Identification of proline metabolic pathway as a molecular target for experimental regulation of apoptosis/autophagy in breast cancer MCF-7 cells. (N/NCN/OP/15/002/2214), Medical University in Bialystok

       The aim of the research project was to assess the role of proline metabolism in the regulation of apoptosis / autophagy in MCF-7 breast cancer cells in order to identify the molecular target of potential pharmacotherapy of breast cancer. The studies were focused on mitochondrial proline dehydrogenase / proline oxidase (PRODH / POX), the enzyme involved in proline degradation; prolidase, a cytoplasmic enzyme providing free proline for metabolic processes; and collagen biosynthesis, a process utilizing this amino acid. It is known that proline degradation by PRODH / POX contributes to the production of ATP by promoting autophagy (pro-survival processes) or ROS inducing apoptosis. Since the mechanism of apoptosis / autophagy switching is not known the hypothesis on the regulation of this process is presented. The basis of this hypothesis is a complex regulation of proline metabolism, involving prolidase, PRODH / POX and collagen biosynthesis, which can be the apoptotic / autophagy interface. Verification of the hypothesis was carried out on an in vitro model using MCF-7 wild-type breast cancer cells and the cells with constitutively silenced PRODH / POX expression using a complementary shRNA fragment of this gene (MCF-7shPRODH / POX), patent application No. P.421954.

       This model was used to evaluate the effect of intracellular proline availability on PRODH / POX-dependent apoptosis. The results of these studies indicate the potentially important role of intracellular proline concentration in the mechanism of its participation in the PRODH / POX-dependent process of apoptosis / autophagy. The effect of selected natural compounds influencing the availability of free proline (betulin and 28-O-propylbetulin and polyphenol propolis) on the PRODH / POX-dependent apoptosis in endometrial adenocarcinoma and squamous cell carcinoma (CAL-27) was verified. It has been found that both betulin and its derivative and polyphenols of propolis induced PRODH / POX-dependent apoptosis in cancer cells.

       Based on this research project, we present a new strategy for the experimental induction of apoptosis involving the inhibition of collagen biosynthesis, increase of proline concentration in the cytoplasm and its utilization in mitochondria with the participation of PRODH / POX. The research suggests that high PRODH / POX activity promotes induction of apoptosis, while low PRODH / POX activity promotes pro-survival metabolic pathways (autophagy) in cancer cells. These conclusions have become the attitude of further hypotheses to improve the strategy of antitumor therapy by stimulation of PRODH / POX expression in cancer cells. These studies broaden the knowledge on the role of proline and PRODH / POX in cancer cell survival and apoptosis. The implementation of this project also resulted in establishing cooperation with outstanding scientists from Italy and USA.

1. Zareba I, Palka J: Prolidase-proline dehydrogenase/proline oxidase/collagen biosynthesis axis as a potential interface of apoptosis/autophagy. Biofactors 2016;42:341-348.
2. Szoka L, Karna E, Hlebowicz-Saratb K, Karaszewski J, Boryczka S, Palka J: Acetylenic derivative of betulin induces apoptosis in endometrial adenocarcinoma cell line. Biomedicine & Pharmacotherapy 2017;95:429–436.
3. Zareba I, Surazynski A, Chrusciel M, Miltyk W, Doroszko M, Rahman N, Palka J: Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX-Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model. Cellular Physiology and Biochemistry 2017;43:670-684.
4. Zareba I, Celinska-Janowicz K, Surazynski A, Miltyk W, Palka J: Silencing Proline Oxidase directs proline-dependent pro-survival pathways in MCF-7 cells. Oncotarget 2018, doi.org/10.18632/oncotarget.24466.
5. Celinska-Janowicz K, Zareba I, Lazarek U, Teul U, Tomczyk M, Palka J, Miltyk W: Constituents of propolis: chrysin, caffeic acid, p-coumaric acid, and ferulic acid induce PRODH/POX-dependent. Frontiers in Pharmacology 2018;9:336, 3 pp.

Identification of the mechanism of antineoplastic activity of metformin, as an experimental approach to improve cancer treatment (2017/25/B/NZ7/02183). Medical University in Bialystok

       Metformin is currently the first-line drug in the treatment of type II diabetes. Antineoplastic activity of metformin has been documented in pharmaco-epidemiological study in 2005 showing a reduced risk of cancer in patients with type II diabetes who received metformin. Numerous experimental and clinical studies are currently underway. However, the molecular mechanism of anti-cancer activity of metformin is not yet known.

       One of the effects of metformin is activation of AMP kinase (AMPK). When the AMP / ATP ratio increases, AMPK is activated to restore normal ATP levels and inhibit cell energy expenditure. AMPK is therefore regulated especially in conditions of energy shortage (eg during starvation) and hypoxia (low oxygen level). On the one hand, AMPK inhibits anabolic processes and stimulates catabolism on the other. One of the energy substrates under glucose deficiency is proline, derived from protein degradation products, mainly collagen. Proline is degraded in mitochondria only by proline dehydrogenase / proline oxidase (PRODH / POX). Particularly interesting in this context is observation of the induction of PRODH / POX by AMPK. The main process of proline utilization is the collagen biosynthesis, which may limit the availability of proline to its degradation in mitochondria. On the other hand, prolidase activity (proline releasing enzyme from imidodipeptides) is an important regulator of free proline concentration in the cytoplasm.

       PRODH / POX is a mitochondrial enzyme catalyzing the conversion of proline to pyrrolidine-5-carboxylic acid (P5C). During the conversion of proline to P5C, electrons are transported to the respiratory chain producing ATP or reactive oxygen species (ROS) are generated. In the first case, activation of PRODH / POX leads to the production of ATP for survival, and in the second case, ROS induces apoptosis. Although the mechanism of switching PRODH / POX from inhibitory to stimulatory for growth of tumor cells is not known, it seems that proline availability plays important role in this process. The above data suggest potentially important role of proline availability in the regulation of apoptosis / autophagy.

       The link between AMPK, PRODH / POX and proline with apoptosis / autophagy in tumor cells and stimulatory effect of metformin on AMPK allows to present a hypothesis on the mechanism of metformin-induced apoptosis / autophagy. The intracellular concentration of proline and its conversion to P5C may play a key role in this process. For this reason, proline metabolism may play an important role in the regulation of apoptosis / autophagy. It can be regulated by some growth factor receptors and transcription factors such as HIF-1α, NF-κB.

       The purpose of the research project is to identify the mechanism of anticancer activity of metformin as an apoptosis inducer in breast cancer cells by analyzing the expression of some receptors, transcription factors, signaling proteins, AMPK, PRODH / POX, and apoptosis and autophagy markers, under controlled conditions of cytoplasmic proline level.

       In order to carry out the project, clones of MCF-7 breast cancer cells with modified PRODH / POX and prolidase expressions will be prepared. The effect of metformin on some metabolic processes in these cells will be studied. It is planned to evaluate the effect of metformin on cell proliferation, collagen biosynthesis, prolidase activity, expression of AMPK, PRODH/POX, some growth factor receptors, transcription factors, as well as proteins of signaling pathways by RT-qPCR technique, and Western immunoblot. Protein expression levels will be assessed by immunocytochemistry using confocal microscopy and flow cytometry. The impact of metformin on the cell cycle will also be assessed. Analysis of amino acids by high performance liquid chromatography or gas chromatography coupled with mass spectrometry (LC- MS / GC-MS) will be performed at the final stage of the study, evaluating the effect of metformin on the amino acid profile of PRODH / POX-modified MCF-7 cells.

       This project aims to clarify the molecular mechanism of anti-cancer action of metformin in the experimental model of breast cancer cells. The biological effects of metformin on breast cancer cells will be assessed with respect to the potential use in development of a new targeted cancer pharmacotherapy. Knowing the target of antineoplastic action of metformin allows to improve the pharmacotherapy of cancer. The scientific results of the research project will be documented in journals with a high impact factor and posters presented at national and international scientific conferences.

1. Huynh Thi Yen Ly, Zaręba Ilona, Baszanowska Weronika, Lewoniewska Sylwia, Pałka Jerzy. Understanding the role of key amino acids in regulation of proline dehydrogenase/proline oxidase (prodh/pox)-dependent apoptosis/autophagy as an approach to targeted cancer therapy. Molecular and Cellular Biochemistry, 2020, 466, s. 35-44; Impact Factor: 3.396, Punktacja MEiN: 70.000, DOI: 10.1007/s11010-020-03685-y.
2. Patent: Dwuniciowy kwas nukleinowy do wyciszania ekspresji genu kodującego białko PRODH/POX i jego zastosowania, wektor ekspresyjny, komórka gospodarza, klon komórkowy, kompozycja farmaceutyczna, sposób in vitro wyciszania ekspresji genu kodującego białko PRODH/POX, jednoniciowy kwas nukleinowy do wyciszania ekspresji genu kodującego białko PRODH/POX i jego zastosowania. Przyznane patenty nr 240375, nr 240376, nr 239994.
3. Karna Ewa, Szoka Łukasz Marek, Huynh Thi Yen Ly, Pałka Jerzy. Proline-dependent regulation of collagen metabolism. Cellular and Molecular Life Sciences 2020 : 77, 10, s. 1911-1918, Impact Factor: 9.261, Punktacja MEiN: 140.000, DOI: 10.1007/s00018-019-03363-3.
4. Pałka Jerzy, Ościłowska Ilona, Szoka Łukasz Marek. Collagen metabolism as a regulator of proline dehydrogenase/proline oxidase-dependent apoptosis/autophagy. Amino Acids, 2021, 9 pp; Impact Factor: 3.520; Punktacja MEiN: 100.000, DOI: 10.1007/s00726-021-02968-y.
5. Zaręba Ilona, Huynh Thi Yen Ly, Kazberuk Adam, Teul Joanna, Klupczyńska Agnieszka, Matysiak Jan, Surażyński Arkadiusz, Pałka Jerzy. Overexpression of prolidase induces autophagic death in MCF-7 breast cancer cells. Cellular Physiology and Biochemistry, 2020, 54, 875-887; Punktacja MEiN: 140.000, DOI: 10.33594/000000275.
6. Ościłowska Ilona, Huynh Thi Yen Ly, Baszanowska Weronika, Prokop Izabela, Surażyński Arkadiusz, Galli Mauro, Zabielski Piotr, Pałka Jerzy. Proline oxidase silencing inhibits p53-dependent apoptosis in MCF-7 breast cancer cells. Amino Acids, 2021, 14 pp; Impact Factor: 3.520, Punktacja MEiN: 100.000, DOI: 10.1007/s00726-021-03013-8.
7. Klupczyńska Agnieszka, Misiura Magdalena, Miltyk Wojciech, Ościłowska Ilona, Pałka Jerzy, Kokot Zenon, Matysiak Jan. Development of an LC-MS targeted metabolomics methodology to study proline metabolism in mammalian cell cultures. Molecules, 2020: 25, 20, 13 pp, Article ID 4639; Impact Factor: 4.411, Punktacja MEiN: 140.000, DOI: 10.3390/molecules25204639.
8. Huynh Thi Yen Ly, Ościłowska Ilona, Saiz Jorge, Nizioł Magdalena, Baszanowska Weronika, Barbas Coral, Pałka Jerzy. Metformin treatment or PRODH/POX-knock out similarly induces apoptosis by reprograming of amino acid metabolism, TCA, urea cycle and pentose phosphate pathway in MCF-7 breast cancer cells. Biomolecules 2021 : 11, 12, 21 pp, Article ID 1888; Impact Factor: 4.879; Punktacja MEiN: 100.000, DOI: 10.3390/biom11121888
9. Huynh Thi Yen Ly, Ościłowska Ilona, Szoka Łukasz, Piktel Ewelina, Baszanowska Weronika, Bielawska Katarzyna, Bucki Robert, Miltyk Wojciech, Pałka Jerzy. Metformin induces PRODH/POX-dependent apoptosis in breast cancer cells. Frontiers in Molecular Biosciences 2022, 9, 11 pp, Article ID 869413, Impact Factor: 5.246, Punktacja MNiSW: 140.000, DOI: 10.3389/fmolb.2022.869413
10. Ościłowska Ilona, Rółkowski Karol, Baszanowska Weronika, Huynh Thi Yen Ly, Lewoniewska Sylwia, Nizioł Magdalena, Sawicka Magdalena, Bielawska Katarzyna, Szoka Paweł, Miltyk Wojciech, Pałka Jerzy. Proline dehydrogenase/proline oxidase (PRODH/POX) is involved in the mechanism of metformin-induced apoptosis in C32 melanoma cell line.  International Journal of Molecular Sciences 2022 : 23, 4, 15 pp., Article ID: 2354 Impact Factor: 5.924, Punktacja MEiN: 140.000, DOI: 10.3390/ijms23042354
11. Rozprawa doktorska: Karol Rółkowski - Wpływ metforminy na proces POX-zależnej apoptozy/autofagii w komórkach czerniaka C32. Publiczna obrona rozprawy doktorskiej: 30.06.2022
12. Rozprawa doktorska: Thi Yen Ly Huynh - Metabolomic approach to understand the mechanism of metformin-induced PRODH/POX-dependent apoptosis in MCF-7 breast cancer cells. Rozprawa doktorska złożona: 28.04.2022
13. Rozprawa habilitacyjna: Ilona Ościłowska (Zaręba) – Mechanizmy regulujące PRODH/POX-zależną apoptozę w komórkach raka piersi MCF-7. Rozprawa hablilitacyjna złożona do Rady Doskonałości Naukowej: 28.01.2022

The role of estrogens in the regulation of PRODH/POX-dependent apoptosis/autophagy in breast cancer cells MCF-7 (2017/27/N/NZ7/01770 ). Medical University in Bialystok.

       Estrogens mainly estradiol are important regulators of cell proliferation, survival and differentiation in various tissues. One of the metabolites of estradiol is 2-methoxyestradiol (MOE), which inhibits cell proliferation in various types of cancer. An important function of estrogens is the regulation of connective tissue metabolism. Estradiol stimulates collagen turnover by enhancing the biosynthesis and degradation of this protein. An indicator of collagen turnover is increase of prolidase activity (the enzyme releasing proline from imidodipeptides for collagen resynthesis).

       PRODH/POX is a mitochondrial enzyme catalyzing the conversion of proline to pyrrolidine-5-carboxylic acid (P5C). During the conversion of proline to P5C, electrons are transported to the respiratory chain, producing ATP or reactive oxygen species (ROS). In the first case, activation of PRODH / POX leads to the production of ATP for survival, in the second one, ROS induces apoptosis. The mechanism of switching the PRODH / POX function from inhibitory to stimulatory for tumor cell growth is not known. Probably the availability of proline in this process may play an important role in the mechanism of regulation of apoptosis/autophagy.

       The link between estradiol and its metabolite (MOE), PRODH/POX and proline with apoptosis/autophagy in tumor cells allows to present a hypothesis on the role of estradiol and its metabolite (MOE) in regulation of apoptosis/autophagy. The intracellular concentration of proline and its conversion to P5C may play a key role in this process. It contributes to induction apoptosis in cancer cells by activation of signaling pathways including PRODH/POX activity and some transcriptional factors.

       The purpose of the research project is to verify the hypothesis that the activation of estrogen receptor stimulates proline metabolism (increase in collagen biosynthesis and proline utilization in this process) leading to a reduction in the amount of free proline (as a substrate for PRODH/POX-dependent apoptosis) and creating a pro-survival phenotype of breast cancer cells (MCF-7). In contrast, the estradiol metabolite, MOE, by impaired collagen biosynthesis leads to increase in intracellular proline concentration and inducing PRODH/POX-dependent apoptosis. The impairment of estrogen receptor function through MOE should therefore contribute to the induction of the pro-apoptotic phenotype of the cell.

       In order to carry out the project, clones of MCF-7 breast cancer cells with modified PRODH / POX expressions will be prepared. The effect of estradiol and its metabolite (MOE) on some metabolic processes in these cells will be studied. It is planned to evaluate the effect of estradiol and MOE on cell proliferation, collagen biosynthesis, prolidase activity, expression of some apoptosis and autophagy markers, PRODH/POX, some growth factor receptors, transcription factors, as well as proteins of signaling pathways by RT-qPCR technique, and Western immunoblot. Protein expression will be also assessed by immunocytochemistry using confocal microscopy and flow cytometry. The impact of estradiol and MOE on the cell cycle will also be assessed. Analysis of amino acids by high performance liquid chromatography or gas chromatography coupled with mass spectrometry (LC- MS / GC-MS) will be performed at the final stage of the study, evaluating the effect of estradiol and MOE on the amino acid profile MCF-7 cells with modified PRODH/POX expression.

       This project aims to clarify the molecular impact of estradiol and its metabolite (MOE) on PRODH/POX-dependent apoptosis/autophagy in the experimental model of breast cancer cells. The biological effects of estrogen and MOE on breast cancer cells will be assessed with respect to the potential use in development of a new targeted cancer pharmacotherapy. Understanding the role of estradiol and MOE on PRODH/POX-dependent apoptosis/autophagy control help to improve the pharmacotherapy of cancer. The scientific results of the research project will be documented in journals with a high impact factor and posters presented at national and international scientific conferences.

1. Lewoniewska Sylwia, Ościłowska Ilona, Forlino Antonella, Pałka Jerzy. Understanding the role of estrogen receptor status in PRODH/POX-dependent apoptosis/survival in breast cancer cells. Biology 2021: 10, 12, 15 pp, Article ID 1314; Impact Factor: 5.079; Punktacja MEiN: 100.000, DOI: 10.3390/biology10121314
2. Lewoniewska Sylwia, Ościłowska Ilona, Huynh Thi Yen Ly, Prokop Izabela, Baszanowska Weronika, Bielawska Katarzyna, Pałka Jerzy. Troglitazone-induced PRODH/POX-dependent apoptosis occurs in the absence of estradiol or ERβ in ER-negative breast cancer cells. Journal of Clinical Medicine 2021: 10, 20, 18 pp., Article ID: 4641; Impact Factor: 4.242; Punktacja MEiN: 140.000, DOI: 10.3390/jcm10204641.
3. Ościłowska Ilona, Huynh Thi Yen Ly, Baszanowska Weronika, Prokop Izabela, Surażyński Arkadiusz, Galli Mauro, Zabielski Piotr, Pałka Jerzy. Proline oxidase silencing inhibits p53-dependent apoptosis in MCF-7 breast cancer cells. Amino Acids, 2021, 14 pp; Impact Factor: 3.520, Punktacja MEiN: 100.000, DOI: 10.1007/s00726-021-03013-8.
4. Rozprawa doktorska: Sylwia Lewoniewska – The role of estrogen receptor status in proline dehydrogenase/proline oxidase-dependent apoptosis in breast cancer cells. Rozprawa doktorska złożona: 13.06.2022
5. Rozprawa habilitacyjna: Ilona Ościłowska (Zaręba) – Mechanizmy regulujące PRODH/POX-zależną apoptozę w komórkach raka piersi MCF-7. Rozprawa hablilitacyjna złożona do Rady Doskonałości Naukowej: 28.01.2022