Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

2-methoxyestradiol (2ME 2 ) is an endogenous metabolite of estradiol with estrogen-receptor-independent antitumor and antiangiogenic activity. We examined the effects of 2ME 2 on the cellular proliferation of 8 human melanoma cell lines. We show that 2ME 2 inhibited cell proliferation by inducing apoptosis and an arrest in the G 2 /M phase, and the mechanism of action involved microtubules, mitochondrial damage and caspase activation. In male SCID mice, 2ME 2 was effective in reducing primary tumor weight and the number of liver metastases after intrasplenic injection of human melanoma cells. In the metastases, we found a significantly higher rate of apoptotic cells after 2ME 2 treatment. These findings on the antitumor effect of 2ME 2 in cell culture as well as in an animal model may have implications for designing alternative treatment options for patients with advanced malignant melanoma. © 2004 Wiley-Liss, Inc. Key words: 2-methoxyestradiol; melanoma; apoptosis; metastasisThe possibility that endocrine factors may influence the clinical course of human malignant melanoma is suggested by the higher survival rate in premenopausal vs. postmenopausal women or men of any ages. 1,2 The epidemiological data were supported by studies using melanoma cell lines grown in experimental animals. 3,4 Our previous experiments showed that intrasplenic injection of human melanoma cells resulted in a significantly higher number of liver metastases in male than in female SCID mice. 5 On the other hand, investigations on the sex hormone receptor status of human cutaneous melanomas yielded conflicting results; while earlier studies applying biochemical methods for the detection of estrogen receptors (ER) gave positive results in some cases, other approaches using monoclonal anti-ER antibodies generally failed to demonstrate the presence of type I receptors in melanoma tissues. 1,6 It has been suggested that low-affinity type II ERs may be responsible for the observed hormone binding. Nevertheless, most investigators have shown the lack of an effect of estrogens on the proliferative activity of human melanoma cells. 7,8 There are less data available on the presence of other sex hormone receptors (progesterone and androgens) and their effect on the biological behavior of melanoma cells. 1,9 One possible mechanism behind the observed female superiority in survival of melanoma patients is the tumor growth inhibitory effect of 2-methoxyestradiol (2ME 2 ), an endogenous metabolite of estradiol exerting its activities independently of the ERs. 2ME 2 is formed by the sequential hydroxylation and methylation at the 2-position; it is produced during the normal route of detoxification, especially in the liver, and excreted through the urinary system. 10 -12 This is the only estradiol metabolite devoid of estrogenic activity in vivo and has no known physiological function. Its affinity to ER␣ and to ER␤ is 500-fold and 3,200-fold lower than that of estradiol, respectively, and its activity is independent of the presence of estrogen receptor...

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“…The remaining DNA content was measured using a FACStar flow cytometer (Becton Dickinson, Sunnyvale, CA). 32,33 …”

Section: Flow Cytometric Apoptosis Assay and Cell Cycle Analysismentioning

confidence: 99%

In vitro and in vivo antitumor effect of 2‐methoxyestradiol on human melanoma

Dobos

Tı́már

Bocsi

et al. 2004

Intl Journal of Cancer

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“…Apoptotic cells were detected as a sub-G1 fraction after propidium iodide staining and analysis using a Becton Dickinson FACScan. 19,20 Expression of cell surface Fas GC 3 /c1, RKO and Caco2 cells were plated at a density of 200 000 cells/well in six-well plates. After overnight attachment, cells were cultured for 72 h in the presence or absence of 5-azadC (1 mM).…”

Section: Apoptosis Assaysmentioning

confidence: 99%

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma

Peták¹,

Danam

Tillman³

et al. 2003

Cell Death Differ

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Expression of the cell surface receptor Fas is frequently lost or decreased during tumor progression in human colon carcinomas. The methylation status of a 583 bp CpG-rich region within the Fas promoter (À575 to +8) containing 28 CpG sites was determined in human colon carcinoma cell lines. In Caco2 (no Fas expression), 82-93% of CpG sites were methylated, whereas none were methylated in GC 3 /c1 (high Fas expression). In RKO (intermediate level of Fas), a single CpG site, located at -548, was 100% methylated. The inhibitor of methylation, 5-aza-2 0 -deoxycytidine (5-azadC), upregulated Fas expression in four of eight cell lines, and sensitized RKO cells to recombinant FasL-induced apoptosis. The p53-binding region in the first intron of the Fas gene was partially methylated in Caco2, and 5-azadC potentiated Ad-wtp53-induced upregulation of Fas expression. Methylation-specific PCR of the first intron detected partial methylation in four out of 10 colon carcinoma tumor samples in vivo. The data suggest that DNA hypermethylation is one mechanism that contributes to the downregulation of Fas expression and subsequent loss of sensitivity to Fas-induced apoptosis in colon carcinoma cells.

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“…Cells were stained with 10 AL of Annexin V-antigen presenting cells (Becton Dickinson and Co., San Jose, CA) and 10 AL of propidium iodide (50 ng/mL) according to the instructions of the manufacturer, incubated for 15 minutes at room temperature in the dark, and immediately analyzed by flow cytometry. Alternatively apoptotic cells were detected as a sub-G 1 (hypodiploid nuclei) fraction after propidium iodide staining and analysis using a Becton Dickinson FACScan (14). Cells were also pretreated with the caspase inhibitor zVAD-fmk (50 Amol/L) for 1 hour before TRAIL treatment.…”

Section: Introductionmentioning

confidence: 99%

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

et al. 2005

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The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in #W m and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of #W m and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrialderived ROS, TRAIL induced mitochondrial release of Smac/ DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3. (Cancer Res 2005; 65(16): 7436-45)

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Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

Cited by 16 publications

References 16 publications

In vitro and in vivo antitumor effect of 2‐methoxyestradiol on human melanoma

In vitro and in vivo antitumor effect of 2‐methoxyestradiol on human melanoma

Hypermethylation of the gene promoter and enhancer region can regulate Fas expression and sensitivity in colon carcinoma

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

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