Restoring Endocrine Response in Breast Cancer Cells by Inhibition of the Sphingosine Kinase-1 Signaling Pathway

Abstract

We previously demonstrated that sphingosine kinase-1 (SphK1) is an important mediator in the cytoplasmic signaling of estrogens, including Ca(2+) mobilization, ERK1/2 activation, and the epidermal growth factor receptor transactivation. Here we report for the first time that SphK1 activity is causally associated with endocrine resistance in MCF-7 human breast cancer cells. Enforced overexpression of human SphK1 in MCF-7 cells resulted in enhanced cell proliferation and resistance to tamoxifen-induced cell growth arrest and apoptosis. Tamoxifen-resistant (TamR) MCF-7 cells selected by prolonged exposure to 4-hydroxytamoxifen, exhibited higher levels in SphK1 expression and activity, compared with the control cells. Inhibition of SphK1 activity by either specific pharmaceutical inhibitors or the dominant-negative mutant SphK1(G82D) restored the antiproliferative and proapoptotic effects of tamoxifen in the TamR cells. Furthermore, silencing of SphK1, but not SphK2, expression by the specific small interference RNA also restored the tamoxifen responsiveness in the TamR cells. Thus, blockade of the SphK1 signaling pathway may reprogram cellular responsiveness to tamoxifen and abrogate antiestrogen resistance in human breast cancer cells.

Combination of FTY720 with cisplatin exhibits antagonistic effects in ovarian cancer cells: Role of autophagy

“…Cells (3,000 cell/well) were seeded in three to four replication into 96-well plates and incubated with or without FTY720 at the indicated concentration for a desired time period. After the indicated treatments, cell viability was assessed using the MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Sigma) assay as described previously (15). The absorbance intensity of the MTS product is directly proportional to the number of viable cells in culture when cell number is between 2,000 and 200,000; otherwise the exponential dependence was determined.…”

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

“…After the indicated treatments, cell viability was assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt; Sigma, M2003) assay as described previously. 42 The absorbence intensity of the MTS product is directly proportional to the number of viable cells in culture when cell number is between 2,000 and 200,000; otherwise the exponential dependence was determined. The cell viability was expressed as a percentage of absorbance in cells with indicated treatments to that in cells with vehicle control treatment.…”

“…In addition to the aforementioned MTS assay, cell death was quantitatively and qualitatively analyzed by the following approaches: (i) morphological analysis under phase-contrast microscopy; (ii) annexin V/propidium iodide (PI) staining and flow cytometry analysis as described previously; 42 and (iii) analysis of caspase 3 cleavage by western blot assays.…”

High Expression of Sphingosine 1-Phosphate Receptors, S1P1 and S1P3, Sphingosine Kinase 1, and Extracellular Signal-Regulated Kinase-1/2 Is Associated with Development of Tamoxifen Resistance in Estrogen Receptor-Positive Breast Cancer Patients

“…These data are consistent with the finding that enforced expression of SK1 in MCF-7 cells induces tamoxifen resistance. 16 …”