Study on the Mechanism of the Adrenaline-Evoked Procoagulant Response in Human Platelets.

Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E1, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.

NSAIDs Induce Proline Dehydrogenase/Proline Oxidase-Dependent and Independent Apoptosis in MCF7 Breast Cancer Cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered in cancer therapy for their inhibitory effect on cyclooxygenase-2 (COX-2), which is overexpressed in most cancers. However, we found that NSAIDs as ligands of peroxisome proliferator-activated receptor-γ (PPARγ)-induced apoptosis independent of the COX-2 inhibition, and the process was mediated through activation of proline dehydrogenase/proline oxidase (PRODH/POX)-dependent generation of reactive oxygen species (ROS). This mitochondrial enzyme converts proline to ∆1-pyrroline-5-carboxylate (P5C) during which ATP or ROS is generated. To confirm the role of PRODH/POX in the mechanism of NSAID-induced apoptosis we obtained an MCF7 CRISPR/Cas9 PRODH/POX knockout breast cancer cell model (MCF7POK-KO). Interestingly, the studied NSAIDs (indomethacin and diclofenac) in MCF7POK-KO cells contributed to a more pronounced pro-apoptotic phenotype of the cells than in PRODH/POX-expressing MCF7 cells. The observed effect was independent of ROS generation, but it was related to the energetic disturbances in the cells as shown by an increase in the expression of AMPKα (sensor of cell energy status), GLUD1/2 (proline producing enzyme from glutamate), prolidase (proline releasing enzyme), PPARδ (growth supporting transcription factor) and a decrease in the expression of proline cycle enzymes (PYCR1, PYCRL), mammalian target of rapamycin (mTOR), and collagen biosynthesis (the main proline utilizing process). The data provide evidence that the studied NSAIDs induce PRODH/POX-dependent and independent apoptosis in MCF7 breast cancer cells.

Nonsteroidal Anti-Inflammatory Drugs as PPARγ Agonists Can Induce PRODH/POX-Dependent Apoptosis in Breast Cancer Cells: New Alternative Pathway in NSAID-Induced Apoptosis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are considered to be therapeutics in cancer prevention because of their inhibitory effect on cyclooxygenases (COX), which are frequently overexpressed in many types of cancer. However, it was also demonstrated that NSAIDs provoked a proapoptotic effect in COX knocked-out cancer cells. Here, we suggest that this group of drugs may provoke antineoplastic activity through the activation of PPARγ, which induces proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that catalyzes proline degradation, during which ATP or reactive oxygen species (ROS) are generated. We have found that NSAIDs induced PRODH/POX and PPARγ expressions (as demonstrated by Western Blot or immunofluorescence analysis) and cytotoxicity (as demonstrated by MTT, cytometric assay, and DNA biosynthesis assay) in breast cancer MCF7 cells. Simultaneously, the NSAIDs inhibited collagen biosynthesis, supporting proline for PRODH/POX-induced ROS-dependent apoptosis (as demonstrated by an increase in the expression of apoptosis markers). The data suggest that targeting proline metabolism and the PRODH/POX-PPARγ axis can be considered a novel approach for breast cancer treatment.

P5C as an Interface of Proline Interconvertible Amino Acids and Its Role in Regulation of Cell Survival and Apoptosis.

Studies of cancer metabolism have focused on the production of energy and the interconversion of carbons between cell cycles. More recently, amino acid metabolism, especially non-essential amino acids (NEAAs), has been investigated, underlining their regulatory role. One of the important mediators in energy production and interconversion of carbons in the cell is Δ1-pyrroline-5-carboxylate (P5C)-the physiological intracellular intermediate of the interconversion of proline, ornithine, and glutamate. As a central component of these conversions, it links the tricarboxylic acid cycle (TCA), urea cycle (UC), and proline cycle (PC). P5C has a cyclic structure containing a tertiary nitrogen atom (N) and is in tautomeric equilibrium with the open-chain form of L-glutamate-γ-semialdehyde (GSAL). P5C is produced by P5C synthase (P5CS) from glutamate, and ornithine via ornithine δ-amino acid transferase (δOAT). It can also be converted to glutamate by P5C dehydrogenase (P5CDH). P5C is both a direct precursor of proline and a product of its degradation. The conversion of P5C to proline is catalyzed by P5C reductase (PYCR), while proline to P5C by proline dehydrogenase/oxidase (PRODH/POX). P5C-proline-P5C interconversion forms a functional redox couple. Their transformations are accompanied by the transfer of a reducing-oxidizing potential, that affect the NADP+/NADPH ratio and a wide variety of processes, e.g., the synthesis of phosphoribosyl pyrophosphate (PRPP), and purine ribonucleotides, which are crucial for DNA synthesis. This review focuses on the metabolism of P5C in the cell as an interconversion mediator of proline, glutamate, and ornithine and its role in the regulation of survival and death with particular emphasis on the metabolic context.

MM-129 as a Novel Inhibitor Targeting PI3K/AKT/mTOR and PD-L1 in Colorectal Cancer.

BACKGROUND AND AIMS: The purpose of the present study was to examine the pharmacodynamics features of MM-129 (1,2,4-triazine derivative) as a novel promising drug candidate against colon cancer. METHODS: MM-129 was assessed for antitumor activity through an in vivo study on Cby.Cg-Foxn1nu/cmdb mice. The mechanistic studies investigated cellular affinity of a new 1,2,4-triazine derivative by measuring levels of intracellular/extracellular signal molecules participating in tumorigenesis. RESULTS: The results revealed that MM-129 significantly reduced tumor growth in mice challenged with DLD-1 and HT-29 cells. It exerted the ability to inhibit intracellular molecules promoting tumorigenesis and inducing cell cycle arrest, like Akt, mTOR, and CDK2. Simultaneously, it was able to downregulate PD-L1 expression, which involves immunological self-tolerance. Combined administration of MM-129 and 5-fluorouracil (5-FU) additionally amplified these effects, which were manifest as an increase population of cells in the G0/G1 phase. CONCLUSIONS: A novel 1,2,4-triazine derivative with a dual mechanism of antitumor activity-MM-129, may act as a chemosensitizer, overcoming chemoresistance against 5-FU, the first-line agent in the chemotherapy of colon cancer.

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

A novel plausible mechanism of NSAIDs-induced apoptosis in cancer cells: the implication of proline oxidase and peroxisome proliferator-activated receptor.

Although pharmaco-epidemiological studies provided evidence for the anticancer potential of non-steroidal anti-inflammatory drugs (NSAIDs), the mechanism of their anti-cancer activity is not known. Several lines of evidence suggest that proline dehydrogenase/proline oxidase (PRODH/POX) may represent a target for NSAIDs-dependent anti-cancer activity. PRODH/POX catalyzes conversion of proline into Δ1-pyrroline-5-carboxylate releasing ATP or reactive oxygen species for autophagy/apoptosis. Since NSAIDs are ligands of peroxisome proliferator-activated receptor (PPARs) and PPARs are implicated in PRODH/POX-dependent apoptosis we provided a hypothesis on the mechanism of NSAIDs-induced apoptosis in cancer cells.

Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer.

BACKGROUND AND PURPOSE: Bruton's tyrosine kinase (Btk) is a non-receptor tyrosine kinase involved in the activation of signalling pathways responsible for cell maturation and viability. Btk has previously been reported to be overexpressed in colon cancers. This kind of cancer is often accompanied by anaemia, which is treated with an erythropoietin supplement. The goal of the present study was to assess the effects of combination therapy with erythropoietin ß (Epo) and LFM-A13 (Btk inhibitor) on colon cancer in in vitro and in vivo models. EXPERIMENTAL APPROACH: DLD-1 and HT-29 human colon adenocarcinoma cells were cultured with Epo and LFM-A13. Cell number and viability, and mRNA and protein levels of Epo receptors, Btk and Akt were assessed. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13. KEY RESULTS: The combination of Epo and LFM-A13 mostly exerted a synergistic inhibitory effect on colon cancer cell growth. The therapeutic scheme used effectively killed the cancer cells and attenuated the Btk signalling pathways. Epo + LFM-A13 also prevented the normal process of microtubule assembly during mitosis by down-regulating the expression of Polo-like kinase 1. The combination of Epo and LFM-A13 significantly reduced the growth rate of tumour cells, while it showed high safety profile, inducing no nephrotoxicity, hepatotoxicity or changes in the haematological parameters. CONCLUSION AND IMPLICATIONS: Epo significantly enhances the antitumour activity of LFM-A13, indicating that a combination of Epo and LFM-A13 has potential as an effective therapeutic approach for patients with colorectal cancer.