Journal: Chemical research in toxicology

Article Title: Δ(9)-Tetrahydrocannabinol disrupts estrogen-signaling through up-regulation of estrogen receptor β (ERβ).

doi: 10.1021/tx4000446

Figure Lengend Snippet: Figure 4. Δ9-THC inhibits ERα-mediated transcriptional activities in concert with overexpression of ERβ or addition of ICI 182,780. (A) MCF-7 cells were transiently transfected with a luciferase reporter gene construct-containing three copies of an ERE and expression plasmid for ERβ. After transfection, cells were treated with vehicle (−/−) or Δ9-THC (5 or 25 μM). ERβ transfection (−) indicates mock-transfected groups. After 24 h, cells were harvested and assayed for luciferase activity, and all transfections were normalized for efficiency using the internal Renilla control plasmid. Data are expressed as the percent of vehicle-treated control (indicated as −/−), as the mean ± SD (n = 5). *Significantly different (p < 0.05) from the vehicle-treated group. (B) MCF-7 cells were transiently transfected with a luciferase reporter gene construct containing three copies of an ERE and expression plasmid for human ERβ. After transfection, cells were treated with vehicle (−/−/−) or Δ9-THC (25 μM) in the presence or absence of ICI 182,780 (ICI, 0.1−2.5 μM). After 24 h, cells were harvested and assayed for luciferase activity, and all transfections were normalized for efficiency using the internal Renilla control plasmid. Data are expressed as the percent of vehicle-treated control (indicated as −/−/−), as the mean ± SD (n = 5). N.D., not detectable due to complete inhibition. (C) Effects of Δ9-THC and E2 on ERα- or ERβ-mediated transcription activities. ERα-negative MDA- MB-231 cells were transiently transfected with a luciferase reporter gene construct-containing three copies of ERE and expression plasmids for human ERα or ERβ. After transfection, cells were treated with vehicle (−/−/−/−) or E2 (100 pM) or Δ9-THC (25 μM). ERα/ β transfection (−) indicates mock-transfected groups. After 24 h, cells were harvested and assayed for luciferase activity, and all transfections

Article Snippet: To construct human ERα and ERβ expression plasmids, we first obtained cDNAs of human ERα (catalog #: RC213277) and ERβ (catalog #: RC218519) from OriGene (Rockville, MD, USA).

Techniques: Over Expression, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Control, Inhibition

Journal: Chemical research in toxicology

Article Title: Δ(9)-Tetrahydrocannabinol disrupts estrogen-signaling through up-regulation of estrogen receptor β (ERβ).

doi: 10.1021/tx4000446

Figure Lengend Snippet: Figure 3. Δ9-THC up-regulates ERβ. (A) Results of DNA microarray analysis. Data are expressed as fold induction vs vehicle-treated groups. MCF-7 cells were treated with vehicle or 25 μM Δ9-THC for 48 h, followed by mRNA isolation. Details of microarray conditions are described under Materials and Methods. (B) RT-PCR analysis of ERα, ERβ, and Ki-67 transcript levels after treatment with 5 or 25 μM Δ9- THC or without Δ9-THC (indicated as −). β-Actin was used as an RNA normalization control. A 100-bp DNA ladder marker was also loaded. A representative data image is shown. (C) Western immunoblot analysis of ERβ. MCF-7 cells were treated with 5 μM or 25 μM Δ9-THC (indicated as 5 or 25) or vehicle (indicated as −) for 48 h. The cell lysates derived from transient transfection of human ERβ cDNA-expression plasmid (transfected plasmids: 0.0025, 0.25, and 2.5 μg) were also loaded. Total cell lysates were prepared, and Western immunoblot analyses were performed using antibodies specific for ERβ and β-actin, respectively. The band intensity of ERβ (−/−lane as 1.0), which was quantified by using NIH Image, version 1.61, software, is shown beneath the blot image. β-Actin was used an internal loading control.

Article Snippet: To construct human ERα and ERβ expression plasmids, we first obtained cDNAs of human ERα (catalog #: RC213277) and ERβ (catalog #: RC218519) from OriGene (Rockville, MD, USA).

Techniques: Microarray, Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Marker, Western Blot, Derivative Assay, Transfection, Expressing, Plasmid Preparation, Software

Journal: Chemical research in toxicology

Article Title: Δ(9)-Tetrahydrocannabinol disrupts estrogen-signaling through up-regulation of estrogen receptor β (ERβ).

doi: 10.1021/tx4000446

Figure Lengend Snippet: Figure 5. Δ9-THC behaves an activator for ERβ in ERα-negative MDA-MB-231 cells. (A) MDA-MB-231 cells were transiently transfected with a luciferase reporter gene construct containing three copies of an ERE and expression plasmid for human ER. After transfection, cells were treated with vehicle (−/−) or Δ9-THC (5, 10, or 25 μM). ERβ transfection (−) indicates mock-transfected groups. After 24 h, cells were harvested and assayed for luciferase activity, and all transfections were normalized for efficiency using the internal Renilla control plasmid. Data are expressed as the percent of vehicle- treated control (indicated as −/−), as the mean ± SD (n = 5). *Significantly different (p < 0.05) from the vehicle-treated group. Inset: RT-PCR analysis of ERα and ER mRNA basal levels in MDA- MB-231 cells. β-Actin was used as an RNA normalization control. A 100-bp DNA ladder marker was also loaded. (B) MDA-MB-231 cells were transiently transfected with a luciferase reporter gene construct containing three copies of an ERE and expression plasmid for human ERβ. After transfection, cells were treated with vehicle (−/−/−) or Δ9-THC (25 μM) in the presence or absence of ICI 182,780 (ICI, 2.5 μM) or PHTPP (2.5 μM). After 24 h, cells were harvested and assayed for luciferase activity, and all transfections were normalized for efficiency using the internal Renilla control plasmid. Data are expressed as the percent of vehicle-treated control (indicated as −/−/−), as the mean ± SD (n = 5). *Significantly different (p < 0.05) from the vehicle-treated group (−/−/−). **Significantly different (p < 0.05) from the Δ9-THC/ICI-treated groups (−/−/−). N.S., not significant.

Article Snippet: To construct human ERα and ERβ expression plasmids, we first obtained cDNAs of human ERα (catalog #: RC213277) and ERβ (catalog #: RC218519) from OriGene (Rockville, MD, USA).

Techniques: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Control, Reverse Transcription Polymerase Chain Reaction, Marker