Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells

Jost, L.; Kirkwood, John M.; Whiteside, Theresa L.

doi:10.1016/s0022-1759(12)80003-2

Cited by 171 publications

(99 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Specific cytotoxicity was monitored by an XTT (2,3-bis (2-methoxy-4-nitro-5sulphonyl)-5((phenyl-amino)carbonyl)-2H-tetrazolium hydroxide) reagent-based colorimetric assay, according to Jost. 38 Briefly, engineered and mock-transduced T cells were cocultivated with ErbB2 + or ErbB2 -tumor cells. After 48 h, XTT (1 mg ml -1 ) (Cell Proliferation Kit II; Roche Diagnostics, GmbH, Mannheim, Germany) was added and incubated for 30 to 90 min at 37 1C.…”

Section: Car-mediated Activation Of Engineered T Cellsmentioning

confidence: 99%

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

2010

View full text Add to dashboard Cite

Adoptive immunotherapy of cancer using chimeric antigen receptor (CAR)-engineered T cells with redirected specificity showed efficacy in recent trials. In preclinical models, 'second-generation' CARs with CD28 costimulatory domain in addition to CD3z performed superior in redirecting T-cell effector functions and survival. Whereas CD28 costimulation sustains physiological T-cell receptor (TCR)-CD3 activation of naïve T cells, the impact of CD28 cosignalling on the threshold of CAR-mediated activation of pre-stimulated T cells without B7-CD28 recruitment remained unclear. Using CARs of different binding affinities, but same epitope specificity, we demonstrate that CD28 cosignalling neither lowered the antigen threshold nor the binding affinity for redirected T-cell activation. 'Affinity ceiling' above which increase in affinity does not increase T-cell activation was not altered. Accordingly, redirected tumor cell killing depended on the binding affinity but was likewise effective for CD3z and CD28-CD3z CARs. In contrast to CD3z, CD28-CD3z CAR-driven activation was not increased further by CD28-B7 engagement. However, CD28 cosignalling, which is required for interleukin-2 induction could not be replaced by high-affinity CD3z CAR binding or high-density antigen engagement. We conclude that CD28 CAR cosignalling does not alter the activation threshold but redirects T-cell effector functions.

show abstract

Section: Car-mediated Activation Of Engineered T Cellsmentioning

confidence: 99%

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

2010

View full text Add to dashboard Cite

show abstract

“…After preincubation for 24 hr, the medium was replaced with endothelial-serum free medium without growth factors for HUVECs and serum-free RPMI-1640 media for LLC cells containing with various concentrations of OXO. The cultures were incubated for 12 hr (LLC cells) or 24 hr (HUVECs, LLC cells) and then metabolically viable cell fraction was determined by 2,3-bis[l2-methoxy-4-nitro-5-sulfo]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric method, 23 which measures mitochondrial ability to metabolize the redox sensitive dye as described in a previous report. 24 Mitogen-stimulated HUVEC proliferation assay HUVECs (5 3 10 3 ) were seeded in complete medium onto 0.1% gelatin coated 96-well and incubated in a humidified incubator for 24 hr.…”

Section: Cells and Culture Conditionsmentioning

confidence: 99%

6‐(1‐Oxobutyl)‐5,8‐dimethoxy‐1,4‐naphthoquinone inhibits lewis lung cancer by antiangiogenesis and apoptosis

Lee

Song

et al. 2007

Intl Journal of Cancer

View full text Add to dashboard Cite

Lung cancer is a leading cause of cancer mortality worldwide. Novel and nontoxic agents targeting angiogenesis and tumor cell proliferation and survival are desirable for lung cancer chemoprevention and treatment. Previously we have reported that 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO) exhibits anti-tumor activity against S-180 sarcoma in vitro and in vivo. Here we studied the anti-angiogenic and apoptogenic attributes of OXO in vitro and in vivo targeting lung cancer. In human umbilical vein endothelial cells (HUVECs), we show that OXO more potently inhibited VEGF-stimulated than basic bFGF-stimulated HUVEC proliferation and capillary differentiation. In Lewis lung carcinoma (LLC) cells, OXO not only induces S-phase arrest and mitochondria/caspase-9 pathway mediated apoptosis, but also effectively down-regulated the hypoxia-induced expression of HIF-1a and VEGF at mRNA and protein levels in LLC and decreased VEGF secretion into conditioned culture media. OXO significantly reduced in vivo functional angiogenesis in the mouse Matrigel plug assay. Furthermore, OXO potently inhibited the growth of LLC cells inoculated on the flank of syngenic mice at dosages that did not affect their body weight. The in vivo anti-cancer effect was associated with decreased HIF-1a and VEGF expression, decreased microvessel density as well as a reduction of tumor cell proliferation and increased tumor cell apoptosis. Taken together, these results demonstrate that OXO exerts anti-cancer activity through anti-angiogenesis and tumor cell cycle arrest and apoptosis. These findings warrant additional studies of OXO as a novel agent for the chemoprevention and treatment of lung cancer. ' 2007 Wiley-Liss, Inc.Key words: angiogenesis; HIF-1 alpha; VEGF; apoptosis; lung cancer Lung cancer is the leading cause of cancer mortality worldwide, 1 including the United States (US). 2 The survival benefit of current cytotoxic cancer therapeutic drugs is very dismal in spite of serious drug side effects. As in many other solid cancers, lung cancer development (carcinogenesis) is a multiple step process that involves alterations of many molecular and cellular pathways regulating cancer cell proliferation, survival (anti-apoptosis) as well as neo-angiogenesis. Induction of apoptosis by chemopreventive agents can lead to the elimination of pre-neoplastic cells, affording a permanent protection against carcinogenesis. 3,4 Most anticancer therapy drugs induce apoptosis to achieve therapeutic efficacy. Novel and nontoxic agents that exert selective multi-targeting actions on cancer cell cycle progression and apoptosis as well as neo-angiogenesis will be important to combat this deadly disease through chemoprevention and innovative treatment.Much work in the past decades has convincingly supported the obligatory need of angiogenesis for solid tumor growth and progression. 3,[5][6][7] Hypoxia commonly develops within solid tumors because the rate of tumor cell proliferation is out-pacing the rate of blood vessel formation. 8 Hypoxia is a potent ...

show abstract

“…To determine cell viability, XTT assay was performed in 96-well plate format as described previously (Jost et al, 1992). Briefly, 1x10 4 cells were seeded in each well in 100 µL culture medium.…”

Section: Cell Viabilitymentioning

confidence: 99%

Antitumorigenicity of xanthones-rich extract from Garcinia mangostana fruit rinds on HCT 116 human colorectal carcinoma cells

Aisha¹,

Abu‐Salah²,

Nassar³

et al. 2011

Rev. bras. farmacogn.

View full text Add to dashboard Cite

This study aimed to investigate the antitumorigenicity of xanthones-rich extract from Garcinia mangostana L., Clusiaceae, fruit rinds which was obtained by a simple recrystallization of 75% ethanolic extract. α-Mangostin content of the extract was determined qualitatively by TLC and quantitatively by HPLC, and total xanthones content was quantified by UV spectrophotometry. The extract was evaluated for cytotoxicity, apoptosis and antitumorigenicity on HCT 116 human colorectal carcinoma cells. α-Mangostin was found to be the main constituent of the extract which was 71.2±0.1%, and the total xanthones content was 95±4.8% (wt/ wt). The extract showed potent dose dependent cytotoxicity with IC50 value 9.2 µg/ mL. Apoptosis studies revealed activation of caspases 3 and 7, DNA fragmentation, chromatin condensation and loss of mitochondrial membrane potential. Studies on cell migration and colony formation indicate reduced cell migration ability and clonogenicity of treated HCT 116 cells at sub-inhibitory concentrations. Taken together, the cytotoxic effect of the xanthones extract is mediated through the mitochondrial pathway of apoptosis. The reduced cell migration and clonogenicity of HCT 116 cells might prevent both primary and metastatic tumor growth in vivo which will be the topic of our future work using the metastatic orthotopic colon cancer model.

show abstract

Improved short- and long-term XTT-based colorimetric cellular cytotoxicity assay for melanoma and other tumor cells

Cited by 171 publications

References 9 publications

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

6‐(1‐Oxobutyl)‐5,8‐dimethoxy‐1,4‐naphthoquinone inhibits lewis lung cancer by antiangiogenesis and apoptosis

Antitumorigenicity of xanthones-rich extract from Garcinia mangostana fruit rinds on HCT 116 human colorectal carcinoma cells

Contact Info

Product

Resources

About