Article Figures & Data
Figures
- Figure 1.
6-SHO inhibits cell survival and induces apoptosis in human prostate cancer cells. A, LNCaP, DU145, and PC-3 cells were treated with the indicated concentrations of 6-SHO for 24, 48, and 72 hours and cell survival was measured by MTT assay. The data are presented as mean±SEM. B, cells were treated with vehicle or 40 μmol/L 6-SHO for 48 hours and apoptosis was measured by Annexin V staining. The data are presented as mean±SEM. C, cells were treated with vehicle or 6-SHO for 24 hours and Western blot analysis was performed for apoptosis markers. a, significantly different (P < 0.05) compared with the control group and (b) compared with the other treated groups. Changes in relative band intensities (normalized to β-actin) for Western blot data in C are given at the top of each column and represent the average of two separate experiments.
- Figure 2.
Reduction of constitutive and IL-6 induced STAT3 activation by 6-SHO in human prostate cancer cells. A–E, cell lines were cultured as indicated in Materials and Methods. Western blot analysis was performed to determine STAT3 status and level (pSTAT3Y705, pSTAT3S727, and total STAT3) in whole-cell lysates collected after the indicated treatments. A, cell lysates were prepared from untreated cultures of LNCaP, DU145, and PC-3 cells. B, LNCaP and DU145 cells were treated with 20 or 40 μmol/L 6-SHO and subjected to Western blot analysis. C, time course of the effect of 6-SHO on total and phosphorylated STAT3 in LNCaP and DU145 cells. Cells were treated with vehicle or 40 μmol/L 6-SHO for 1, 3, or 6 hours and Western blot analysis performed. D, LNCaP and DU145 cells were pretreated with 20 and 40 μmol/L 6-SHO for 2 hours followed by IL-6 for 30 minutes. E, LNCaP and DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by IL-6 treatment for 0.5, 1, or 3 hours. F, DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours and the nuclear localization of STAT3 (red) was measured by immunofluorescence staining. G, LNCaP and DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by IL-6 for 30 minutes. pJAK2 and pSrc was determined by Western blotting. Changes in relative band intensities (normalized to β-actin and total protein) for Western blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation is ≥40%.
- Figure 3.
Inhibition of constitutive and TNF-α induced NF-κB activity by 6-SHO in human prostate cancer cells. A, DU145 and PC-3 cells were treated with vehicle or 40 μmol/L 6-SHO and subjected to Western blot analyses. B, cells were treated with 20 and 40 μmol/L 6-SHO for 2 hours followed by TNF-α for 30 minutes and pNF-κBp65, NF-κBp65, and pIκBα levels were measured by Western blot analysis. Changes in relative band intensities (normalized to β-actin and total protein) for Western blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation is ≥ 40%. C, LNCaP and DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by TNF-α for 30 minutes and the nuclear localization of NF-κBp65 (green) was measured by immunofluorescence staining.
- Figure 4.
Effect of 6-SHO on STAT3 and NF-κB targets in human prostate cancer cells. A, LNCaP and DU145 cells were treated with vehicle, 20 or 40 μmol/L 6-SHO and the level of survivin was measured by Western blot analysis. B, LNCaP and DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 1 or 3 hours and the level of cyclin D1, survivin, and cMyc was measured by Western blot analysis. C, LNCaP and DU145 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by IL-6 and the level of cyclin D1, survivin, and cMyc was measured by Western blot analysis. D, LNCaP, DU145, and PC-3 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by TNF-α and were subjected to Western blot analysis for cMyc protein. Changes in relative band intensities (normalized to β-actin) for Western blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in protein level is ≥ 40%. E, LNCaP and DU145 cells were treated with vehicle, 20 or 40 μmol/L 6-SHO for 24 hours. Gene expression for CCL5, IL-7, BCL2, BAX, p21, p27, SOCS1, and IRF-1 was measured by quantitative PCR. a, significantly different (P < 0.05) compared with the control group and b, compared with the other treated groups.
- Figure 5.
Effect of 6-SHO on mouse prostate cancer HMVP2 cells. A, HMVP2 cells were treated with the indicated concentrations of 6-SHO for 24, 48, and 72 hours and cell survival was measured by MTT assay. The data are presented as mean±SEM. B, HMVP2 cells were treated with vehicle or 40 μmol/L 6-SHO for 48 hours and apoptosis was measured by Annexin V staining. The data are presented as mean±SEM. C, HMVP2 cells were treated with 40 μmol/L 6-SHO for 30 and 60 minutes and Western blot analysis was performed for pSTAT3Tyr705, STAT3, and β-actin (top two); HMVP2 cells were treated with 20 and 40 μmol/L 6-SHO for 2 hours followed by IL-6 for 30 minutes and Western blot analysis was performed for pSTAT3Tyr705, STAT3, and β-actin (bottom two). D, HMVP2 cells were treated with vehicle or 40 μmol/L 6-SHO followed by TNF-α treatment for 30 minutes and Western blot analysis was performed for pNF-κBp65, NF-κBp65, and β-actin (top); HMVP2 cells were treated with 20 and 40 μmol/L 6-SHO for 2 hours followed by TNF-α for 30 minutes and Western blot analysis was performed for pNF-κBp65, NF-κBp65, pIκBα, and β-actin (bottom). E, HMVP2 cells were treated with vehicle, 20 or 40 μmol/L 6-SHO for 2 hours followed by IL-6 and the level of cyclin D1 was measured by Western blot analysis. F, HMVP2 cells were treated with vehicle or 40 μmol/L 6-SHO for 2 hours followed by TNF-α and the level of cMyc was measured by Western blot analysis. Changes in relative band intensities (normalized to β-actin and total protein) for Western blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation or protein level is ≥40%. G, LNCaP, DU145, PC-3, and HMVP2 cells were treated with the indicated concentrations of 6-PAR, 6-GIN, or 6-SHO for 48 hours and cell survival was measured by MTT assay. The data are presented as mean±SEM. a, significantly different (P < 0.05) compared with the control group and b, compared with the other treated groups of the same compound.
- Figure 6.
6-SHO reduces HMVP2 allograft tumor growth in vivo. Spheroids from HMVP2 cells were injected subcutaneously into the flank of male FVB mice. Two weeks after injection, mice were treated intraperitoneally with 6-SHO (50 or 100 mg/kg body weight, BW) every other day for the duration of the experiment. A, tumor volume is expressed as average tumor volume/mouse (mm3). B, tumor weight is presented as average tumor weight (mg)/mouse. C, average mouse BW (g) for each group. D, average feed consumption/mouse/day. Data are mean±SEM; n = 5 mice per group; *, P < 0.05 compared with the control group. Lysates of allograft tumor tissues were subjected to Western blot analysis for pSTAT3Tyr705 and STAT3 (E) and cyclinD1 and survivin (F) and β-actin was used as control for both sets of blots. Changes in relative band intensities (normalized to β-actin and total protein) for Western blot data are given at the top of each column. The results are significant (P < 0.05) where a decrease in phosphorylation or protein level is ≥40%.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Information - PDF - 191K, Tert-Butyldimethylsilyl ether-protected Zingerone.
- Supplementary Figure 1 - PDF - 231K, Supplemental Figure 1. Reduction of constitutive and IL?6 induced STAT3 activation by 6?SHO in human PCa cells.
- Supplementary Figure 2 - PDF - 119K, Supplemental Figure 2. G-SHO does not reduce survival of NMVP cells. Cells were treated with the indicated concentrations of G-SHO for 48 or 72 h and cell survival was measured by MTT assay. The data are presented as mean(plus/minus)SEM.
- Supplementary Figure 3 - PDF - 248K, Supplemental Figure 3. Effects of ginger components, 6-GIN and 6-PAR, on human and mouse PCa cells.
- Supplementary Figure 4 - PDF - 231K, Supplemental Figure 4. Effects of ginger components, 6-GIN and 6-PAR, on human and mouse PCa cells. DU145 and PC-3 cells were treated with 6-PAR, 6-GIN or 6-SHO followed by TNF-a and the levels of phospho IICBa, phospho NFICBp65 and NF-ICBp65 were measured by Western blot.
- Supplementary Figure 5 - PDF - 267K, Supplemental Figure 5. Representative HMVP2 tumors from untreated mice.
Volume 7, Issue 6
