships, rhabdoid tumor cell line activity