Publications by authors named "Azmi Yerlikaya"

24 Publications

  • Page 1 of 1

The Ubiquitin-Proteasome Pathway and Epigenetic Modifications in Cancer.

Anticancer Agents Med Chem 2021 ;21(1):20-32

Kutahya Health Sciences University, Faculty of Medicine, Department of Medical Biology, Kütahya, Turkey.

Background: The ubiquitin-proteasome pathway is involved in almost all cellular processes (cell cycle, gene transcription and translation, cell survival and apoptosis, cell metabolism and protein quality control) mainly through the specific degradation of the majority of intracellular proteins (>80%) or partial processing of transcription factors (e.g., NF-κB). A growing amount of evidence now indicates that epigenetic changes are also regulated by the ubiquitin-proteasome pathway. Recent studies indicate that epigenetic regulations are equally crucial for almost all biological processes as well as for pathological conditions such as tumorigenesis, as compared to non-epigenetic control mechanisms (i.e., genetic alterations or classical signal transduction pathways).

Objective: Here, we reviewed the recent work highlighting the interaction of the ubiquitin-proteasome pathway components (e.g., ubiquitin, E1, E2 and E3 enzymes and 26S proteasome) with epigenetic regulators (histone deacetylases, histone acetyltransferases and DNA methyltransferases).

Results: Alterations in the regulation of the ubiquitin-proteasome pathway have been discovered in many pathological conditions. For example, a 2- to 32-fold increase in proteasomal activity and/or subunits has been noted in primary breast cancer cells. Although proteasome inhibitors have been successfully applied in the treatment of hematological malignancies (e.g., multiple myeloma), the clinical efficacy of the proteasomal inhibition is limited in solid cancers. Interestingly, recent studies show that the ubiquitin-proteasome and epigenetic pathways intersect in a number of ways through the regulation of epigenetic marks (i.e., acetylation, methylation and ubiquitylation).

Conclusion: It is therefore believed that novel treatment strategies involving new generation ubiquitinproteasome pathway inhibitors combined with DNA methyltransferase, histone deacetylase or histone acetyltransferase inhibitors may produce more effective results with fewer adverse effects in cancer treatment as compared to standard chemotherapeutics in hematological as well as solid cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1871520620666200811114159DOI Listing
June 2021

The Ubiquitin-Proteasome Pathway and Resistance Mechanisms Developed Against the Proteasomal Inhibitors in Cancer Cells.

Curr Drug Targets 2020 ;21(13):1313-1325

Bursa Uludag University, Faculty of Medicine, Department of Immunology, Bursa, Turkey.

Background: The ubiquitin-proteasome pathway is crucial for all cellular processes and is, therefore, a critical target for the investigation and development of novel strategies for cancer treatment. In addition, approximately 30% of newly synthesized proteins never attain their final conformations due to translational errors or defects in post-translational modifications; therefore, they are also rapidly eliminated by the ubiquitin-proteasome pathway.

Objective: Here, an effort was made to outline the recent findings deciphering the new molecular mechanisms involved in the regulation of ubiquitin-proteasome pathway as well as the resistance mechanisms developed against proteasome inhibitors in cell culture experiments and in the clinical trials.

Results: Since cancer cells have higher proliferation rates and are more prone to translational errors, they require the ubiquitin-proteasome pathway for selective advantage and sustained proliferation. Therefore, drugs targeting the ubiquitin-proteasome pathway are promising agents for the treatment of both hematological and solid cancers.

Conclusion: A number of proteasome inhibitors are approved and used for the treatment of advanced and relapsed multiple myeloma. Unfortunately, drug resistance mechanisms may develop very fast within days of the start of the proteasome inhibitor-treatment either due to the inherent or acquired resistance mechanisms under selective drug pressure. However, a comprehensive understanding of the mechanisms leading to the proteasome inhibitor-resistance will eventually help the design and development of novel strategies involving new drugs and/or drug combinations for the treatment of a number of cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389450121666200525004714DOI Listing
January 2020

An investigation of the mechanisms underlying the proteasome inhibitor bortezomib resistance in PC3 prostate cancer cell line.

Cytotechnology 2020 Feb 20;72(1):121-130. Epub 2019 Dec 20.

Department of Biology, Faculty of Art and Sciences, Dumlupınar University, Kütahya, Turkey.

The phenomenon of acquired resistance to chemotherapeutic agents is a long-standing conundrum in cancer treatment. To help delineate drug resistance mechanisms and pave the way for the development of novel strategies, we generated a PC3 prostate cancer cell line resistant to proteasome inhibitor bortezomib for the first time. The resistant cells were found to have an IC value of 359.6 nM, whereas the IC value of parental cells was 82.6 nM after 24 h of treatment with varying doses of bortezomib. The resistant cells were also partly cross-resistant to the novel proteasome inhibitor carfilzomib; however, they were not resistant to widely used chemotherapeutic agent vincristine sulfate, indicating that enhanced cellular drug efflux via the multidrug resistance (MDR) transporters is not the molecular basis of the resistance. Since both bortezomib and carfilzomib target and inhibit the chymotrypsin-related activity residing in the β5 subunit of the proteasome (PSMB5), we next examined its expression and found surprisingly no significant alteration in the expression profile of the mature form. However, a significant increase in the accumulation of the precursor form of PSMB5 in response to 100 nM bortezomib was observed in the parental cells without a significant accumulation in the resistant cells. The results presented here thus suggest that the molecular mechanisms causing resistance to proteasome inhibitors need to be examined in-depth to overcome the resistance to ubiquitin-proteasome pathway inhibitors in cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10616-019-00362-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002631PMC
February 2020

A novel and effective inhibitor combination involving bortezomib and OTSSP167 for breast cancer cells in light of label-free proteomic analysis.

Cell Biol Toxicol 2019 02 14;35(1):33-47. Epub 2018 Jun 14.

Faculty of Medicine, Department of Medical Biology, Kütahya Health Sciences University, Kütahya, Turkey.

Purpose: The 26S proteasome plays important roles in many intracellular processes and is therefore a critical intracellular cellular target for anticancer treatments. The primary aim of the current study was to identify critical proteins that may play roles in opposing the antisurvival effect of the proteasome inhibitor bortezomib together with the calcium-chelator BAPTA-AM in cancer cells using label-free LC-MS/MS. In addition, based on the results of the proteomic technique, a novel and more effective inhibitor combination involving bortezomib as well as OTSSP167 was developed for breast cancer cells.

Methods And Results: Using label-free LC-MS/MS, it was found that expressions of 1266 proteins were significantly changed between the experimental groups. Among these proteins were cell division cycle 5-like (Cdc5L) and drebrin-like (DBNL). We then hypothesized that inhibition of the activities of these two proteins may lead to more effective anticancer inhibitor combinations in the presence of proteasomal inhibition. In fact, as presented in the current study, Cdc5L phosphorylation inhibitor CVT-313 and DBNL phosphorylation inhibitor OTSSP167 were highly cytotoxic in 4T1 breast cancer cells and their IC values were 20.1 and 43 nM, respectively. Under the same experimental conditions, the IC value of BAPTA-AM was found 19.9 μM. Using WST 1 cytotoxicity assay, it was determined that 10 nM bortezomib + 10 nM CVT-313 was more effective than the control, the single treatments, or than 5 nM bortezomib + 5 nM CVT-313. Similarly, 10 nM bortezomib + 10 nM OTSSP167 was more cytotoxic than the control, the monotherapies, 5 nM bortezomib + 5 nM OTSSP167, or than 5 nM bortezomib + 10 nM OTSSP167, indicating that bortezomib + OTSSP167 was also more effective than bortezomib + CVT-313 in a dose-dependent manner. Furthermore, the 3D spheroid model proved that bortezomib + OTSSP167 was more effective than the monotherapies as well as bortezomib + CVT-313 and bortezomib + BAPTA-AM combinations. Finally, the effect of bortezomib + OTSSP167 combination was tested on MDA-MB-231 breast cancer cells, and it similarly determined that 20 nM bortezomib +40 nM OTSSP167 combination completely blocked the formation of 3D spheroids.

Conclusions: Altogether, the results presented here indicate that bortezomib + OTSSP167 is a novel and effective combination and may be tested further for cancer treatment in vivo and in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10565-018-9435-zDOI Listing
February 2019

Correction to: Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

Breast Cancer Res Treat 2018 07;170(2):251-256

Department of Medical Biology, School of Medicine, Dumlupınar University, Kütahya, Turkey.

In the original publication of the article, Acknowledgement section was missed out and Table 1 was published incompletely. The Acknowledgment and complete table 1 are given in this correction. The original article has been corrected.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10549-018-4781-3DOI Listing
July 2018

Secretomes reveal several novel proteins as well as TGF-β1 as the top upstream regulator of metastatic process in breast cancer.

Breast Cancer Res Treat 2018 Jul 20;170(2):235-250. Epub 2018 Mar 20.

Department of Medical Biology, School of Medicine, Dumlupınar University, Kütahya, Turkey.

Purpose: Metastatic breast cancer is resistant to many conventional treatments and novel therapeutic targets are needed. We previously isolated subsets of 4T1 murine breast cancer cells which metastasized to liver (4TLM), brain (4TBM), and heart (4THM). Among these cells, 4TLM is the most aggressive one, demonstrating mesenchymal phenotype. Here we compared secreted proteins from 4TLM, 4TBM, and 4THM cells and compared with that of hardly metastatic 67NR cells to detect differentially secreted factors involved in organ-specific metastasis.

Method And Results: Label-free LC-MS/MS proteomic technique was used to detect the differentially secreted proteins. Eighty-five of over 500 secreted proteins were significantly altered in metastatic breast cancer cells. Differential expression of several proteins such as fibulin-4, Bone Morphogenetic Protein 1, TGF-β1 MMP-3, MMP-9, and Thymic Stromal Lymphopoietin were further verified using ELISA or Western blotting. Many of these identified proteins were also present in human metastatic breast carcinomas. Annexin A1 and A5, laminin beta 1, Neutral alpha-glucosidase AB were commonly found at least in three out of six studies examined here. Ingenuity Pathway Analysis showed that proteins differentially secreted from metastatic cells are involved primarily in carcinogenesis and TGF-β1 is the top upstream regulator in all metastatic cells.

Conclusions: Cells metastasized to different organs displayed significant differences in several of secreted proteins. Proteins differentially altered were fibronectin, insulin-like growth factor-binding protein 7, and Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1. On the other hand, many exosomal proteins were also common to all metastatic cells, demonstrating involvement of key universal factors in distant metastatic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10549-018-4752-8DOI Listing
July 2018

Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line.

Cytotechnology 2017 Aug 9;69(4):711-724. Epub 2017 Apr 9.

Vatan Clinic, Medical School, İstanbul Medipol University, Istanbul, Turkey.

p38 mitogen-activated protein kinase (MAPK) belongs to the MAPK superfamily, phosphorylating serine and/or threonine residues of the target proteins. The activation of p38 MAPK leads to cell growth, differentiation, inflammation, survival or apoptosis. In this study, we tested the effect of two highly specific and potent inhibitors of p38 MAPK (namely, SB203580 and SB202190) on human breast cancer cell line MDA-MB-231 to elucidate the controversial role of p38 MAPK on cell proliferation and/or cell migration/metastasis further. It was determined that the IC value of SB203580 was 85.1 µM, while that of SB202190 was 46.6 µM, suggesting that SB202190 is slightly more effective than SB203580. To verify the effect of each inhibitor on cell proliferation and cytotoxicity, the cells were treated with various doses of SB203580 and SB202190 and examined using iCELLigence system. No significant effect of 1 and 5 µM of both inhibitors were seen on cell proliferation as compared to the DMSO-treated control cells for up to 96 h. On the other hand, both SB203580 and SB202190 significantly prevented cell proliferation at a concentration of 50 µM. SB202190 was again more effective than SB203580. Afterwards, we tested the effect of each inhibitor on cell migration using wound assay. Both SB203580 and SB202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 µM. However, interestingly it was observed that a low and noncytotoxic dose of 5 µM of SB203580 and SB202190 also did cause significant cell migration inhibition at 48 h of the treatment, corroborating the fact that p38 MAPK pathway has a critical role in cell migration/metastasis. Then, we tested whether each p38 MAPK inhibitor has any effect on cell adhesion during a treatment period of 3 h using iCELLigence system. A concentration of only 50 µM of SB202190 reduced cell adhesion for about 1.5 h (p < 0.001); after that period of time, cell adhesion in 50 µM SB202190-treated cells returned to the level of the control cells. To determine the mechanism of growth and cell migration inhibitory effects of p38 MAPK inhibitors, the activation/inactivation of various proteins and enzymes was subsequently analyzed by PathScan Intracellular Signaling Array kit. The ERK1/2 phosphorylation level was not modified by low concentrations (1 or 5 µM) of SB202190 and SB203580; while a high concentration (50 µM) of both inhibitors caused significant reductions in the ERK1/2 phosphorylation. In addition, it was determined that both p38 MAPK inhibitors caused significant increases on the Ser15 phosphorylation of mutant p53 in MDA-MB-231 under these experimental conditions; while SB202190 was more potent than SB203580.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10616-017-0079-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507849PMC
August 2017

A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib.

Oncol Lett 2016 Jul 17;12(1):323-330. Epub 2016 May 17.

Department of Pediatrics, Faculty of Medicine, Dumlupınar University, Kütahya 43100, Turkey.

Glucose-regulated protein 78 kDa/binding immunoglobulin protein (GRP78/BIP) is a well-known endoplasmic reticulum (ER) chaperone protein regulating ER stress by facilitating protein folding, assembly and Ca binding. GRP78 is also a member of the heat shock protein 70 gene family and induces tumor cell survival and resistance to chemotherapeutics. Bortezomib is a highly specific 26S proteasome inhibitor that has been approved as treatment for patients with multiple myeloma. The present study first examined the dose- and time-dependent effects of bortezomib on GRP78 expression levels in the highly metastatic mouse breast cancer 4T1 cell line using western blot analysis. The analysis results revealed that GRP78 levels were significantly increased by bortezomib at a dose as low as 10 nM. Time-dependent experiments indicated that the accumulation of GRP78 was initiated after a 24 h incubation period following the addition of 10 nM bortezomib. Subsequently, the present study determined the half maximal inhibitory concentration of intracellular calcium chelator BAPTA-AM (13.6 µM) on 4T1 cells. The combination effect of BAPTA-AM and bortezomib on the 4T1 cells was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and WST-1 assays and an iCELLigence system. The results revealed that the combination of 10 nM bortezomib + 5 µM BAPTA-AM is more cytotoxic compared with monotherapies, including 10 nM bortezomib, 1 µM BAPTA-AM and 5 µM BAPTA-AM. In addition, the present results revealed that bortezomib + BAPTA-AM combination causes cell death through the induction of apoptosis. The present results also revealed that bortezomib + BAPTA-AM combination-induced apoptosis is associated with a clear increase in the phosphorylation of stress-activated protein kinase/Jun amino-terminal kinase SAPK/JNK. Overall, the present results suggest that bortezomib and BAPTA-AM combination therapy may be a novel therapeutic strategy for breast cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ol.2016.4597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4906623PMC
July 2016

Bortezomib and etoposide combinations exert synergistic effects on the human prostate cancer cell line PC-3.

Oncol Lett 2016 May 16;11(5):3179-3184. Epub 2016 Mar 16.

Department of Medical Biology, Faculty of Medicine, Dumlupınar University, Kütahya 43100, Turkey.

Novel treatment modalities are urgently required for androgen-independent prostate cancer. In order to develop an alternative treatment for prostate cancer, the cytotoxic effects of the 26S proteasome inhibitor bortezomib, either alone or in combination with the two commonly used chemotherapeutic agents irinotecan and etoposide, on the human prostate cancer cell line PC-3 were evaluated in the present study. The PC-3 cell line was maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and treated with various doses of bortezomib, irinotecan, etoposide or their combinations. The growth inhibitory and cytotoxic effects were determined by water-soluble tetrazolium (WST)-1 assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or iCELLigence system. The combination index values were determined by the Chou-Talalay method. The half maximal inhibitory concentration (IC50) value of bortezomib on the PC-3 cell line was determined to be 53.4 nM by WST-1 assay, whereas the IC50 values of irinotecan and etoposide were determined to be 2.1 and 26.5 µM, respectively. These results suggest that the 26S proteasome inhibitor bortezomib is more potent, compared with irinotecan and etoposide, in the androgen-insensitive and tumor protein p53-null cell line PC-3. The combined effects of bortezomib+irinotecan and bortezomib+etoposide were also tested on PC-3 cells. The effect of bortezomib+irinotecan combination was not significantly different than that produced by either monotherapy, according to the results of iCELLigence system and MTT assay. However, 40 nM bortezomib+5 µM etoposide or 40 nM bortezomib+20 µM etoposide combinations were observed to be more effective than each drug tested alone. The results of the current study suggest that bortezomib and etoposide combination may be additionally evaluated in clinical trials for the treatment of hormone-refractory prostate cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ol.2016.4340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4841005PMC
May 2016

Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib.

Data Brief 2014 Dec 19;1:56-9. Epub 2014 Oct 19.

Department of Medical Biochemistry, Uludağ University, Bursa, Turkey.

This data article contains data related to the research article entitled, "A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line" by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dib.2014.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459767PMC
December 2014

A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line.

J Proteomics 2015 Jan 8;113:315-25. Epub 2014 Oct 8.

Department of Medical Biochemistry, Uludağ University, Bursa, Turkey.

Unlabelled: The 26S proteasome is a proteolytic enzyme found in both cytoplasm and nucleus. In this study, we examined the differential expression of proteasome inhibitor bortezomib-induced proteins in p53-deficient 4T1 cells. It was found that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24h. Next, we analyzed the expression of intracellular proteins in response to treatment with 100nM bortezomib for 24h by label-free LC-MS/MS. These analyses showed that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The proteins identified by label-free LC-MS/MS were then analyzed by Ingenuity Pathway Analysis (IPA) Tool to determine biological networks affected by inhibition of the 26S proteasome. The analysis results showed that post-translational modifications, protein folding, DNA replication, energy production and nucleic acid metabolism were found to be among the top functions affected by the 26S proteasome inhibition. The biological network analysis indicated that ubiquitin may be the central regulator of the pathways modulated after bortezomib-treatment. Further investigation of the mechanism of the proteins modulated in response to the proteasomal inhibition may lead to the design of more effective and novel therapeutic strategies for cancer.

Biological Significance: Although the proteasome inhibitor bortezomib is approved and used for the treatment of human cancer (multiple myeloma), the mechanism of action is not entirely understood. A number of studies showed that proteasome inhibitors induced apoptosis through upregulation of tumor suppressor protein p53. However, the role of tumor suppressor protein p53 in bortezomib-induced apoptosis is controversial and not well-understood. The tumor suppressor p53 is mutated in at least 50% of human cancers and is strongly induced by proteasomal inhibition. Some also reported that the proteasome inhibitor can induce apoptosis in a p53-independent manner. Also, it is reported that Noxa, a target of p53, is induced in response to proteasomal inhibition in a p53-independent manner. However, we have also previously reported that neither Puma nor Noxa are induced by proteasomal inhibition in p53-null 4T1 breast cancer cells, which is commonly used for in vivo breast cancer tumor models. The current results provided additional targets of proteasome inhibitor bortezomib and may therefore help in understanding the p53-independent mechanism of apoptosis induction by proteasome inhibitors. In addition, the results presented in this current study report for the first time that proteasomal subunit Psmd14, anti-apoptotic GRP78, anti apoptotic protein Card10, Dffb, Traf3 and Trp53bp2 are regulated and overexpressed in response to proteasome inhibitor bortezomib in p53-deficient 4T1 cells. Therefore, novel therapeutic strategies targeting these anti-apoptotic or pro-apoptotic proteins as well as inhibiting the proteasome simultaneously may be more effective against cancer cells. The proteins identified here present new avenues for the development of anti-cancer drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2014.09.010DOI Listing
January 2015

Additive enhancement of apoptosis by TRAIL and fenretinide in metastatic breast cancer cells in vitro.

Biomed Pharmacother 2014 May 27;68(4):477-82. Epub 2014 Mar 27.

University of Thessaly, School of Medicine, Department of Pharmacology, Biopolis, Larissa, Greece.

Successful management of metastatic breast cancer still needs better chemotherapeutic approaches. The combination of fenretinide (4-HPR), a synthetic retinoid inducing apoptosis by ROS generation, and TRAIL, a cell death ligand inducing caspase-dependent apoptosis, might result in more powerful cytotoxic activity. We therefore investigated the cytotoxic activity and resulting cell death mode of this combination in MDA-MB-231 cell line as a representative of metastatic state. Cytotoxicity was assessed by the ATP viability assay while the mode of cell death was determined both morphologically using fluorescence microscopy and biochemically using Western blotting and ELISA. The combination resulted in an additive cytotoxic effect at the doses used. Fragmented and/or pyknotic nuclei, which is a feature of apoptosis, were observed after treatment with fenretinide or TRAIL. However, the combinatorial treatment further increased apoptotic figures. Confirming apoptosis, active caspase-3 and cleaved PARP were increased by fenretinide or TRAIL in both western blotting and ELISA. Again, apoptosis was further increased by the combination. The combination warrants further studies due to its superior cytotoxic activity in the metastatic setting of breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biopha.2014.03.013DOI Listing
May 2014

Anticancer agent ukrain and bortezomib combination is synergistic in 4T1 breast cancer cells.

Anticancer Agents Med Chem 2014 Mar;14(3):466-72

Dumlupınar University, Faculty of Medicine, Department of Medical Biology, Kütahya, Turkey.

The identification and in-depth understanding of intracellular signalling pathways led to the synthesis and discovery of many agents targeting cancer cells. In this study, we investigated for the first time the effect of anticancer agent ukrain as a single agent or in combination with cisplatin, etoposide, 5-fluorouracil, quercetin and bortezomib in 4T1 breast cancer and B16F10 melanoma cells. It was found that ukrain is cytotoxic and apoptotic in 4T1 breast carcinoma and B16F10 melanoma cells when given alone. The IC50 value of ukrain in 4T1 cells was found as 40 ± 6.8 μM and that in B16F10 cells as 76 ± 10 μM. It was then found that apoptosis can be induced in 4T1 breast cancer cells in a dose-dependent manner in response to ukrain treatment, based on DNA fragmentation evidence. The induction of apoptosis was corroborated by the analysis of cleavage products of caspase-3 in 4T1 cells using Western blot technique. When ukrain was tested in combination with cisplatin and etoposide, no significant enhancement of cytotoxicity was detected as compared with single agent treatments. Similarly, 5-fluorouracil and quercetin also did not potentiate the cytotoxic effects of ukrain in 4T1 cells. Finally, we examined the effect of various concentrations of ukrain in combination with 10 nM bortezomib in 4T1 cells. Determination of combination index values showed that bortezomib potentiated the effect of ukrain. And the combination was found to cause synergistic cell death. The lowest combination index detected was 0.57 which was obtained when the cells were treated with 10 nM bortezomib + 100 μM ukrain. Likewise, when cells were treated with different doses of bortezomib in the presence of 25 μM ukrain, synergism was similarly detected between the two drugs in a dose-dependent manner. Altogether, the results presented here suggest that the combination of ukrain + bortezomib may be further evaluated and tested in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/18715206113139990318DOI Listing
March 2014

Effect of bortezomib in combination with cisplatin and 5‑fluorouracil on 4T1 breast cancer cells.

Mol Med Rep 2013 Jul 8;8(1):277-81. Epub 2013 May 8.

Department of Medical Biology, Faculty of Medicine, Dumlupınar University, Kütahya 43100, Turkey.

Bortezomib is a highly selective and reversible inhibitor of the 26S proteasome. It has been approved for the treatment of patients with relapsed and refractory multiple myeloma. A number of studies have been conducted to evaluate the activity and safety of bortezomib either alone or in combination with several cytotoxic agents and radiation. In the current study, the efficacy of bortezomib alone or in combination with cisplatin and 5‑fluorouracil was evaluated in 4T1 breast cancer cells, a highly metastatic murine cancer cell line. Using MTT assay, IC50 values of cisplatin and 5‑fluorouracil were determined to be 14.2 and 8.9 µM for cisplatin and 5‑fluorouracil, respectively. The effects of different concentrations of cisplatin and 5‑fluorouracil in combination with two different concentrations of bortezomib were examined in the 4T1 cells. Statistically significant differences were found when 1 or 5 µM cisplatin was combined with 10 or 50 nM bortezomib. Similarly, 1 µM 5‑fluorouracil or 5 µM 5‑fluorouracil in combination with 10 nM bortezomib caused significant cell death as compared to treatment with single agents. However, 1 or 5 µM 5‑fluorouracil did not potentiate the effects of higher concentrations of bortezomib (50 nM). The effect of the combination of cisplatin, 5‑fluorouracil and bortezomib was determined by soft agar assay. It was confirmed that a combination of cisplatin and bortezomib was more effective than each drug as a monotherapy. Therefore, the combination of cisplatin and bortezomib should be tested further in clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mmr.2013.1466DOI Listing
July 2013

The significance of ubiquitin proteasome pathway in cancer development.

Recent Pat Anticancer Drug Discov 2013 Sep;8(3):298-309

Department of Biology, Art and Science Faculty, Dumlupınar University, Kütahya, 43100, Turkey.

The ubiquitin proteasome pathway is the most significant intracellular proteolytic pathway. The target proteins are usually ubiquitinated prior to degradation by the proteasome; however, ubiquitin-independent targeting mechanisms have also been reported (e.g., the antizyme-mediated degradation of ornithine decarboxylase). Aberrations in the components of the ubiquitin proteasome pathway are commonly observed in many cancers, and uncontrolled growth of cancer cells can result either from stabilization of oncoproteins (e.g., c-jun) or increased degradation of tumor suppressor proteins (e.g., p53). In addition, due to the pleiotropic functions of the ubiquitin proteasome pathway in cells, there is great interest in developing inhibitors to specifically block this pathway for cancer treatment. This review summarizes the recent literature and several patented inventions on the ubiquitin proteasome pathway with respect to its role in cancer development and treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1574891x113089990033DOI Listing
September 2013

Expression of heme oxygenase-1 in response to proteasomal inhibition.

Authors:
Azmi Yerlikaya

Protein Pept Lett 2012 Dec;19(12):1330-3

Dumlupınar University, Art and Science Faculty, Department of Biology, Kütahya, Turkey.

Heme oxygenase-1 (HO-1) is an antioxidant, antiapoptotic and cytoprotective enzyme, catalysing the degradation of heme to carbon monoxide, biliverdin and ferrous iron. Recent studies indicated that expression of HO-1 is under the control of proapoptotic transcription factor p53 and antioxidant transcription factor Nrf2. Whether each of these transcription factors act independently or there is a cooperation between them in inducing HO-1 expression remains to be elucidated. In this study, we examined the expression of HO-1 in B16F10 melanoma and 4T1 breast cancer cells after cell exposure to proteasome inhibitors. We found that HO-1 protein level is increased by about 70% in p53-wt B16F10 cells in response to proteasome inhibitor MG132 after 6 h. Likewise, a 6.8 fold increase in HO-1 level was observed after cell exposure to the highly specific proteasome inhibitor bortezomib after 6 h of treatment in B16F10 cells. Whereas no induction of HO-1 was observed in p53-null 4T1 cells after treatment with bortezomib for 6 h. Next, we aligned HO-1 untranslated region with a consensus p53-responsive element. This bioinformatic analysis identified a p53-responsive element within the untranslated region of HO-1. Then, we examined HO-1 expression after a prolonged exposure to bortezomib in both B16F10 and 4T1 cell. These analyses similarly indicated that HO-1 is strongly induced in B16F10 cells in a dosedependent; contrary to our expectations, a strong induction of HO-1 is also observed in 4T1 cells. Therefore, it is concluded that HO-1 expression is under the control of p53 during early time points of proteasomal inhibition. However, during prolonged incubation with proteasome inhibitors, HO-1 expression can be induced in a p53-independent manner, suggesting participation of other protein(s) with longer half-lives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/092986612803521657DOI Listing
December 2012

The p53-independent induction of apoptosis in breast cancer cells in response to proteasome inhibitor bortezomib.

Tumour Biol 2012 Oct 4;33(5):1385-92. Epub 2012 Apr 4.

Art and Science Faculty, Department of Biology, Dumlupınar University, Kütahya, Turkey.

An important hallmark of cancer cells is acquired resistance toward apoptosis. The apoptotic pathway is the most well-defined cell death program and is characterized by several morphological and biochemical features. The tumor suppressor protein p53 is a critical regulator of apoptosis in many cell types. p53 stimulates a wide network of signals that act through either extrinsic or intrinsic pathways of apoptosis. However, a number of studies have shown that apoptosis can be induced in a p53-independent manner as well. In this study, we examined the mechanism of apoptosis in p53-null breast cancer cells in response to the proteasome inhibitor bortezomib. Initially, we determined the p53 status of 4T1 breast carcinoma and 4THMpc (a highly mestatic derivative of 4T1) cells and verified that both cells are p53 deficient. It was subsequently shown that apoptosis can be induced in both cells in a dose-dependent manner in response to bortezomib treatment, based on DNA fragmentation evidence. Western blot analyses of ubiquitin-protein conjugates additionally showed that the proteasome is potently inhibited by bortezomib in p53-null 4T1 and 4THMpc cells. The results presented in the current study also show that caspase-3 is significantly activated in response to the treatment with bortezomib, implying that induction of apoptosis in these p53-deficient cells is occurring via caspase-3. The additional results presented here suggest that the pro-apoptotic proteins Bad, Noxa, and Puma are not critical regulators of apoptosis induction in p53-null 4T1 and 4THMpc cells. Similarly, there was no difference in the expression level of Mcl-1 in treated cells, suggesting that this anti-apoptotic protein is also uninvolved in the apoptotic response resulting from bortezomib treatment. In contrast, a very significant upregulation of the anti-apoptotic protein Hsp25/27 was detected in these p53-deficient cells after treatment with bortezomib. If the increased expression of Hsp25/27 protein levels are muting the apoptotic effects of the bortezomib treatment, then the apoptosis-inducing effects of such proteasome inhibitors might be increased with approaches simultaneously inhibiting Hsp25/27 protein in p53-deficient cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13277-012-0386-3DOI Listing
October 2012

Combined effects of the proteasome inhibitor bortezomib and Hsp70 inhibitors on the B16F10 melanoma cell line.

Mol Med Rep 2010 Mar-Apr;3(2):333-9

Department of Biology, Art and Science Faculty, Dumlupinar University, Kütahya 43100, Turkey.

Studies have shown that the 26S proteasome is involved in cell cycle control, transcription, DNA repair, immune response and protein synthesis. In the present study, we investigated the antiproliferative effects of the proteasome inhibitor bortezomib and heat shock protein (Hsp)70 inhibitors on the B16F10 melanoma cell line. The IC50 value of bortezomib was found to be 2.46 nM, while that of the Hsp70 inhibitor quercetin was 45 µM in the B16F10 cells. This indicates that bortezomib is more effective than quercetin in inhibiting cell growth. In response to treatment with 10 nM bortezomib for 24 h, cells underwent rounding, shrinkage and detachment. Unexpectedly, such morphological changes were not observed in cells treated with 20 µM quercetin alone, nor in cells treated with bortezomib + quercetin, indicating that quercetin inhibited the cytotoxic effects of bortezomib. Quantitation of cell viability also indicated that quercetin interfered with the cytotoxic effects of bortezomib. However, the combination of quercetin with another proteasome inhibitor, MG132, caused significant cell death as compared to single-agent treatment. A DNA ladder assay also confirmed the inhibitory effect of quercetin on the apoptosis-inducing effect of bortezomib. However, quercetin did not prevent the induction of apoptosis by MG132; on the contrary, it potentiated the apoptosis-inducing effect of MG132. These results suggest that the combination of quercetin with clinically beneficial proteasome inhibitors (except bortezomib) may have increased efficacy in the treatment of cancer. We also tested the combination of two other Hsp70 inhibitors, KNK-437 and schisandrin-B, in combination with bortezomib. Neither of these combinations was more effective than single-agent treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/mmr_00000262DOI Listing
August 2013

Investigation of the eIF2alpha phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells.

Mol Biol (Mosk) 2010 Sep-Oct;44(5):859-66

Dumlupinar University, Art and Science Faculty, Department of Biology, Kütahya, Turkey.

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2alpha) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2alpha in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2alpha. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2alpha phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2alpha phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2alpha phosphorylation; futhermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eLF2alpha. Zinc (II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2alpha phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2010

Differential sensitivity of breast cancer and melanoma cells to proteasome inhibitor Velcade.

Int J Mol Med 2008 Dec;22(6):817-23

Dumlupinar University, Art and Science Faculty, Department of Biology, Kütahya, Turkey.

Velcade (also known as PS-341 or Bortezomib) is a highly selective and reversible inhibitor of the 26S proteasome and is approved for the treatment of patients with advanced multiple myeloma. Here we investigated the anti-proliferative effect of Velcade on 4T1 breast cancer and B16F10 melanoma cells and evaluated the mechanism of action. It was found that two cell lines are differentially sensitive to proteasome inhibitor Velcade. The IC50 concentrations for B16F10 and 4T1 were 2.5 nM and 71 nM, respectively, indicating that B16F10 cells are more sensitive to proteasomal inhibition. Velcade was equally potent in inhibiting the chymotrypsin-like activity of the proteasome in both cell lines. It was determined that B16F10 cells proliferate more rapidly than 4T1 cells; doubling time (Td) =14.2 h versus Td =22.9 h, suggesting that a rapid proliferation rate may be an important factor in cellular resistance towards proteasomal inhibition. We observed for the first time that p53 and p21 proteins were increased in B16F10 cells but not in 4T1 following Velcade-treatment, demonstrating that p53 and p21 may enhance Velcade sensitivity. Furthermore, it was observed that caspase-3 proenzyme was reduced by approximately 20% in B16F10 melanoma cells, but not in 4T1 cells in response to 26S proteasomal inhibition by Velcade. Altogether, we concluded that p53 protein plays a central role in higher sensitivity of B16F10 cells to Velcade by inducing the accumulation of p21, a cell cycle inhibitor, as well as by stimulating the mitochondrial pathway of apoptosis through caspase-3 activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2008

Phosphorylation of eIF2alpha in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase.

Biochem J 2008 Jun;412(3):579-88

Department of Biology, Faculty of Science and Arts, University of Dumlupinar, Kutahya, Turkey.

The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2alpha (eukaryotic translational initiation factor 2alpha) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55-60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2alpha, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2alpha, increased phosphorylation of eIF2alpha still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2alpha. In contrast, in a fourth MEF knockout cell line, HRI(-/-) cells lacking the HRI kinase failed to increase eIF2alpha phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20080324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2842126PMC
June 2008

Structural basis for the inactivation of AdoMetDC K12R mutant.

Protein Pept Lett 2006 ;13(3):313-7

Dumlupinar University, Art and Science Faculty, Department of Biology, Kütahya, Turkey.

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the alpha (30.7 kDa) and beta (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45-->Arg and Lys56-->Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/092986606775338489DOI Listing
April 2006

Influence of dietary oils on liver and blood lipid peroxidation.

Saudi Med J 2005 Mar;26(3):442-6

Department of Biology, Art and Science Faculty, University of Dumlupinar, Kutahya, Turkey.

Objective: Diets high in unsaturated fatty acids have been recommended to lower the risk of cardiovascular disease. However, these lipids are more susceptible to lipid peroxidation than saturated fatty acids. The aim of the present study described herein was to investigate the effects of dietary oils (differing in their degree of saturated and unsaturated fatty acids) on liver and blood lipid peroxidation in chicks.

Methods: The experiments were conducted at the laboratories of University of Dumlupinar, Kutahya, Turkey and Osmangazi University, Eskisehir, Turkey between November 2002 and December 2003. The animals were randomly divided into 5 groups of 30 and fed dietary butter, margarine, olive oil, sunflower oil or corn oil for 7 weeks. Liver malondialdehyde level, blood superoxide dismutase activity (SOD) and glutathione peroxidase activity (GPx), serum vitamin E, and total antioxidant (AOA) levels were measured to determine the effects of the dietary oils on lipid peroxidation.

Results: No significant differences were observed in SOD and GPx activities, or vitamin E and AOA levels between the experimental groups. However, the results indicated that the corn oil feeding caused significant increases in liver malondialdehyde (a genotoxic byproduct of lipid peroxidation) level as compared with the other oils.

Conclusion: The results demonstrate that corn oil feeding increases lipid peroxidation significantly and thus may raise the susceptibility of tissues to free radical oxidative damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
March 2005

S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination.

J Biol Chem 2004 Mar 12;279(13):12469-78. Epub 2004 Jan 12.

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M312625200DOI Listing
March 2004
-->