Fig. 7
Effects of 2′,3′,4′-THC on ERα degradation and MAPK activity. A 2′,3′,4′-THC does not compete for [3H]-E2 binding in MCF-7 breast cancer cells. Competitive binding of 2′,3′,4′-THC in the cells treated with 5 nM [3H]-E2 and increasing doses of 2′,3′,4′-THC for 1 h at 37 °C. Specific [3H]-E2 binding was measured with a scintillation counter and calculated by subtracting non-specific binding from total binding. The error bars are mean ± SE. B, C 2′,3′,4′-THC does not alter ERα degradation, whereas SERMs prevent ERα degradation in MCF-7 cells. B Western blot of MCF-7 cells treated with 10 nM E2 in the absence and presence of 5 μM 2′,3′,4′-THC, 10 μM tamoxifen (Tam), or 1 μM raloxifene (Ral) for the indicated time and then ERα and β-actin protein levels were determined. C Quantification of ERα and β-actin protein levels. The error bars are means ± SE. The differences among various treatments were analyzed with one-way ANOVA. The asterisk over the bars indicates the significant difference between E2 alone and E2 in combination with 2′,3′,4′-THC, Tam, or Ral at various times. D 2′,3′,4′-THC inhibits E2 activation of MAPK. Active phospho-p44/42 MAPK (top panel) and inactive p44/42 MAPK (bottom panel) were measured by western blotting after the cells were treated with 10 nM E2, 5 μM 2′,3′,4′-THC alone or in combination for various times. E 2′,3′,4′-THC does not alter EGF activation of MAPK. Active phospho-p44/42 MAPK (top panel) and inactive p44/42 MAPK (bottom panel) were measured by western blotting after the cells were treated with 100 ng/ml EGF alone or in combination with 2′,3′,4′-THC for various times. F Quantification of phospho-p44/42 and p44/42 protein levels in E2-treated cells. The asterisk over the bars indicates the significant difference between E2 alone and E2 in combination with 2′,3′,4′-THC. G Quantification of phospho-p44/42 and p44/42 protein levels in EGF-treated cells. Quantification of protein levels of western blots was done by ImageJ. The asterisks indicate a significant difference between the control and EGF-treated cells