A real-time, bioluminescent annexin V assay for the assessment of apoptosis

Kupcho, Kevin R.; Shultz, John; Hurst, Robin; Hartnett, Jim; Zhou, Wenhui; Machleidt, Thomas; Grailer, Jamison; Worzella, Tracy J; Riss, Terry; Lazar, Dan; Cali, James J.; Niles, Andrew L.

doi:10.1007/s10495-018-1502-7

Cited by 69 publications

(59 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Briefly, in this assay, compounds that induce apoptosis will produce time- and dose-dependent increases in luminescence—annexin V fusion protein binding to exposed phosphatidylserine (PS)], which precede temporal increases in fluorescence due to secondary necrosis (loss of membrane integrity). This kinetic difference in the emergence of the signals is the hallmark of an apoptotic phenotype [ 28 ]. Our results from this assay indicated that the apoptotic phenotype of cell death is characteristic of the malignant melanoma A375 cell line, treated with both vanicosides.…”

Section: Discussionmentioning

confidence: 99%

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1

Nawrot-Hadzik

Choromańska

Abel

et al. 2020

IJMS

View full text Add to dashboard Cite

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines—keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

show abstract

Section: Discussionmentioning

confidence: 99%

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1

Nawrot-Hadzik

Choromańska

Abel

et al. 2020

IJMS

View full text Add to dashboard Cite

show abstract

“…To determine the apoptosis induced in the presence of PFD and PFD-D-Lip, the Real Time-Glo Annexin V apoptosis assay was employed. This assay measures the exposure of phosphatidylserine on the outer leaflet of cell membranes during the apoptotic process through annexin-V binding detected by luminescence, which indicates early apoptosis [52]. It was found that both PFD and PFD-D-Lip (0.5 mg/mL) induced a luminescence signal at the 3-h time point, which increased further until the 6-h time point.…”

Section: Real-time Annexin Apoptosis Assaymentioning

confidence: 99%

Systematic Development and Optimization of Inhalable Pirfenidone Liposomes for Non-Small Cell Lung Cancer Treatment

et al. 2020

View full text Add to dashboard Cite

Non-small cell lung cancer (NSCLC) is a global disorder, treatment options for which remain limited with resistance development by cancer cells and off-target events being major roadblocks for current therapies. The discovery of new drug molecules remains time-consuming, expensive, and prone to failure in safety/efficacy studies. Drug repurposing (i.e., investigating FDA-approved drug molecules for use against new indications) provides an opportunity to shorten the drug development cycle. In this project, we propose to repurpose pirfenidone (PFD), an anti-fibrotic drug, for NSCLC treatment by encapsulation in a cationic liposomal carrier. Liposomal formulations were optimized and evaluated for their physicochemical properties, in-vitro aerosol deposition behavior, cellular internalization capability, and therapeutic potential against NSCLC cell lines in-vitro and ex-vivo. Anti-cancer activity of PFD-loaded liposomes and molecular mechanistic efficacy was determined through colony formation (1.5- to 2-fold reduction in colony growth compared to PFD treatment in H4006, A549 cell lines, respectively), cell migration, apoptosis and angiogenesis assays. Ex-vivo studies using 3D tumor spheroid models revealed superior efficacy of PFD-loaded liposomes against NSCLC, as compared to plain PFD. Hence, the potential of inhalable liposome-loaded pirfenidone in NSCLC treatment has been established in-vitro and ex-vivo, where further studies are required to determine their efficacy through in vivo preclinical studies followed by clinical studies.

show abstract

“…Due to its subcellular resolution capabilities, optical microscopy methods have shown great promise in identifying, tracking, and studying cell death processes in living cells [9,10]. In particular, several nonlinear optical microscopy techniques have been developed to directly probe the metabolic state of cells with high-sensitivity based on endogenous contrast from metabolic co-enzymes [11].…”

Section: Introductionmentioning

confidence: 99%

Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy

Bower

Sorrells

et al. 2019

Biomed. Opt. Express

View full text Add to dashboard Cite

Programmed cell death, or apoptosis, is an essential process in development and homeostasis, and disruptions in associated pathways are responsible for a wide variety of diseases such as cancer, developmental abnormalities, and Alzheimer's disease. On the other hand, cell death, in many cases, is the desired outcome of therapeutic treatments targeting diseases such as cancer. Recently, metabolic imaging based on two-photon fluorescence microscopy has been developed and shown to be highly sensitive to certain cell death processes, most notably apoptosis, thus having the potential as an advanced label-free screening tool. However, the typically low acquisition rates of this imaging technique have resulted in a limited throughput approach, allowing only a small population of cells to be tracked at well-separated time points. To address this limitation, a high-speed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) platform capable of video-rate imaging is applied to study and further characterize the metabolic dynamics associated with cell death. Building upon previous work demonstrating the capabilities of this system, this microscope is utilized to study rapid metabolic changes during cell death induction, such as dose-dependency of metabolic response, response in invasive vs. noninvasive cancer cells, and response in an apoptosis-resistant cell line, which is further shown to undergo autophagy in response to toxic stimuli. Results from these experiments show that the early apoptosis-related metabolic dynamics are strongly correlated with important cellular parameters including responsiveness to apoptosis-inducing stimuli. The high speed and sensitivity of the presented imaging approach enables new investigations into this highly dynamic and complex process.

show abstract

A real-time, bioluminescent annexin V assay for the assessment of apoptosis

Cited by 69 publications

References 36 publications

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1

Systematic Development and Optimization of Inhalable Pirfenidone Liposomes for Non-Small Cell Lung Cancer Treatment

Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy

Contact Info

Product

Resources

About