Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP)

Ludwig, Lindsey M.; Maxcy, Katrina; LaBelle, James L.

doi:10.1007/978-1-4939-8861-7_5

Cited by 10 publications

(5 citation statements)

References 34 publications

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“…The percentage of each apoptotic marker positive cells was measured by flow cytometry (FACScan; Becton Dickinson). Acquired data were analyzed by the use of Cell Quest software [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

Enhancement of Cisplatin Cytotoxicity by Cu(II)–Mn(II) Schiff Base Tetradentate Complex in Human Oral Squamous Cell Carcinoma

et al. 2020

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Oral squamous cell carcinoma (SCC) is one of the most predominant tumors worldwide and the present treatment policies are not enough to provide a specific solution. We aimed to assess the cytotoxic effect of Cu(II)–Mn(II) Schiff base tetradentate complex alone or in combination with cisplatin against squamous cell carcinoma cell line (SCCs) in vitro. Oral-derived gingival mesenchymal stem cells (GMSCs) were used as control. The cell viability was assessed by MTT assay. IC50 values were calculated. Evaluation of apoptosis and DNA damage were performed. In addition, the expression of pro-apoptotic and anti-apoptotic genes and proteins were tested. IC50 values indicated less toxicity of the Schiff base complex on GMSCs compared to cisplatin. Schiff base complex treatment resulted in up-regulation of p53 and Bax genes expression and down-regulation of Bcl2 gene expression in SCCs paralleled with increased protein expression of caspase-3 and Bax and down-regulation of Bcl-2 protein. Annexin V-FITC apoptosis kit showed a higher apoptotic effect induced by a Schiff base complex compared to the cisplatin-treated group. These effects were markedly increased on the combination of Schiff base and cisplatin. The present study established that Cu(II)–Mn(II) Schiff base tetradentate complex might induce a cytotoxic effect on SCCs cells via induction of the apoptotic pathway. Moreover, this Schiff base complex augments the anticancer effect of cisplatin.

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“…The percentage of each apoptotic marker positive cells was measured by flow cytometry (FACScan; Becton Dickinson). Acquired data were analyzed by the use of Cell Quest software [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

Enhancement of Cisplatin Cytotoxicity by Cu(II)–Mn(II) Schiff Base Tetradentate Complex in Human Oral Squamous Cell Carcinoma

et al. 2020

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“…BH3 profiling is a functional assay that can identify selective antiapoptotic protein dependencies (BCL-2, BCL-xL, or MCL1) in different T-ALL cell lines or primary cells. In this assay, mitochondrial outer membrane permeabilization (MOMP) is a readout for apoptotic activation, which we assessed as described previously ( 18 ). In plate-based fluorimetry method, BH3 peptides were plated in triplicate on a black 384-well plate at following concentrations: BAD, HRK, and MS1 were used at 1 μmol/L concentration, whereas BIM and BID were also plated at different concentrations for titration assays (0–100 μmol/L).…”

Section: Methodsmentioning

confidence: 99%

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia

Saygin,

Giordano,

Shimamoto

et al. 2023

Clinical Cancer Research

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Purpose: Relapsed T-acute lymphoblastic leukemia (T-ALL) has limited treatment options. We investigated mechanisms of resistance to BH3 mimetics in T-ALL in order to develop rational combination strategies. We also looked at the preclinical efficacy of NWP-0476, a novel BCL-2/BCL-xL inhibitor, as single agent and combination therapy in T-ALL. Methods: We used BH3 profiling as a predictive tool for BH3 mimetic response in T-ALL. Using isogenic control, venetoclax-resistant (ven-R) and NWP-0476-resistant (NWP-R) cells, phosphokinase array was performed to identify differentially regulated signaling pathways. Results: Typical T-ALL cells had increased dependence on BCL-xL, while early T-precursor (ETP)-ALL cells had higher BCL-2 dependence for survival. BCL-2/BCL-xL dual inhibitors were effective against both subtypes of T-lineage ALL. A 71-protein human phosphokinase array showed increased LCK activity in ven-R cells, and increased ACK1 activity in ven-R and NWP-R cells. We hypothesized that pre-TCR and ACK1 signaling pathways are drivers of resistance to BCL-2 and BCL-xL inhibition, respectively. First, we silenced LCK gene in T-ALL cell lines, which resulted in increased sensitivity to BCL-2 inhibition. Mechanistically, LCK activated NF-κB pathway and the expression of BCL-xL. Silencing ACK1 gene resulted in increased sensitivity to both BCL-2 and BCL-xL inhibitors. ACK1 signaling up-regulated AKT pathway, which inhibited the pro-apoptotic function of BAD. In a T-ALL patient-derived xenograft model, combination of NWP-0476 and dasatinib demonstrated synergy without major organ toxicity. Conclusions:LCK and ACK1 signaling pathways are critical regulators of BH3 mimetic resistance in T-ALL. Combination of BH3 mimetics with tyrosine kinase inhibitors might be effective against relapsed T-ALL.

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“…THP-1 cells were cultured for 24 h before harvest and analyzed for intracellular protein using the published protocol by Ludwig and colleagues [ 60 ]. Briefly, cells were fixed and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Platelet Microparticles Protect Acute Myelogenous Leukemia Cells against Daunorubicin-Induced Apoptosis

Cacic

Reikvam

Nordgård

et al. 2021

Cancers

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The role of platelets in cancer development and progression is increasingly evident, and several platelet–cancer interactions have been discovered, including the uptake of platelet microparticles (PMPs) by cancer cells. PMPs inherit a myriad of proteins and small RNAs from the parental platelets, which in turn can be transferred to cancer cells following internalization. However, the exact effect this may have in acute myelogenous leukemia (AML) is unknown. In this study, we sought to investigate whether PMPs could transfer their contents to the THP-1 cell line and if this could change the biological behavior of the recipient cells. Using acridine orange stained PMPs, we demonstrated that PMPs were internalized by THP-1 cells, which resulted in increased levels of miR-125a, miR-125b, and miR-199. In addition, co-incubation with PMPs protected THP-1 and primary AML cells against daunorubicin-induced cell death. We also showed that PMPs impaired cell growth, partially inhibited cell cycle progression, decreased mitochondrial membrane potential, and induced differentiation toward macrophages in THP-1 cells. Our results suggest that this altering of cell phenotype, in combination with decrease in cell activity may offer resistance to daunorubicin-induced apoptosis, as serum starvation also yielded a lower frequency of dead and apoptotic cells when treated with daunorubicin.

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Flow Cytometry-Based Detection and Analysis of BCL-2 Family Proteins and Mitochondrial Outer Membrane Permeabilization (MOMP)

Cited by 10 publications

References 34 publications

Enhancement of Cisplatin Cytotoxicity by Cu(II)–Mn(II) Schiff Base Tetradentate Complex in Human Oral Squamous Cell Carcinoma

Enhancement of Cisplatin Cytotoxicity by Cu(II)–Mn(II) Schiff Base Tetradentate Complex in Human Oral Squamous Cell Carcinoma

Dual Targeting of Apoptotic and Signaling Pathways in T-Lineage Acute Lymphoblastic Leukemia

Platelet Microparticles Protect Acute Myelogenous Leukemia Cells against Daunorubicin-Induced Apoptosis

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