Enhanced angiogenesis of human umbilical vein endothelial cells via THP-1-derived M2c-like macrophages and treatment with proteasome inhibitors 'bortezomib and ixazomib'.

The leading cause of cancer-related death is lung cancer, with metastasis being the most common cause of death. To elucidate the role of macrophages in lung cancer and angiogenesis processes, we established an in vitro co-culture model of A549 or HUVEC with THP-1 cells that polarized to M2c macrophages with hydrocortisone. The proteasome inhibitors bortezomib and ixazomib were investigated for their effects on proliferation, invasion, migration, metastasis, and angiogenesis pathways. The effects of bortezomib and ixazomib on gene expression in gene panels, including crucial genes related to angiogenesis and proteasomes, were investigated after the co-culture model to determine these effects at the molecular level. In conclusion, bortezomib and ixazomib showed antiproliferative effects in both cells, as well as in M2c macrophage co-culture. M2c macrophages also increased invasion in A549 cells and both invasion and migration in HUVEC. mRNA expression upregulation, specifically in the NFKB and VEGF genes, supported the metastatic and angiogenic effects found in A549 and HUVEC with M2c macrophage co-culture. Additionally, bortezomib inhibited the VEGFB pathway in HUVEC and NFKB1 in A549 cells. The significant findings obtained as a result of this study will provide information regarding angiogenesis induced by M2 macrophages.

The investigation of cytotoxic and apoptotic activity of Cl-amidine on the human U-87 MG glioma cell line.

BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.

Proteasome inhibitor immunotherapy for the epithelial to mesenchymal transition: assessing the A549 lung cancer cell microenvironment and the role of M1, M2a and M2c 'hydrocortisone-polarised' macrophages.

BACKGROUND: Lung cancer is a leading cause of cancer-related deaths, primarily as a result of metastases. In this metastasis, the epithelial-to-mesenchymal transition (EMT) is essential. Interaction with the cancer cell microenvironment is primarily dependent on M1- and M2-polarized macrophage. METHODS AND RESULTS: In this study, we revealed the EMT-associated activity of M1, M2a and M2c macrophages in A549 lung cancer cells. We established a co-culture model of A549 lung cancer cells utilizing THP-1-derived M1/M2 polarised macrophages to explore the involvement of macrophages in the immune response, apoptosis, and EMT in lung cancer. Although multiple polarising agents are routinely used for M1 and M2 conversion, we assessed a new possible polarising agent, hydrocortisone. CONCLUSIONS: M1 increased A549 cell sensitivity to proteasome inhibitors and decreased A549 cell viability by inducing apoptosis. EMT was induced in the presence of M2c macrophages in A549 cells by the levels of vimentin, fibronectin, E-cadherin, NF-kB, CCL-17. We also revealed the antiproliferative effects of bortezomib and ixazomib on A549 cells in both 2D and 3D cultures. Our findings could help develop an immunotherapeutic strategy by shedding light on a previously undiscovered part of the EMT pathway. Furthermore, additional investigation may reveal that polarising tumour-associated macrophages to M1 and eliminating the M2a or particularly the M2c subtype are effective anti-cancer strategies.

Investigation of the neuroprotective and neuritogenic effects of halotolerant Penicillium flavigenum-derived sorbicillin-like compounds on PC-12 Adh cells.

Parkinson's disease (PD) is an adult-onset neurodegenerative condition caused by oxidative stress and mitochondrial malfunction. In this study, the neuroprotective and neuritogenic activity of water fraction (Sw-fr) containing sorbicillin-like active metabolites of halotolerant P. flavigenum isolated from Salt Lake in Konya, Turkey were investigated on a 6-hydroxydopamine (6-OHDA)-induced PD in vitro PC-12 Adh cell model. Firstly, Sw-fr containing sorbicillin-like active metabolites were extracted from P. flavigenum and was compared with a sorbicillin standard by liquid chromatography-mass spectrometry (LC-MS). Then, the effects of non-cytotoxic concentrations of Sw-fr on the 6-OHDA-induced PD cell model were investigated via real-time cell proliferation analysis using the RTCA DP instrument. The effects of these concentrations on mitochondrial membrane integrity, caspase-3 were investigated by flow cytometry. Neurite outgrowth analysis and immunofluorescence staining were used to explore the neuritogenic effects of neuroprotective doses. By improving PC-12 Adh cell viability, decreasing reactive oxygen species production, and reducing apoptotic cell death, 1 and 10 µg/mL Sw-fr and sorbicillin standard proved neuroprotective against 6-OHDA-induced neurotoxicity. Furthermore, 1 and 10 µg/mL Sw-fr significantly induced neurite outgrowth. As a result, sorbicillin-like active metabolites containing Sw-fr were found to have neuroprotective and neuritogenic effects. Sorbicillin-like metabolites obtained from fungi may be novel natural medicines for neurodegenerative diseases. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00498-9.