Differential Effects of In Vitro Treatment with Cinobufotalin on Three Types of Ovarian Cancer Cells.

BACKGROUND/AIM: Cinobufotalin (CINO), a cardiotonic steroid, has been used as an anticancer agent. This study assessed the cell-specific effect of CINO on SK-OV-3, CRL-1978 and CRL-11731 ovarian cancer cells which differ in terms of their respective karyotypes. MATERIALS AND METHODS: Cell cultures were treated with CINO (0.1, 1, 5 and 10 µM) for 24, 48, and 72 h. Cell proliferation, migration, and invasion were measured using CellTiter, Cytoselect, and FluoroBlock assays, respectively. Expression of proliferating cell nuclear antigen (PCNA) was evaluated by western blot analysis. Cell viability was determined by fluorescence-activated cell sorting. Immunofluorescence was performed using Annexin-V staining and fluorescein isothiocyanate (FITC). Mitochondrial membrane potential (MMP) was measured using MitoTracker™ Red. RESULTS: CINO at 0.5 µM inhibited SK-OV-3, CRL-1978, and CRL-11731 proliferation, migration, and invasion. Each cell type differed in response to CINO doses for PCNA, Annexin-V expression and MMP. CONCLUSION: The antineoplastic property of CINO is consistent, but its mode of action varies among cell lines.

Differential Mechanism of Action of 3,4',7-O-trimethylquercetin in Three Types of Ovarian Cancer Cells.

BACKGROUND/AIM: 3,4',7-O-trimethylquercetin (34'7TMQ), a derivative of quercetin, inhibited ovarian cancer cell migration and invasion without affecting proliferation. In this study, the apoptotic effect of 34'7TMQ on three cancer cell lines (CRL-1978, CRL-11731, SK-OV-3) was evaluated. MATERIALS AND METHODS: Expression of pro-apoptotic proteins such as Bax/Bcl-2 ratio, p38 MAP kinase, and caspase-9 were measured by western blot analysis. Annexin-V staining was performed to visualize apoptotic signaling. RESULTS: Caspase-9 was up-regulated in all three cell lines. Bax/Bcl-2 ratio was up-regulated in CRL-1978 and SK-OV-3 but down-regulated in CRL-11731. The p38 MAPK was down-regulated in CRL-1978, up-regulated in SK-OV-3, and had differential expression in CRL-11731. Annexin V staining indicated that 34'7TMQ at 6.25 µM induced apoptotic signaling in the CRL-1978 ovarian cancer cell line. CONCLUSION: 34'7TMQ induced apoptosis in three types of cancer cell lines but it appears to have a different mechanism of action in each cell line.

3,4',7-O-trimethylquercetin Inhibits Invasion and Migration of Ovarian Cancer Cells.

BACKGROUND/AIM: Methylquercetin, 3,4',7-O-trimethylquercetin (34'7TMQ), has been reported to inhibit metastasis. Recently, we demonstrated that 34'7TMQ inhibited the in vitro melanoma B16 cell metastatic activity. We evaluated the effect of 34'7TMQ on three ovarian cancer cells (SK-OV-3, CRL11731 and CRL1978). MATERIALS AND METHODS: Proliferation, migration and invasion were measured in 34'7TMQ-treated ovarian cancer cells by commercially available kits. We also evaluated the expression of proliferating cell nuclear antigen (PCNA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and matrix metalloproteinase (MMP)-2 by western blot analysis. RESULTS: 34'7TMQ inhibited ovarian cancer cell migration and invasion without effecting proliferation. Furthermore, 34'7TMQ inhibited the expression of uPA and MMP-2; however, it had no effect on PAI-1 and PCNA. CONCLUSION: 34'7TMQ significantly regulates the expressions of protein to inhibit metastasis in ovarian cancers, while the regulatory effects of 34'7TMQ vary between different ovarian cancer cell lines.

Synthetic Receptors Induce Anti Angiogenic and Stress Signaling on Human First Trimester Cytotrophoblast Cells.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

Structure-Activity Relationships of Methylquercetin on Anti-migration and Anti-proliferation Activity in B16 Melanoma Cells.

BACKGROUND: Melanoma is a malignant skin tumor and quercetin has been reported to inhibit metastasis. A quercetin glycoside and 7 methylquercetins were synthesized from rutin to investigate structure-activity relationships. MATERIALS AND METHODS: We evaluated the anti-proliferative and anti-migration activity of quercetin glycoside and 7 methylquercetins in murine B16 melanoma cells by commercially available kits. We also examined the effect of these compounds on growth of human fibroblast cells to evaluate cytotoxicity. RESULTS: 3-O-methylquercetin (2) exhibited 30, 38, 45% migration activity at 25, 12.5, 6.25 µM respectively. Furthermore, 3,4',7-O-trimethylquercetin (5) and 3,4'-O-dimethylquercetin (3) inhibited migration more potent than 2 with no cytotoxicity. 3-hydroxymethylquercetin and quercetin glycoside exhibited no anti-migration activity. Furthermore, the 3 and 4 inhibited melanoma proliferation with no cytotoxicity. CONCLUSION: The methoxyl group at the C-3 position plays an important role in inhibiting the migration of B16 melanoma cells.

Attenuation of hyperglycemia-induced apoptotic signaling and anti-angiogenic milieu in cultured cytotrophoblast cells.

OBJECTIVE: Preeclampsia (preE) is a hypertensive disorder that occurs 20% in diabetic pregnancy. We have shown that hyperglycemia impairs cytotrophoblast cell (CTB) function. In this study, we assess apoptotic and anti-angiogenic signaling in excess glucose-induced CTBs. STUDY DESIGN: Human extravillous CTBs (Sw. 71) were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48 h. Some cells were pretreated with a p38 inhibitor (SB203580) or a peroxisome proliferator-activated receptor gamma (PPARγ) ligand (rosiglitazone) or with D-mannitol. Cell lysates were utilized to measure p38 MAPK phosphorylation, PPARγ, Bcl-2-associated-X protein (Bax), anti-apoptotic Bcl-2, caspase-9, and cyclooxygenase-2 (Cox-2) expression by western blot. Levels of the vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. RESULTS: p38 phosphorylation and PPARγ were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL). Expressions of Bax/Bcl-2, Cox-2, and caspase-9 were upregulated (p < 0.05) in CTBs treated with ≥150 mg/dL glucose. Secretion of sFlt-1, sEng, and IL-6 was increased while VEGF and PIGF were decreased in CTB-treated ≥150 mg/dl of glucose (*p < 0.01 for each). SB203580 or rosiglitazone pretreatment significantly attenuated hyperglycemia-induced apoptotic and anti-angiogenic signaling. D-Mannitol had no effect. CONCLUSION: Hyperglycemia induced apoptotic and anti-angiogenic signaling in CTBs. The observed diminution of hyperglycemia-induced signaling by SB203580 or rosiglitazone pretreatment suggests the involvement of apoptotic and anti-angiogenic signaling in CTB dysfunction.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.