Transforming growth factor beta 1 (TGF β1) is a protein that in most cells control proliferation and differentiation. One of the best characterized functions of TGF β1 is stimulation of collagen biosynthesis that may lead to tissue fibrosis. Several reports suggest that prolidase, through regulation of expression of growth factors and transcription factors, e.g. vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1 α) may be important in many physiologic and pathophysiologic processes like: wound healing, inflammation and angiogenesis. We found that inhibitors of prolidase activity (N-benzyloxycarbonyl-l-proline, Cbz-Pro and phosphoenolopyruvate, PEP) induced decrease in TGF β1 and its receptor expressions. On the other hand, products of prolidase catalytic activity, proline (Pro) and hydroxyproline (HyPro) induced increase in the amount of TGF β1 and TGF β receptors. Simultaneously, inhibitors of prolidase induced down-regulation of expression of the phospho-AKT. An addition of Pro or HyPro to the cells induced increase in the expression of phospho-AKT. An important transcription factor involved in signal induced by TGF β receptor is mammalian target of rapamycin (mTOR). We found that prolidase inhibitors induced decrease in the expression of phospho-mTOR, while Pro or HyPro counteracted the effect. Rapamycin (pharmacological inhibitor of mTOR) resulted in decrease in prolidase activity. The down-regulation of phospho-mTOR by rapamycin contributed to down-regulation of prolidase activity suggesting its important role in prolidase-dependent function. It seems, that products of prolidase activity, Pro or HyPro may act as an interface between mTOR and phospho-mTOR in regulation of numerous TGF β receptor-dependent functions.

Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process.

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 β (Epo) and LFM-A13 (Btk inhibitor) on colon cancer in in vitro and in vivo models.

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.

The effect of Amaranthus cruentus L. seed oil (AmO) on collagen biosynthesis and wound healing was studied in cultured human dermal fibroblasts exposed to UVA radiation. It was found that UVA radiation inhibited collagen biosynthesis, prolidase activity, and expression of the β1-integrin receptor, and phosphorylated ERK1/2 and TGF-β, while increasing the expression of p38 kinase. The AmO at 0.05-0.15% counteracted the above effects induced by UVA radiation in fibroblasts. UVA radiation also induced the expression and nuclear translocation of the pro-inflammatory NF-κB factor and enhanced the COX-2 expression. AmO effectively suppressed the expression of these pro-inflammatory factors induced by UVA radiation. Expressions of β1 integrin and IGF-I receptors were decreased in the fibroblasts exposed to UVA radiation, while AmO counteracted the effects. Furthermore, AmO stimulated the fibroblast's migration in a wound healing model, thus facilitating the repair process following exposure of fibroblasts to UVA radiation. These data suggest the potential of AmO to counteract UVA-induced skin damage.

Degenerative joint diseases are related to the excessive degradation of collagen and proteoglycans in cartilage. One of the potent inflammatory mediators of cartilage metabolism is interleukin-1 (IL-1) that has been implicated in the pathogenesis of degenerative joint diseases. Peripheral benzodiazepine receptor ligands have anti-inflammatory activity in rheumatoid arthritis. The present study shows that 4'-chlorodiazepam (Ro-54864), an agonist of peripheral benzodiazepine receptors, counteract inhibition of collagen and DNA biosynthesis, induced by IL-1. Pk-1195, an antagonist of peripheral benzodiazepine receptors did not restore inhibitory effects of IL-1. The mechanism of collagen biosynthesis and cell division regulation involves insulin-like growth factor-I receptor signaling. We found that IL-1 inhibited expression of IGF-IR, while Ro-54864 stimulated the expression of this receptor. Increase in the expression of this receptor was accompanied by increase in mTOR expression and AKT phosphorylation while it had no effect on Ras-Raf-mitogen activated protein kinase (MAPK) pathway. Although IL-1 caused activation of apoptosis in chondrocytes, an addition of Ro-54864 to the cells inhibited the process as detected by annexin V cell staining followed by flow cytometry. The mechanism of this process may be related to protective effect of signal induced by IGF-I receptor. The data suggest that the mechanism of the protective effects of Ro-54864 on IL-1-induced effects in chondrocytes undergoes through mTOR and AKT signaling. It suggest that peripheral benzodiazepine receptor agonist may be considered as a potential pharmacotherapeutical agents in the treatment of inflammatory diseases.

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.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and in women. The impact of the new pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide (MM-129) was evaluated against human colon cancer in vitro and in zebrafish xenografts. Our results show that this new synthesised compound effectively inhibits cell survival in BTK-dependent mechanism. Its effectiveness is much higher at a relatively low concentration as compared with the standard chemotherapy used for CRC, i.e. 5-fluorouracil (5-FU). Flow cytometry analysis after annexin V-FITC and propidium iodide staining revealed that apoptosis was the main response of CRC cells to MM-129 treatment. We also found that MM-129 effectively inhibits tumour development in zebrafish embryo xenograft model, where it showed a markedly synergistic anticancer effect when used in combination with 5-FU. The above results suggest that this novel heterofused 1,2,4-triazine derivative may be a promising candidate for further evaluation as chemotherapeutic agent against CRC.

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.

Recombinant human erythropoietin (Epo) is an effective and convenient treatment for cancer-related anaemia. In our study for the first time, we evaluated the effect of simultaneous use of Epo and Bruton's tyrosine kinase (BTK) inhibitor LFM-A13 on the viability and tumour development of breast cancer cells. The results demonstrated that Epo significantly intensifies the anticancer activity of LFM-A13 in MCF-7 and MDA-MB-231. The featured therapeutic scheme efficiently blocked the tumour development in zebrafish experimental cancer model. Epo and LFM-A13 administered together resulted in effective cell killing, accompanied by attenuation of the BTK signalling pathways, loss of mitochondrial membrane potential (MMP), accumulation of apoptotic breast cancer cells with externalised PS, a slight increase in phase G0/G1 and a reduction in cyclin D1 expression. Simultaneous use of Epo with LFM-A13 inhibited early stages of tumour progression. This therapeutic scheme may be rationale for further possible research.

Proline oxidase (POX) is mitochondrial proline-degrading enzyme of dual apoptosis/survival function. POX expression and proline availability are considered an underlying mechanism for differential POX functions. The mechanism for POX-dependent regulation of cell death/survival was studied in wild-type (MCF-7WT) and shRNA POX-silenced breast cancer cells (MCF-7iPOX). Proline concentration and proteomic analyses were determined by LC/MS/QTOF and LC/MS/ORBITRA, respectively. Inhibition of collagen biosynthesis (proline utilizing process) by 2-methoxyestradiol (2ME) contributed to induction of apoptosis in MCF-7WT cells, as detected by increase in the expression of active caspase-3, -9 and p53. The process was not shown in MCF-7iPOX. In MCF-7iPOX cells prolidase activity and expression as well as proline concentration were drastically increased, compared to MCF-7WT cells. Down-regulation of p53 in MCF-7iPOX cells was corroborated by proteomic analysis showing decrease in the expression of p53-related proteins. The mechanism for down-regulation of p53 expression in MCF-7iPOX cells was found at the level of p53-PEPD complex formation that was counteracted by hydrogen peroxide treatment. In this study, we found that silencing POX modulate pro-survival phenotype of MCF-7 cells and suggest that the mechanism of this process undergoes through down-regulation of p53-dependent signaling.