Journal: Hormones & Cancer

Article Title: Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps

doi: 10.1007/s12672-017-0292-7

Figure Lengend Snippet: Effect of drug treatment on cell proliferation in growth hormone receptor knockdown (GHRKD) SK-MEL-28 cells. a SK-MEL-28 cells were exposed to either DMSO (a), or 0.5 um cisplatin (b), or 10 nM doxorubicin (c), or 0.5 uM oridonin (d), or 1 nM paclitaxel (e), or 15 nM vemurafenib (f) for 24 h. Treatments were done 48 h post-transfection with either scr-siRNA (1 and 2) or GHR-siRNA (3 and 4). Panels 1 and 3 show cellular DNA stained with DAPI while panels 2 and 4 show fluorescence signals from AF488-tagged anti-Ki67 antibody. Picture was taken at ×40 magnification; scale bar represents 500 um. Similar profiles for MALME-3M (Fig S2), MDA-MB-435 (Fig S3), and SK-MEL-5 (Fig S4) melanoma cells are in supplementary. b–e Quantitation of Ki67+ and DAPI+ cells in drug-treated GHRKD and control cells in SK-MEL-28 (b), MALME-3M (c), MDA-MB-435 (d), and SK-MEL-5 (e) using ImageJ. The Ki67+/DAPI+ ratio represents proliferating cells normalized to cell number. GHRKD significantly reduced cell proliferation in combination with drug treatment. [*p < 0.05, one-way ANOVA, n = 3]

Article Snippet: Pre-designed siRNA duplex against human GHR (Origene #SR301794, Rockville, MD) at 20 nM was used (siRNA-B: AGCUAGAAUUGAGUGUUUAAAGUTC) that resulted in a >80% decrease in GHR transcript in all four melanoma cells while a universal scrambled siRNA duplex (Origene #SR30004) was used as control.

Techniques: Knockdown, Transfection, Staining, Fluorescence, Quantitation Assay, Control

Journal: Hormones & Cancer

Article Title: Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps

doi: 10.1007/s12672-017-0292-7

Figure Lengend Snippet: Effect of GHRKD on ABC efflux pump expression following drug treatment in human melanoma cells. a Significant downregulation of ABC-transporter expressions in GHRKD SK-MEL-28 cells following treatment with anti-cancer drugs. b Heat-map showing the statistically significant variations in RNA expressions of ABC transporters following GHRKD, in all four human melanoma cell lines, following treatment with all five drugs (35 combinations per cell line, or 28 combinations per drug). Detailed comparison for SK-MEL-28 (Fig S5), MALME-3M (Fig S6), SK-MEL-5 (Fig S7), and MDA-MB-435 (Fig S8) melanoma cells are in supplementary. Experiments were conducted in presence of 50 ng/mL hGH and 0.5% final concentration of DMSO was used as control. In all cases, drug treatment was for 24 h starting 48 h post-transfection. RNA expressions were quantified by RT-qPCR and normalized against expression of ACTB and GAPDH as reference genes [*p < 0.05, Wilcoxon sign rank test, n = 3]. c Changes in protein expressions of ABCC1 and ABCB8 were analyzed. Western blot comparison was done for protein extracted from all four melanoma cells, 60 h post-transfection with GHR-or scr-siRNA. Blots were quantified using ImageJ software and mean of three blots per sample was taken. Expressions were normalized against expression of ACTB (β-actin) [*p < 0.05, Student’s t test, n = 3]

Article Snippet: Pre-designed siRNA duplex against human GHR (Origene #SR301794, Rockville, MD) at 20 nM was used (siRNA-B: AGCUAGAAUUGAGUGUUUAAAGUTC) that resulted in a >80% decrease in GHR transcript in all four melanoma cells while a universal scrambled siRNA duplex (Origene #SR30004) was used as control.

Techniques: Expressing, Comparison, Concentration Assay, Control, Transfection, Quantitative RT-PCR, Western Blot, Software

Journal: Hormones & Cancer

Article Title: Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps

doi: 10.1007/s12672-017-0292-7

Figure Lengend Snippet: List of ABC-transporter pumps with significantly downregulated RNA expression following 24 h exposure to anti-tumor compounds in GHR-siRNA-transfected melanoma cells compared to corresponding scr-siRNA-transfected controls

Article Snippet: Pre-designed siRNA duplex against human GHR (Origene #SR301794, Rockville, MD) at 20 nM was used (siRNA-B: AGCUAGAAUUGAGUGUUUAAAGUTC) that resulted in a >80% decrease in GHR transcript in all four melanoma cells while a universal scrambled siRNA duplex (Origene #SR30004) was used as control.

Techniques: RNA Expression, Expressing

Journal: Hormones & Cancer

Article Title: Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps

doi: 10.1007/s12672-017-0292-7

Figure Lengend Snippet: Effect of GH treatment in vemurafenib resistance in human melanoma cells. SK-MEL-28 and SK-MEL-5 cells grown in normal media (C), or in presence of 20 ng/mL GH (CGH), or with 3-day cycles of 20 nM vemurafenib (V) or with 3-day cycles of 20 nM vemurafenib in presence of 20 ng/mL GH (VGH). EC-50 of all four groups per cell line was measured and the ratio to untreated control (C) was expressed as EC50 ratio for SK-MEL-28 (a) and SK-MEL-5 (b). Relative RNA expression in SK-MEL-28 (c) and SK-MEL-5 (d) cells of ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, ABCG2 and GH, GHR, CDH1, CDH2, and vimentin. e Heat-map showing the statistically significant variations in RNA expressions of ABC transporters in three human melanoma cell lines, following treatment with all five drugs, in presence of 50 ng/mL GH for 24 h (e1) or 20 ng/mL GH treatment for 8-weeks (e2). Detailed comparison for SK-MEL-28 (Fig S9), MALME-3M (Fig S10), and SK-MEL-5 (Fig S11) melanoma cells are in supplementary. In all cases, drug treatment was for 24 hr with 0.5% final DMSO concentration. RNA expressions were quantified by RT-qPCR and normalized against expression of ACTB and GAPDH as reference genes [*p < 0.05, Wilcoxon sign rank test, n = 3]

Article Snippet: Pre-designed siRNA duplex against human GHR (Origene #SR301794, Rockville, MD) at 20 nM was used (siRNA-B: AGCUAGAAUUGAGUGUUUAAAGUTC) that resulted in a >80% decrease in GHR transcript in all four melanoma cells while a universal scrambled siRNA duplex (Origene #SR30004) was used as control.

Techniques: Control, RNA Expression, Comparison, Concentration Assay, Quantitative RT-PCR, Expressing

Journal: Hormones & Cancer

Article Title: Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps

doi: 10.1007/s12672-017-0292-7

Figure Lengend Snippet: GHRKD resulted in increased drug retention and drastically reduced proliferation of melanoma cells. Changes in amounts of calcein retained inside cells following treatment with calcein-AM ester was analyzed by the fluorescence readout from intracellular calcein. Increased abundance of transporter pumps is reflected by decreased levels of intracellular calcein. a Significantly lower calcein retention in human melanoma cells compared to human melanocyte ST-MEL. b Human melanoma cells exhibit significantly higher levels of intracellular calcein following GHRKD. Assays were performed 48 h post-transfection with either scr-siRNA or GHR-siRNA. Effect of GHRKD on cell proliferation following 24 h exposure to EC50 levels of cisplatin and paclitaxel was tested. c SK-MEL-28 and d MALME-3M cells were exposed to DMSO (vehicle), or 10 um cisplatin (Cis), or 5 nM paclitaxel (Pac) for 24 h. Treatments were done 48 h post-transfection with either scr-siRNA (scr) or GHR-siRNA (GHR). Mean of three independent experiments performed in triplicate was taken [*p < 0.05, Student’s t test, n = 3]

Article Snippet: Pre-designed siRNA duplex against human GHR (Origene #SR301794, Rockville, MD) at 20 nM was used (siRNA-B: AGCUAGAAUUGAGUGUUUAAAGUTC) that resulted in a >80% decrease in GHR transcript in all four melanoma cells while a universal scrambled siRNA duplex (Origene #SR30004) was used as control.

Techniques: Fluorescence, Transfection