DNA Damage-Inducing 10-Methoxy-canthin-6-one (Mtx-C) Promotes Cell Cycle Arrest in G2/M and Myeloid Differentiation of Acute Myeloid Leukemias and Leukemic Stem Cells.

Synthetic 10-methoxy-canthin-6-one (Mtx-C), an alkaloid derivative, exhibits cytotoxic effects against acute myeloid cells (AMLs) and leukemic stem cells (LSCs) at a concentration of approximately 60 µM. However, the antitumor mechanism of Mtx-C in AMLs and LSCs remains elusive. Using Mtx-C at concentrations with low cytotoxicity (2-4 µM) for 72 h, we observed cell arrest with the accumulation of cells in the G2/M phase of the cell cycle. This effect was controlled by cyclin B1 expression and induction of the DNA damage cascade characterized by ATM, ATR, Chk1/2, p53, and H2A.X phosphorylation. Molecular docking analysis confirmed Mtx-C as a DNA intercalator. Moreover, the expression of inhibitors of cyclin-dependent kinases, including p21 (Cip1) and p27 (Kip1), increased. In addition, several miRNAs that are considered oncosuppressors were regulated by Mtx-C in Kasumi-1 cells. Finally, concomitant with cell cycle arrest, the underlying molecular mechanisms of Mtx-C in AML cells include myeloid differentiation, as evidenced by the increased expression of PU.1, myeloperoxidase, CD15, CD11b, and CD14 in the AML and LSC populations with the participation of p38 mitogen-activated protein kinase. Thus, we showed that Mtx-C simultaneously induced cell cycle arrest and myeloid differentiation in AML lineages and in the LSC population, providing insights into new therapeutic alternatives for the treatment of AML based on naturally occurring molecules.

SARS-CoV-2 infection and replication kinetics in different human cell types: The role of autophagy, cellular metabolism and ACE2 expression.

AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.

Primary Role for Kinin B1 and B2 Receptors in Glioma Proliferation.

This study investigated the role of kinins and their receptors in malignant brain tumors. As a first approach, GL-261 glioma cells were injected (2 × 105 cells in 2 µl/2 min) into the right striatum of adult C57/BL6 wild-type, kinin B1 and B2 receptor knockout (KOB1R and KOB2R) and B1 and B2 receptor double knockout mice (KOB1B2R). The animals received the selective B1R (SSR240612) and/or B2R (HOE-140) antagonists by intracerebroventricular (i.c.v.) route at 5, 10, and 15 days. The tumor size quantification, mitotic index, western blot analysis, quantitative autoradiography, immunofluorescence, and confocal microscopy were carried out in brain tumor samples, 20 days after tumor induction. Our results revealed an uncontrolled tumor growing in KOB1R or SSR240612-treated mice, which was blunted by B2R blockade with HOE-140, suggesting a crosstalk between B1R and B2R in tumor growing. Combined treatment with B1R and B2R antagonists normalized the upregulation of tumor B1R and decreased the tumor size and the mitotic index, as was seen in double KOB1B2R. The B1R was detected on astrocytes in the tumor, indicating a close relationship between this receptor and astroglial cells. Noteworthy, an immunohistochemistry analysis of tumor samples from 16 patients with glioma diagnosis revealed a marked B1R immunopositivity in low-grade gliomas or in older glioblastoma individuals. Furthermore, the clinical data revealed a significantly higher immunopositivity for B1R, when compared to a lower B2R immunolabeling. Taken together, our results show that blocking simultaneously both kinin receptors or alternatively stimulating B1R may be of therapeutic value in the treatment of brain glioblastoma growth and malignancy.