Vitamin D inhibits proliferation of human uterine leiomyoma cells via catechol-O- methyltransferase  Chakradhari Sharan, Ph.D., Sunil K. Halder, Ph.D., Chandrasekhar Thota, Ph.D., Tarannum Jaleel, Sangeeta Nair, D.V.M., M.Sc., Ayman Al-Hendy, M.D., Ph.D.  Fertility and Sterility  Volume 95, Issue 1, Pages 247-253 (January 2011) DOI: 10.1016/j.fertnstert.2010.07.1041 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of vitamin D on proliferation of HuLM cells. 2 × 103 human uterine leiomyoma (HuLM) cells were seeded in 96-well plates and treated with vitamin D. Ethanol vehicle was added to the control cells. Relative cell numbers were assessed at each time point using methylthiazolyl tetrazolium assay. Individual data points are the mean ± SE of triplicate determinations. ∗P<.05 compared with corresponding control. Fertility and Sterility 2011 95, 247-253DOI: (10.1016/j.fertnstert.2010.07.1041) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of vitamin D on proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase (CDK) 1 expression as well as antiapoptotic BCL-2 and BCL-w in human uterine leiomyoma (HuLM) cells. HuLM cells were treated with different doses of vitamin D for 48 hours as indicated. Lysates prepared from both treated and untreated control cells were analyzed by Western blotting with (A) anti-PCNA and (B) anti-CDK1 antibodies. The intensity of each protein signal was quantified and normalized with corresponding β-actin. ∗P<.05 compared with untreated control. (C) HuLM cells were serum starved and treated with vitamin D for 8 hours as indicated. Cell lysates were prepared and were analyzed by Western blotting using anti–phosphorylated extracellular signal–regulated kinase (P-ERK) and anti-ERK antibodies (left). β-Actin was used as loading control. The signal intensity of P-ERK and total ERK were quantified and normalized as above (right). ∗P<.05 compared with untreated control. (D and E) HuLM cells were treated with vitamin D for 48 hours as indicated. Lysates from both vitamin D–treated and –untreated control cells were analyzed by Western blotting with anti–BCL-2, anti–BCL-xL, and anti–BCL-w antibodies. The intensity of each band was normalized with corresponding β-actin as above (bottom). ∗P<.05 compared with untreated control. Fertility and Sterility 2011 95, 247-253DOI: (10.1016/j.fertnstert.2010.07.1041) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of vitamin D on COMT mRNA and protein expression, and enzyme activity. (A) Quantitative analysis of COMT mRNA expression levels with real time reverse-transcription polymerase chain reaction after treatment of human uterine leiomyoma (HuLM) cells with different doses of vitamin D (1 nM to 1 μM) for 48 hours. Expression levels of COMT mRNA are shown as the mean ratio of target-to-reference gene ± SE relative to the vehicle-treated control. Results are expressed as mean ± SE of three independent experiments. ∗P<.05 compared with untreated control. (B) HuLM cells were treated with vitamin D for 48 hours, and cell lysates were prepared and analyzed by western blotting with anti-COMT antibody. The intensity of each protein signal was quantified and normalized with corresponding β-actin. ∗P<.05 compared with untreated control. (C) COMT enzyme activity was assayed after the treatment of HuLM cells with different doses of vitamin D as indicated. COMT enzyme activity was expressed in units per milligram of total protein. ∗P<.05 compared with untreated control. Fertility and Sterility 2011 95, 247-253DOI: (10.1016/j.fertnstert.2010.07.1041) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of endogenous COMT silencing on vitamin D–mediated inhibition of human uterine leiomyoma (HuLM) cell proliferation. (A) The vector control HuLM cells and COMT-deficient HuLM cell lines (shRNA-COMT) were cultured and cell lysates prepared. Equal amounts of each cell lysate from representative stably transfected colonies were analyzed for endogenous COMT expression by Western blotting using anti-COMT antibody. Endogenous levels of membrane-bound (mCOMT) and soluble (sCOMT) catechol-O-methyltransferase were determined in wildtype HuLM cells, a vector control clone, and an shRNA-COMT clone that expresses significantly lower levels of COMT using Western blot analyses with anti-COMT antibody (top). The intensity of COMT signals (mCOMT and sCOMT) was quantified and normalized with corresponding β-actin as above (bottom). ∗P<.05 compared with untreated control. (B) 2 × 103 cells from each pool of the above vector control HuLM cells and the shRNA-COMT clone and in addition primary LM298 cells were seeded in 96-well plates and treated with vitamin D for MTT proliferation assay as described previously (43). Relative cell numbers were assessed at 120 hours, and individual data points are the mean ± SE of triplicate determinations. ∗P<.05 compared with corresponding control. Fertility and Sterility 2011 95, 247-253DOI: (10.1016/j.fertnstert.2010.07.1041) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions