Duocarmycin SA Reduces Proliferation and Increases Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.

Neutrophil Extracellular DNA Traps in Response to Infection or Inflammation, and the Roles of Platelet Interactions.

Neutrophils present the host's first line of defense against bacterial infections. These immune effector cells are mobilized rapidly to destroy invading pathogens by (a) reactive oxygen species (ROS)-mediated oxidative bursts and (b) via phagocytosis. In addition, their antimicrobial service is capped via a distinct cell death mechanism, by the release of their own decondensed nuclear DNA, supplemented with a variety of embedded proteins and enzymes. The extracellular DNA meshwork ensnares the pathogenic bacteria and neutralizes them. Such neutrophil extracellular DNA traps (NETs) have the potential to trigger a hemostatic response to pathogenic infections. The web-like chromatin serves as a prothrombotic scaffold for platelet adhesion and activation. What is less obvious is that platelets can also be involved during the initial release of NETs, forming heterotypic interactions with neutrophils and facilitating their responses to pathogens. Together, the platelet and neutrophil responses can effectively localize an infection until it is cleared. However, not all microbial infections are easily cleared. Certain pathogenic organisms may trigger dysregulated platelet-neutrophil interactions, with a potential to subsequently propagate thromboinflammatory processes. These may also include the release of some NETs. Therefore, in order to make rational intervention easier, further elucidation of platelet, neutrophil, and pathogen interactions is still needed.

Local and Systemic Effects of Porphyromonas gingivalis Infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106  CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

Platelet plug formation in whole blood is enhanced in the presence of Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative anaerobic bacterium and an etiologic agent of adult periodontitis. By inducing a dysbiotic state within the host microbiota it contributes to a chronic inflammatory environment in the oral cavity. Under some circumstances, the oral bacteria may gain access to systemic circulation. While the most widely recognized function of platelets is to reduce hemorrhage in case of vascular damage, it is known that platelets are also involved in the hematologic responses to bacterial infections. Some pathogenic bacteria can interact with platelets, triggering their activation and aggregation. The aim of this study was to assess platelet responses to the presence of P. gingivalis in whole blood. Human whole blood was pretreated with P. gingivalis and then platelet plug formation was measured under high shear conditions using the PFA-100. In the presence of P. gingivalis, time for a platelet plug to occlude the aperture in the collagen/ADP cartridge was shortened in a manner dependent on bacterial concentration and the duration of bacterial preincubation of blood. P. gingivalis enhances thrombus forming potential of platelets in whole blood.