Кафедра лабораторної діагностики біологічних систем
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Browsing Кафедра лабораторної діагностики біологічних систем by Author "Kolybo, Denys"
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Item Alternative routes for HB-EGF mitogenic effects inhibition(2023) Siromolot, Andriy; Kolybo, DenysHeparin-binding epidermal growth factor–like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathophysiological functions. Also, HB-EGF plays a pivotal role in distinct tumors progression. HB-EGF is highly expressed in certain cancer cells, such as ovarian and breast cancers, that makes it beneficial for the development of antitumor therapeutic agents. Neutralizing agents against HB-EGF can act on two strategies: sterically blocking the binding of HB-EGF with receptors (monoclonal or polyclonal antibodies, diphtheria toxin derivatives) or capable preventing the formation of soluble HB-EGF from proHB-EGF (ectodomain shedding) with MMPs or ADAMs inhibitors. Recombinant analogs of Corynebacterium diphtheriae toxin fragments (subunit B) and it toxoid CRM197 are able to block the binding of fluorescent derivative HB-EGF (HB-EGF-mCherry) with receptors HER1 and HER4 on the surface of A431 and Vero cell lines. It has been shown that immune serum-containing polyclonal antibodies against HB-EGF is able to inhibit HBEGF binding to surface cellular receptors and block the mitogenic activation with HB-EGF of cells by deactivating Ras-MAPK/ERK1/2. Mitogenic activity of sHB-EGF is significantly enhanced by membrane-associated heparan sulfate proteoglycans (HSPG). Interaction of the N-terminal heparin-binding region of HB-EGF with HSPG leads to stabilization of the EGF-like domain of the molecule in complex with the receptor. The ability of heparin to block proliferation of A431 and Vero cells by exogenous HB-EGF has been demonstrated, probably, due to binding with heparin-binding domain of HB-EGF. Moreover, leupeptin and a broad-spectrum metalloproteinase blocker GM6001, and EDTA significantly decrease paracrine activation of cells by HB-EGF. Thus, searching and development of effective neutralizing agents against sHB-EGF is important and perspective research direction for establishing of a new targeted antitumor therapy.Item HB-EGF as a Targeted Carrier for Drug Delivery in Some Cancers(2025) Gamaliia, Inna; Siromolot, Andriy; Kolybo, DenysCell-penetrating peptides (CPPs) facilitate efficient biomolecule delivery with low immunogenicity and cytotoxicity, making them ideal for in vivo drug delivery. Heparin-binding epidermal growth factorlike growth factor (HB-EGF), a ligand of the epidermal growth factor receptor (EGFR), is overexpressed in tumors and can promote angiogenesis. Doxorubicin (DOX), a chemotherapeutic, treats various cancers but has limited use with HB-EGF as a carrier. Aim. In this study we focused on the HB-EGF’s potential in enhancing DOX delivery and antitumor effects. Methods. Recombinant sHB-EGF was expressed in E. coli, purified by IMAC, and loaded with DOX with further dialysis of the complex from unbound antibiotics. Binding to cell surfaces (A431, 3T3, Vero), as well as ROS production using DCFH-DA, were analyzed by flow cytometry. Cell viability was assessed with MTT assays after 48 h. Results. sHB-EGF fluorescent derivatives effectively bound to A431 cells, enhancing DOX delivery to squamous-cell carcinoma and significantly reducing cell viability. Conclusions. HB-EGF efficiently delivers DOX into cells, suggesting its potential as a targeted drug carrier for EGFR/ErbB-1 overexpressed cancers.Item Heparin-binding EGF-like growth factor as a prospective mediator of tissue repair and regeneration(2024) Didan, A.; Siromolot, Andriy; Kolybo, DenysHeparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family. It contains EGF-like and heparin-binding domains allowing interactions with heparin and cell-surface heparin sulfate. [1] It can activate human epidermal growth factor receptors 1 and 4 resulting in a multitude of cell responses, including cellular proliferation, migration, adhesion, and differentiation. HB-EGF levels can increase in response to different forms of injuries as well as extracellular stimuli, such as lysophosphatidic acid, retinoic acid, and 17-estradiol [2]. HB-EGF plays a critical role in tissue repair and regeneration. It promotes cutaneous wound healing, hepatocyte proliferation after partial hepatectomy, intestinal anastomosis strength, alveolar regeneration after pneumonectomy, neurogenesis after ischemic injury, bladder wall thickening in response to urinary tract obstruction, and protects against ischemia/reperfusion injury in many cell types [1, 3, 4]. Aim. To obtain bioactive recombinant human HB-EGF and investigate its proliferation capacity.Item Production and in vitro Evaluation of Recombinant Human HB-EGF for Wound Healing and Targeted Therapy(2025) Vovk, I.; Didan, A.; Zhukova, Dariia; Dronko, Liliia; Rebriev, Andrew; Rybalko, A.; Legach, E.; Gorbatiuk, Oksana; Usenko, Mariia; Skvarchynskyi, Andrii; Dovbymchuk, T.; Siromolot, Andriy; Romaniuk, Svitlana; Kolybo, DenysAim. The goal of the study was to evaluate the biological activity of recombinant human heparinbinding EGF-like growth factor (rhHB-EGF) on mouse fibroblasts in vitro and the possibility of its use as a potential agent for promoting wound healing and tissue regeneration. Methods. The study employed a scratch assay to evaluate the migration of mouse fibroblasts (L929 and NIH-3T3), the MTT test to assess cell proliferation and/or metabolic activity, MALDI-TOF mass spectrometry for protein identification, and flow cytometry to determine cell viability. Results. In the concentration range of 500–1000 ng/ml rhHB-EGF, no cytotoxic effect was recorded, but an increase in proliferation and/or metabolic activity, as well as migration of fibroblasts, was detected, with a maximum effect at 500 ng/ml rhHB-EGF in the cell incubation medium. A 30% overgrowth of the wound surface of fibroblasts was demonstrated in the scratch assay test under the influence of rhHB-EGF compared to the corresponding control. Conclusions. rhHB-EGF at a concentration of 500 ng/ml can be used in preparations to stimulate wound healing and tissue regeneration due to its ability to stimulate proliferation/metabolic activity and migration of fibroblasts, as well as the lack of cytotoxicity. Further, in vivo studies are needed for a comprehensive evaluation of this possibility.Item Synergistic anti-tumour effect of doxorubicin-hydrochloride with CRM197, an inhibitor of HB-EGF, in squamous-cell carcinoma(2024) Radevych, І.; Siromolot, Andriy; Kolybo, DenysСancer is one of the greatest threats to public health, its lethality comparable only to that of cardiovascular diseases, with the approach to its prevention and therapy evolving from populationbased and epidemiological studies to targeted delivery and immunological approaches. Recent studies have shown that cross reacting material (CRM197), a nontoxic variant of diphtheria toxin may play an important role in treating cancers with poor prognoses by inhibiting heparin-binding EGF-like growth factor (HB-EGF) [1, 2]. Doxorubicin-hydrochloride (DOX) is an antineoplastic prescription medicine approved by the U.S. Food and Drug Administration for the treatment of certain types of cancer, including ovarian cancer and multiple myeloma [3]. Considering that CRM197 is a known carrier in targeted delivery, CRM197-DOX complexes might be a step towards targeted therapy and reduced overall toxicity.