Journal: Breast Cancer Research : BCR

Article Title: Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

doi: 10.1186/bcr3616

Figure Lengend Snippet: Dickkopf-1 is a target of zoledronic acid and atorvastatin in breast cancer cells in vitro . (A) Results from a WNT PCR array define Dickkopf-1 (DKK-1) as a target of zoledronic acid. MDA-231 cells were treated with zoledronic acid (100 μM) for 24 hours and assessed by a WNT array. A heat map of all tested genes is shown on the right: green, increased expression compared with control; red, decreased expression compared with control; grey, nondetectable gene. All genes that were regulated at least twofold are separately depicted on the left. (B) Basal expression of DKK-1 in different breast cancer cell lines assessed by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and western Blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a loading control. (C) , (D) Suppression of DKK-1 by zoledronic acid (ZOL; 100 μM) and atorvastatin (ATO; 10 μM) after 24 hours of exposure was confirmed in multiple cell lines. Assessment of unfarnesylated RAS (upper band) and ungeranylated RAP1A confirmed mevalonate pathway inhibition. Representative blots are shown. GAPDH is used as a loading control. Data for PCR and ELISA are shown as mean ± standard deviation of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: RNA (500 ng) was reverse transcribed using Superscript II (Life Technologies, Darmstadt, Germany) and used for SYBR green-based real-time polymerase chain reaction (PCR) using a standard protocol (Applied Biosystems).

Techniques: In Vitro, Expressing, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Inhibition, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

doi: 10.1186/bcr3616

Figure Lengend Snippet: Dickkopf-1 is regulated via inhibited geranylgeranylation. (A) , (B) MDA-231 cells were treated with either zoledronic acid (ZOL; 100 μM) or atorvastatin (ATO; 10 μM) for 24 hours alone or concurrently with mevalonate substrates (geranyl-geranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP) or mevalonate (MEV)). Dickkopf-1 (DKK-1) was analyzed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). (C) Specific inhibitors of geranylgeranylation GGTI-298 (5 μM) and farnesylation FTI-277 (100 nM) were used for further pathway clarification, and treatment was for 24 hours. Results of ELISA and PCR data are presented as the mean ± standard deviation of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. Inhibition of geranylgeranylation by GGTI-298, atorvastatin and zoledronic acid, farnesylation by FTI-277, atorvastatin and zoledronic acid as well as their selective reversal by the substrates was confirmed by assessing unfarnesylated RAS and ungeranylated RAP1A in western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as loading control. Representative blots are shown. (D) Levels of phosphorylated β-catenin decrease following 24 hours of exposure to zoledronic acid and atorvastatin. Levels of total β-catenin remain unchanged. GAPDH is used as a loading control.

Article Snippet: RNA (500 ng) was reverse transcribed using Superscript II (Life Technologies, Darmstadt, Germany) and used for SYBR green-based real-time polymerase chain reaction (PCR) using a standard protocol (Applied Biosystems).

Techniques: Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clarification Assay, Standard Deviation, Inhibition, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

doi: 10.1186/bcr3616

Figure Lengend Snippet: Dickkopf-1 is regulated via Cdc42 and Rho in breast cancer. (A) MDA-231 cells were treated for 48 hours with Clostridium difficile toxin A (250 ng/ml) and assessed for Dickkopf-1 (DKK-1) expression. (B) MDA-231 cells were exposed to zoledronic acid (ZOL) or atorvastatin (ATO) alone or concurrently with a Rho/Rac/Cdc42 activator for 24 hours. Due to the relatively short half-life of the activator, cells were treated every 5 hours with 1 μg/ml of activator until cells were harvested after 15 hours. (C) MDA-231 cells were treated with a number of different GTPase inhibitors for 24 hours and assessed for DKK-1 expression. Concentrations used were 10 μM for Rho#1 and Rho#2, 100 μM for Rac#1, 10 μM for Rac#2 and 50 μM for the Cdc42 inhibitor. (D) MDA-231 cells were transfected with control single interfering RNA (siRNA) or two different siRNAs directed against Cdc42. Sufficient knockdown was verified by western blot. DKK-1 expression was analyzed 48 hours after transfection. (E) Regulation of DKK-1 by Cdc42 and Rho was verified at protein levels, by measuring DKK-1 levels in the supernatant of MDA-231 cells treated with the same inhibitors as in C. (F) The role of Rho signaling was further confirmed by measuring DKK-1 levels following 24 hours of Rho-associated protein kinase inhibition using Y-27632 and H-1152P. Results of enzyme-linked immunosorbent assay and polymerase chain reaction data are presented as the mean ± standard deviation of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: RNA (500 ng) was reverse transcribed using Superscript II (Life Technologies, Darmstadt, Germany) and used for SYBR green-based real-time polymerase chain reaction (PCR) using a standard protocol (Applied Biosystems).

Techniques: Expressing, Transfection, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

doi: 10.1186/bcr3616

Figure Lengend Snippet: Dickkopf-1 inhibits WNT3A-induced osteoblastic differentiation in C2C12 cells. (A) Supernatants of breast cancer cells were harvested after 48 hours. C2C12 cells were cultured in control medium (containing 50% control L-cell medium) or differentiated in WNT3A-containing media (50%) and concurrently treated with breast cancer supernatants (25%) or unconditioned control media (25%). C2C12 cells were treated for 48 hours and assessed for alkaline phosphatase (ALP) expression. (B) C2C12 cells were differentiated with WNT3A media and exposed to increasing concentrations of recombinant Dickkopf-1 (DKK-1; 10, 50, 100 and 250 ng/ml). (C) C2C12 cells were exposed to supernatants of untreated or zoledronic acid-treated MDA-231 cells in the presence of WNT3A. To prevent potential direct effects of zoledronic acid on C2C12 cells, MDA-231 cells were preincubated with zoledronic acid for 24 hours, media were then replaced and zoledronic acid-free supernatants were harvested 48 hours later. Results of polymerase chain reaction data are presented as the mean ± standard deviation of three independent experiments. * P < 0.01; *** P < 0.001.

Article Snippet: RNA (500 ng) was reverse transcribed using Superscript II (Life Technologies, Darmstadt, Germany) and used for SYBR green-based real-time polymerase chain reaction (PCR) using a standard protocol (Applied Biosystems).

Techniques: Cell Culture, Expressing, Recombinant, Polymerase Chain Reaction, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Dickkopf-1 is regulated by the mevalonate pathway in breast cancer

doi: 10.1186/bcr3616

Figure Lengend Snippet: Dickkopf-1 inhibits WNT3A-induced osteoprotegerin production in C2C12 cells. (A) Supernatants of breast cancer cells were harvested after 48 hours. C2C12 cells were cultured in control medium (containing 50% control L-cell medium) or differentiated in WNT3A-containing media (50%) and concurrently treated with breast cancer supernatants (25%) or unconditioned control media (25%). C2C12 cells were treated for 48 hours and assessed for osteoprotegerin (OPG) expression and protein secretion. (B) C2C12 cells were differentiated with WNT3A media and exposed to increasing concentrations of recombinant Dickkopf-1 (DKK-1; 10, 50, 100 and 250 ng/ml). (C) DKK-1 was transiently knocked down in MDA-231 cells using two different single interfering RNAs (siRNAs). Knockdown was verified by quantifying DKK-1 levels secreted into the supernatant 48 hours after transfection. Supernatants of control siRNA and DKK-1 siRNA-treated cells were given to C2C12 cells during WNT3A-induced differentiation in the established ratios of 25% and 50%. OPG levels were determined after 48 hours of exposure. (D) C2C12 cells were exposed to supernatants of untreated or zoledronic acid-treated MDA-231 cells in the presence of WNT3A. To prevent potential direct effects of zoledronic acid on C2C12 cells, MDA-231 cells were preincubated with zoledronic acid for 24 hours, media were then replaced and zoledronic acid-free supernatants were harvested 48 hours later. Suppressed DKK-1 levels in the supernatant were confirmed by enzyme-linked immunosorbent assay (ELISA) prior to use. Results of ELISA and polymerase chain reaction data are presented as the mean ± standard deviation of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: RNA (500 ng) was reverse transcribed using Superscript II (Life Technologies, Darmstadt, Germany) and used for SYBR green-based real-time polymerase chain reaction (PCR) using a standard protocol (Applied Biosystems).

Techniques: Cell Culture, Expressing, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Standard Deviation