A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

Patel, Manish I.; Tuckerman, Rodney; Dong, Qihan

doi:10.1007/s10529-005-5803-x

Cited by 34 publications

(21 citation statements)

References 5 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…LNCaP and PC3 cells were treated with a range of concentrations (0.08-2.5%) of BBP in 10% FCS-containing RPMI for 72 h. The mitochondria active cell numbers were assessed using the [3-(4,5-dimethylthiazol-2-yl)-5-(-3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) as described previously (25). The cell lines were plated in quadruplicate in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Food Extracts Consumed in Mediterranean Countries and East Asia Reduce Protein Concentrations of Androgen Receptor, Phospho-Protein Kinase B, and Phospho-Cytosolic Phospholipase A2α in Human Prostate Cancer Cells

Singh

Xie

et al. 2010

The Journal of Nutrition

Self Cite

View full text Add to dashboard Cite

Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

show abstract

Section: Methodsmentioning

confidence: 99%

Food Extracts Consumed in Mediterranean Countries and East Asia Reduce Protein Concentrations of Androgen Receptor, Phospho-Protein Kinase B, and Phospho-Cytosolic Phospholipase A2α in Human Prostate Cancer Cells

Singh

Xie

et al. 2010

The Journal of Nutrition

Self Cite

View full text Add to dashboard Cite

show abstract

“…After 48 h, cells adherent on 96 well plates were exposed to the indicated treatments for the stated time intervals. Cell growth was assessed using MTS (CellTiter 96 AQ ueous One Solution Cell Proliferation Assay, Promega, Madison, WI) as described previously (17). Values are expressed relative to untreated controls.…”

Section: Methodsmentioning

confidence: 99%

Cytosolic Phospholipase A2-α: A Potential Therapeutic Target for Prostate Cancer

Patel

Singh

Niknami

et al. 2008

Clinical Cancer Research

Self Cite

103

View full text Add to dashboard Cite

Purpose: Cytosolic phospholipase A2-a (cPLA 2 -a) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA 2 -a in prostate cancer cell lines and tissue and the effect of targeting cPLA 2 -a in vitro and in vivo. Experimental Design: The expression of cPLA 2 -a was determined in prostate cancer cells by reverse transcription-PCR,Western blot, and immunocytochemistry. Growth inhibition, apoptosis, and cPLA 2 -a activity were determined after inhibition with cPLA 2 -a small interfering RNA or inhibitor (Wyeth-1). Cytosolic PLA 2 -a inhibitor or vehicle was also administered to prostate cancer xenograft mouse models. Finally, the expression of phosphorylated cPLA 2 -a was determined by immunohistochemistry in human normal, androgen-sensitive and androgen-insensitive prostate cancer specimens. Results: cPLA 2 -a is present in all prostate cancer cells lines, but increased in androgen-insensitive cells. Inhibition with small interfering RNA or Wyeth-1results in significant reductions in prostate cancer cell numbers, as a result of reduced proliferation as well as increased apoptosis, and this was also associated with a reduction in cPLA 2 -a activity. Expression of cyclin D1and phosphorylation of Akt were also observed to decrease. Wyeth-1inhibited PC3 xenograft growth by f33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phosphorylated cPLA 2 -a is increased when hormone refractory is reached. Conclusions: Expression and activation of cPLA 2 -a are increased in the androgen-insensitive cancer cell line and tissue. Inhibition of cPLA 2 -a results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory prostate cancer.Previous studies have shown that the eicosanoid pathway is activated in many types of cancers (1) including prostate (2 -9). Eicosanoids, which are the products of the cyclooxygenase (COX) and lipoxygenase pathways, contribute to cancer progression by promoting cell proliferation, motility, invasion, and angiogenesis (8 -10). Eicosanoids are synthesized from intracellular arachidonic acid, which is released from membrane phospholipids by the action of phospholipase A 2 (PLA 2 ; refs. 11,12). Of the known mammalian PLA 2 enzymes, cytosolic PLA 2 -a (cPLA 2 -a), an 85-kDa protein, is the predominant source of intracellular arachidonic acid for eicosanoid production.We have previously shown that expression of Annexin II, a calcium-dependent phospholipid-binding protein and inhibitory regulator of cPLA 2 -a, is lost in human prostate cancer (13). We have also reported that secretory PLA 2 -IIA (sPLA 2 -IIA), one of the secretory PLA 2 s, is overexpressed in prostate cancer cells including those remaining after androgen ablation therapy (14). The growth-promoting effect of sPLA 2 -IIA seems to be via cPLA 2 -a (14). These data have prompted us to investigate the role of cPLA 2 -a in prostate canc...

show abstract

“…3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega) was done following manufacturer's instructions. Glioma cells were seeded into 96-well plates (14) at a density of 4,000 per well for U87 and U251 and at 12,000 per well for GBM12 1 day before treatment initiation. Dose-response curves were generated using a range of viral multiplicities of infection (MOI) from 0 to 1.0 and different RT doses from 0 to 15 Gy, delivered with a 137 Cs irradiator.…”

Section: Methodsmentioning

confidence: 99%

Combination of Measles Virus Virotherapy and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Liu¹,

Sarkaria

Petell³

et al. 2007

Clinical Cancer Research

View full text Add to dashboard Cite

Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months.We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RTwith MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase^mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality^or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RTcombination^treated cells, as compared with single agent MV-CEA^treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore,TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination^treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/ RT^induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1blocked MV/RT^induced PARP cleavage whereas the Fas agonistic antibody CH11increased PARP cleavage in MV/RTcombination^treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway.These results could have translational implications in glioma therapy.

show abstract

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

Cited by 34 publications

References 5 publications

Food Extracts Consumed in Mediterranean Countries and East Asia Reduce Protein Concentrations of Androgen Receptor, Phospho-Protein Kinase B, and Phospho-Cytosolic Phospholipase A2α in Human Prostate Cancer Cells

Food Extracts Consumed in Mediterranean Countries and East Asia Reduce Protein Concentrations of Androgen Receptor, Phospho-Protein Kinase B, and Phospho-Cytosolic Phospholipase A2α in Human Prostate Cancer Cells

Cytosolic Phospholipase A2-α: A Potential Therapeutic Target for Prostate Cancer

Combination of Measles Virus Virotherapy and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Contact Info

Product

Resources

About