Article Figures & Data
Figures
- Fig. 1.
Chemical structure of melatonin-tamoxifen drug conjugates.
- Fig. 2.
Comparison of potency and efficacy between C2, C4, C5, C9, and C15 conjugates to inhibit BC cell viability. (A and B) The potency (IC50) (A) or efficacy (B) of C2, C4, C5, C9, and C15 on cell viability in MCF-7, MMC, MDA-MB-231, and BT-549 cells. (C and D) The potency (IC50) (C) or efficacy (D) of melatonin, tamoxifen, 4-OH-tamoxifen, unlinked melatonin plus tamoxifen and unlinked melatonin plus 4-OH-tamoxifen on cell viability in MCF-7, MMC, MDA-MB-231, and BT-549 cells. Each bar represents the mean ± S.D. of three independent experiments for each compound (n = 9 for vehicle, since it was run with each experiment conducted). Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: for figure A-a, P < 0.05 vs. C2; b, P < 0.05 vs. C4; c, P < 0.05 vs. C5; d, P < 0.05 vs. C9; for figure B-a, P < 0.05 vs. vehicle; b, P < 0.05 vs. C2; c, P < 0.05 vs. C4; d, P < 0.05 vs. C5, e, P < 0.05 vs. C9; for figure C-a, P < 0.05 vs. melatonin; for figure D-a, P < 0.05 vs. vehicle; b, P < 0.05 vs. melatonin; d, P < 0.05 vs. 4-OH-tamoxifen. Abs, absorbance.
- Fig. 3.
Comparison of potency and efficacy between C2, C4, C5, C9, and C15 conjugates to inhibit BC cell migration. (A and B) The potency (IC50) (A) and efficacy (B) of C2, C4, C5, C9, and C15 on cell migration in MCF-7, MMC, MDA-MB-231, and BT-549 cells. (C and D) The potency (IC50) (C) or efficacy (D) of melatonin, tamoxifen, 4-OH-tamoxifen, unlinked melatonin plus tamoxifen, and unlinked melatonin+4-OH-tamoxifen on cell migration in MCF-7, MMC, MDA-MB-231, and BT-549 cells. Each bar represents the mean ± S.D. of three independent experiments for each compound (n = 9 for vehicle, since it was run with each experiment conducted). Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: for figure A-a, P < 0.05 vs. C2; b, P < 0.05 vs. C4; c, P < 0.05 vs. C5; d, P < 0.05 vs. C9; for figure B-a, P < 0.05 vs. vehicle; b, P < 0.05 vs. C2; c, P < 0.05 vs. C4; d, P < 0.05 vs. C5, e, P < 0.05 vs. C9; for figure C-a, P < 0.05 vs. melatonin; b, P < 0.05 vs. tamoxifen; c, P < 0.05 vs. 4-OH-tamoxifen; for figure D-a, P < 0.05 vs. vehicle; b, P < 0.05 vs. melatonin; c, P < 0.05 vs. tamoxifen; d, P < 0.05 vs. 4-OH-tamoxifen, e, P < 0.05 vs. melatonin+tamoxifen.
- Fig. 4.
Competition of C2, C4, C5, C9, or C15 for [3H]-or [125I]-estradiol binding to ERs expressed in mouse uterus or MCF-7 cells. (A-G) Binding of [3H]-or [125I]-estradiol to ERs expressed in mouse uterus (A and B) and MCF-7 cells by competition binding using tamoxifen (A), 4-OH-tamoxifen (C), or conjugates C2, C4, C5, C9, or C15 (B, D–G) and G1 effects on total [3H]-estradiol binding to ERs expressed in MCF-7 cells (H). Data represent the mean ± S.D. of three independent experiments performed in duplicate. (A) and (B) were taken from Witt-Enderby et al. (2014). Data in (H) were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was indicated with lowercase letters as follows: a, P < 0.05 vs. vehicle; b, P < 0.05 vs. tamoxifen; c, P < 0.05 vs. G1; d, P < 0.05 vs. C4; and e, P < 0.05 vs. C5.
- Fig. 5.
Competition of C2, C4, C5, C9, or C15 for 2-[125I]-iodomelatonin binding to MT1Rs expressed in Chinese hamster ovary (CHO) cells. (A–G) Competition of 2-[125I]-iodomelatonin for binding to MT1Rs expressed in CHO cells in the presence of melatonin (A and B) or conjugates C2, C4, C5, C9, or C15 (C–G). Data represent the mean ± S.D. of three independent experiments performed in duplicate. (B) and (D) were taken from Witt-Enderby et al. (2014).
- Fig. 6.
Effect of C4, C5, melatonin, tamoxifen, 4-OH-tamoxifen, and unlinked melatonin plus tamoxifen on TamR MCF-7 cell viability and cell migration. (A and B) Data in (A) demonstrate the effect of C4, C5, melatonin, tamoxifen, 4-OH-tamoxifen, or unlinked melatonin plus tamoxifen on TamR MCF-7 cell viability and cell migration (B). Data represent the mean ± S.D. of three independent experiments performed in duplicate. Complete characeterization of TamR cells can be found in Supplemental Fig. 6. Abs, absorbance.
- Fig. 7.
Effect of C4, C5, melatonin, tamoxifen, 4-OH-tamoxifen, and unlinked melatonin plus tamoxifen on TU-BcX-4IC cell viability and cell migration and TU-BcX-4QAN tumor tissue. (And B) Data demonstrate the effect of C4, C5, melatonin, tamoxifen, or unlinked melatonin plus tamoxifen on TU-BcX-4IC cell viability (A) and cell migration (B). (C and D) Expression of pERK1/2, pERK5, and NF-κB was analyzed in TU-BcX-4QAN tumor tissue and TU-BcX-4IC cells (C), and total specific binding of 2-[125I]-iodomelatonin or [3H]-estradiol was measured in TU-BcX-4IC cells (D). Data represent the mean ± S.D. of three independent experiments performed in duplicate. Significance was defined as P < 0.05. In (A) and (B), asterisks indicate significance from all treatments except 100 μM treatments for all groups. In (C), “a” indicates significance compared with vehicle and “b” indicates significance compared with 10 μM melatonin. Images of cells in (A) and (B) were exposed to vehicle or 100 μM of the treatment listed within each series of graphs. OD, optical density.
- Fig. 8.
Effect of MEK1/2, MEK5, or PI3K inhibitors on cell viability and migration of C4 or C5 in MCF-7 cells. (A–H) Cell viability (A–D) and migration (E–H) of C4 or C5 in the absence or presence of PD98059, Bix02189, or pictilisib in MCF-7 cells. Each point represents the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Significance of the letters is mentioned in each panel (A-H).
- Fig. 9.
Effect of MEK1/2, MEK5, or PI3K inhibitors on cell viability and migration of C4 or C5 in MMC cells. (A–H) Cell viability (A–D) and migration (E–H) of C4 or C5 in the absence or presence of PD98059, Bix02189, or pictilisib in MMC cells. Each point represents the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Significance of the letters is mentioned in each panel (A-H).
- Fig. 10.
Effect of MEK1/2, MEK5, or PI3K inhibitors on cell viability and migration of C4 or C5 in MDA-MB-231 cells. (A–H) Cell viability (A–D) and migration (E–H) of C4 or C5 in the absence or presence of PD98059, Bix02189, or pictilisib in MDA-MB-231 cells. Each point represents the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Significance of the letters is mentioned in each panel (A-H).
- Fig. 11.
Effect of MEK1/2, MEK5, or PI3K inhibitors on cell viability and migration of C4 or C5 in BT-549 cells. (A–H) Cell viability (A–D) and migration (E–H) of C4 or C5 in the absence or presence of PD98059, Bix02189, or pictilisib in BT-549 cells. Each point represents the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Significance of the letters is mentioned in each panel (A-H).
- Fig. 12.
Summary of mechanisms of action underlying C4 or C5 in BC cells. Depicted is a summary of the mechanisms of action underlying C4 (A, B, D, F, H, J, and K) or C5 (C, E, G, I, J, and L) in MCF-7 (A–C), MMC (D–G), MDA-MB-231 (H and I), or BT-549 (J–L) BC cells. The lines connecting the proteins indicated in black were the relationships deduced from the viability and migration assays, and the lines indicated in gray were derived from the Western blot analyses.
Tables
- TABLE 1
Comparison of potencies between C2, C4, C5, C9, and C15 conjugates to inhibit BC cell viability and migration
Each value represents the mean ± S.D. of potency (IC50) values generated from three to nine individual curves fit by nonlinear regression analysis.
Cell Type 
C2 (n = 1) C4 (n = 3) C5 (n = 4) C9 (n = 8) C15 (n = 14) MCF-7 Viability 69 ± 81 mM 4 ± 4.31 nM 440 ± 331 mM 107 ± 17 mM abcd 34 ± 15 µM Migration 100 ± 156 µM 9 ± 11 mM 4 ± 3.3 µM 218 ± 48 mM abc 1 ± 2 µM MMC Viability 13 ± 8 µM abcd 243 ± 331 nM 6 ± 1.6 µM 6 ± 2.8 µM 333 ± 333 mM Migration 221 ± 65 mM abcd 5 ± 8 mM 227 ± 200 nM 206 ± 25.7 µM abcd 110 ± 149 nM MDA-MB-231 Viability 246 ± 178 µM 37 ± 34 nM 12 ± 11 µM d 145 ± 70 mM 477 ± 674 pM Migration 559 ± 131 mM abcd 11 ± 19 mM d 3 ± 3 µM 269 ± 119 µM 173 ± 157 mM BT-549 Viability 24 ± 8 µM b 6 ± 2 µM b 24 ± 31 µM b 34 ± 34 µM b 2 ± 3 µM b Migration 579 ± 308 mM abcd 9 ± 9 µM 51 ± 62 µM 328 ± 340 mM 18 ± 23 nM Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: a, P < 0.05 vs. C2; b, P < 0.05 vs. C4; c, P < 0.05 vs. C5; d, P < 0.05 vs. C9.
- TABLE 2
Comparison of efficacies between C2, C4, C5, C9, and C15 conjugates to inhibit BC cell viability and migration
Each value represents the mean ± S.D. of maximum inhibitory effect of each drug conjugate represented as the percentage of vehicle for viability and the percentage of baseline for migration assays.
Cell Type C2 C4 C5 C9 C15 MCF-7 Viability 66 ± 2 abcde 29 ± 7 abcde 10 ± 0.3 ab 65 ± 2 abcde 86 ± 2.4 acde Migration 20 ± 11 ab 10 ± 15 ab 2 ± 2 a 19 ± 11 ab −13 ± 5 d MMC Viability 79 ± 6 a 57 ± 22 ab 65 ± 7 ab 79 ± 9 a 97 ± 11 cde Migration 44 ± 34 abcde 7 ± 40 a 1 ± 2.3 a 39 ± 39 abe −32 ± 5 MDA-MB-231 Viability 63 ± 1.4 abd 98 ± 22 ce 75 ± 1.7 ad 68 ± 2 abd 104 ± 6.7 ce Migration 33 ± 13 ab 30 ± 36 ab 10 ± 7 ab 35 ± 10 abcde −43 ± 27 acde BT-549 Viability 80 ± 16 abce 66 ± 4 ab 67 ± 6 ab 88 ± 27 cde 93 ± 7 acde Migration 42 ± 12 abcd 20 ± 4 a 0 ± 0 56 ± 29 abcde −61 ± 14 abcde Values were derived from three to nine individual curves fit by nonlinear regression analysis. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: a, P < 0.05 vs. vehicle; b, P < 0.05 vs. C2; c, P < 0.05 vs. C4; d, P < 0.05 vs. C5; e, P < 0.05 vs. C9.
- TABLE 3
Comparison of potencies between C2, C4, C5, C9, and C15 conjugates and controls to inhibit BC cell viability and migration
Each value represents the mean ± S.D. of potency (IC50) values generated from three to nine individual curves fit by nonlinear regression analysis.
Cell Type n per Group Melatonin Tamoxifen 4-OH- Tamoxifen Melatonin + Tamoxifen Melatonin + 4-OH-Tamoxifen C2 C4 C5 C9 C15 MCF-7 Viability 4 18 ± 18 nM 12 ± 20 mM 34 ± 41 mM 328 ± 222 µM 17 ± 29 mM 69 ± 81 mM 4 ± 4.31 nM 440 ± 331 mM 107 ± 17 mM abcde 34 ± 15 µM Migration 4 309 ± 164 nM 138 ± 117 µM 13 ± 13 mM 11 ± 0.1 nM 131 ± 18 mM abc 100 ± 156 µM e 9 ± 11 mM e 4 ± 3.3 µM e 218 ± 48 mM abce 1 ± 2 µM e MMC Viability 4 427 ± 479 nM 3 ± 3.1 µM 4 ± 1.5 µM 3 ± 2 µM 7 ± 2.3 µM a 13 ± 8 µM abcde 243 ± 331 nM e 6 ± 1.6 µM 6 ± 2.8 µM 333 ± 333 mM Migration 4 231 ± 392 nM 21 ± 23 µM 3 ± 3.6 µM 31 ± 40 µM 68 ± 33 µM ac 221 ± 65 mM abcde 5 ± 8 mM 227 ± 200 nM e 206 ± 25.7 µM abcde 110 ± 149 nM MDA-MB-231 Viability 4 52 ± 89 nM 58 ± 67 mM 26 ± 30 µM 419 ± 354 mM 69 ± 106 nM 246 ± 178 µM 37 ± 34 nM 12 ± 11 µM d 145 ± 70 mM 477 ± 674 pM Migration 4 123 ± 239 µM 30 ± 35 mM 91 ± 84 µM 229 ± 232 mM 5 ± 3 µM 559 ± 131 mM abcde 11 ± 19 mM d 3 ± 3 µM 269 ± 119 µM 173 ± 157 mM BT-549 Viability 3 504 ± 0.1 nM 781 ± 7 mM 30 ± 0.9 µM 11 ± 19 mM 24 ± 11 µM a 24 ± 8 µM b 6 ± 2 µM b 24 ± 31 µM b 34 ± 34 µM b 2 ± 3 µM b Migration 4 4 ± 6 µM 11 ± 18 µM 3 ± 3 µM 24 ± 7 µM 116 ± 93 µM 579 ± 308 mM abcde 9 ± 9 µM 51 ± 62 µM 328 ± 340 mM 18 ± 23 nM Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: a, P < 0.05 vs. melatonin; b, P < 0.05 vs. tamoxifen; c, P < 0.05 vs. 4-OH- tamoxifen; d, P < 0.05 vs. melatonin + tamoxifen; and e, P < 0.05 vs. melatonin + 4-OH- tamoxifen.
- TABLE 4
Comparison of efficacies of C2, C4, C5, C9, and C15 conjugates and controls to inhibit BC cell viability and migration
Each value represents the mean ± S.D. of maximum inhibitory effect of each drug conjugate represented as a percentage of vehicle (n = 9/cell line tested) for viability and a percentage of baseline for migration assays.
Cell Type n per Group Melatonin Tamoxifen 4-OH- Tamoxifen Melatonin + Tamoxifen Melatonin + 4-OH-Tamoxifen C2 C4 C5 C9 C15 MCF-7 Viability 4 87 ± 8 a 13 ± 1.2 ab 14 ± 2.2 ab 11 ± 0.5 ab 12 ± 1.2 ab 66 ± 2 abcdef 29 ± 7 abcdef 10 ± 0.3 ab 65 ± 2 abcdef 86 ± 2.4 acdef Migration 3 −14 ± 6 6 ± 1.6 a 20 ± 10 ab 0 ± 0 a 39 ± 12 abce 20 ± 11 ab 10 ± 15 abf 2 ± 2 af 19 ± 11 ab −13 ± 5 df MMC Viability 3 91 ± 17 65 ± 12 ab 65 ± 13 ab 65 ± 8 ab 66 ± 7 ab 79 ± 6 a 57 ± 22 ab 65 ± 7 ab 79 ± 9 a 97 ± 11 cdef Migration 3 −23 ± 7 a 0.05 ± 0.01 a 0.5 ± 1 a −12 ± 14 a 0.7 ± 9 a 44 ± 34 abcdef 7 ± 40 a 1 ± 2.3 a 39 ± 39 abe −32 ± 5 MDA-MB-231 Viability 3 92 ± 5 76 ± 2 a 107 ± 40 70 ± 5 abd 78 ± 3 abd 63 ± 1.4 abd 98 ± 22 ce 75 ± 1.7 ad 68 ± 2 abd 104 ± 6.7 cef Migration 3 −64 ± 2 9 ± 11 a 3 ± 3 c 0 ± 0 abd 2 ± 2 abd 33 ± 13 ab 30 ± 36 ab 10 ± 7 ab 35 ± 10 abcdef −43 ± 27 acdef BT-549 Viability 3 91 ± 2 66 ± 6 ab 72 ± 6 ab 64 ± 5 ab 65 ± 3 ab 80 ± 16 abcef 66 ± 4 ab 67 ± 6 ab 88 ± 27 cdef 93 ± 7 acdef Migration 3 −15 ± 9 0 ± 0 0 ± 0 15 ± 5 a 32 ± 16 ab 42 ± 12 abcd 20 ± 4 a 0 ± 0 56 ± 29 abcde −61 ± 14 abcdef Values were derived from individual curves fit by nonlinear regression analysis and least squares fit. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Letters denote significance between groups as follows: a, P < 0.05 vs. vehicle; b, P < 0.05 vs. melatonin; c, P < 0.05 vs. tamoxifen; d, P < 0.05 vs. 4-OH- tamoxifen; e, P < 0.05 vs. melatonin + tamoxifen; and f, P < 0.05 vs. melatonin + 4-OH- tamoxifen.
- TABLE 5
The effect of MEK1/2 (PD98059), MEK5 (Bix02189), and PI3K (pictilisib) on C4/C5-mediated effects on pERK1/2, pERK5, pAKT, NF-κB, RUNX2, and β1-INTEGRIN levels in MCF-7, MMC, MDA-MB-231, and BT-549 cell lines
Data represent the mean ± S.D. of three independent experiments.
Cell Type Blot Drug treatments MCF-7 Vehicle (A*) 1 nM C4 (B**) 1 nM C5 (C***) 10 µM Bix02189 1 nM C4 + 10 µM Bix02189 1 nM C5 + 10 µM Bix02189 10 µM PD98059 1 nM C4 + 10 µM PD98059 1 nM C5 + 10 µM PD98059 pERK1/2 0.00092 ± 0.001 NS 0.0093 ± 0.003 (A*) 0.004 ± 0.002 (A*) NS 0.0022 ± 0.001 (C***) 0.00039 ± 0.0005 (A*) NS 0.0021 ± 0.001 (C***) NF-κB 0.045 ± 0.030 NS 0.14 ± 0.043 (A*) NS NS NS NS NS NS Vehicle (A*) 10 µM C4 (B**) 10 µM C5 (C***) 10 µM Bix02189 10 µM C4 + 10 µM Bix02189 10 µM C5 + 10 µM Bix02189 10 µM PD98059 10 µM C4 + 10 µM PD98059 10 µM C5 + 10 µM PD98059 pERK1/2 0.00092 ± 0.0006 0.0042 ± 0.004 (A*) 0.0047 ± 0.003 (*) 0.004 ± 0.002 (A*) NS NS NS NS 0 ± 0 (***) NF-κB 0.045 ± 0.030 0.16 ± 0.085 (A*) 0.13 ± 0.068 (A*) NS NS NS 0.085 ± 0.023 (A*) NS NS 10 µM pictilisib Vehicle (A*) 1 nM C4 (B**) 1 nM C4 + 10 µM pictilisib 10 µM C4 (C***) 10 µM C4 + 10 µM pictilisib pAKT NS 0.00094 ± 0.0008 NS NS 0.0025 ± 0.0012 0.00063 ± 0.0005 (***) RUNX2 NS 0.19 ± 0.04 NS NS 0.15 ± 0.02 (A*) NS MMC Vehicle (A*) 1 nM C4 (B**) 1 nM C5 (C***) 10 µM Bix02189 1 nM C4 + 10 µM Bix02189 1 nM C5 + 10 µM Bix02189 10 µM PD98059 1 nM C4 + 10 µM PD98059 1 nM C5 + 10 µM PD98059 pERK1/2 0.0092 ± 0.004 0.0054 ± 0.001 0.005 ± 0.001 NS NS 0.018 ± 0.007 (C***) NS 0.014 ± 0.001 (B**) NS pERK5 0.041 ± 0.027 0.05 ± 0.018 NS NS 0.093 ± 0.008 (**) NS 0.06 ± 0.035 (*) NS NS NF-κB 0.12 ± 0.051 0.1 ± 0.010 NS NS NS NS NS 0.14 ± 0.016 (B**) NS 10 µM pictilisib Vehicle (A*) 1 nM C4 (B**) 1 nM C4 + 10 µM pictilisib 10 µM C4 (C***) 10 µM C4 + 10 µM pictilisib NF-κB NS 0.36 ± 0.11 NS NS 0.37 ± 0.085 (A*) NS β1-INTEGRIN NS 0.25 ± 0.085 NS NS 0.26 ± 0.077 (*) NS 10 µM pictilisib Vehicle (A*) 1 nM C5 (B**) 1 nM C5 + 10 µM pictilisib 10 µM C5 (C***) 10 µM C5 + 10 µM pictilisib NF-κB NS 0.36 ± 0.11 0.46 ± 0.026 (A*) NS NS NS β1-INTEGRIN NS 0.25 ± 0.085 0.33 ± 0.07 (*) NS NS NS MDA-MB-231 Vehicle (A*) 1 nM C4 (B**) 1 nM C5 (C***) 10 µM Bix02189 1 nM C4 + 10 µM Bix02189 1 nM C5 + 10 µM Bix02189 10 µM PD98059 1 nM C4 + 10 µM PD98059 1 nM C5 + 10 µM PD98059 pERK1/2 0.038 ± 0.017 0.051 ± 0.018 0.063 ± 0.021 NS NS NS NS 0.014 ± 0.005 (B**) 0.012 ± 0.009 (C***) pERK5 0.0028 ± 0.006 NS NS NS NS NS 0.011 ± 0.007 (*) NS NS NF-κB 0.022 ± 0.021 0.032 ± 0.018 NS NS NS NS NS 0.093 ± 0.037 (**) NS Vehicle (A*) 10 µM C4 (B**) 10 µM C5 (C***) 10 µM Bix02189 10 µM C4 + 10 µM Bix02189 10 µM C5 + 10 µM Bix02189 10 µM PD98059 10 µM C4 + 10 µM PD98059 10 µM C5 + 10 µM PD98059 pERK1/2 0.038 ± 0.017 NS 0.033 ± 0.006 NS NS NS NS NS 0.014 ± 0.003 (C***) pERK5 0.0028 ± 0.006 0.004 ± 0.003 NS NS NS NS NS 0.018 ± 0.001 (B**) NS NF-κB 0.022 ± 0.021 0.043 ± 0.055 (*) NS NS NS NS 0.085 ± 0.019 (A*) NS NS 10 µM pictilisib Vehicle (A*) 1 nM C4 (B**) 1 nM C4 + 10 µM pictilisib 10 µM C4 (C***) 10 µM C4 + 10 µM pictilisib pERK1/2 NS 0.0024 ± 0.00069 NS NS 0.0023 ± 0.00055 0.0014 ± 0.00067 (C***) 10 µM pictilisib (d) Vehicle (A*) 1 nM C5 (b** B) 1 nM C5 + 10 µM pictilisib 10 µM C5 (C***) 10 µM C5 + 10 µM pictilisib NF-κB 0.08 ± 0.032 0.093 ± 0.03 0.19 ± 0.072 (a) 0.27 ± 0.063 (bd) NS NS RUNX2 NS 0.16 ± 0.055 0.19 ± 0.025 0.22 ± 0.042 (**) NS NS β1-INTEGRIN NS 0.022 ± 0.009 0.037 ± 0.021 0.11 ± 0.011 (B**) 0.061 ± 0.010 (*) NS BT-549 Vehicle (A*) 10 µM C4 (B**) 10 µM C5 (C***) 10 µM Bix02189 10 µM C4 + 10 µM Bix02189 10 µM C5 + 10 µM Bix02189 10 µM PD98059 10 µM C4 + 10 µM PD98059 10 µM C5 + PD98059 NF-κB 0.058 ± 0.019 NS 0.08 ± 0.039 NS NS NS NS 0.018 ± 0.006 (C***) NS 10 µM pictilisib Vehicle (A*) 1 nM C4 (B**) 1 nM C4 + 10 µM pictilisib 10 µM C4 (C***) 10 µM C4 + 10 µM pictilisib pERK1/2 NS 0.00087 ± 0.0015 0.00018 ± 0.00032 0.0021 ± 0.0003 (B**) NS NS 10 µM pictilisib Vehicle (A*) 1 nM C5 (B**) 1 nM C5 + 10 µM pictilisib 10 µM C5 (C***) 10 µM C5 + 10 µM pictilisib pERK5 NS 0.0051 ± 0.0026 0.0026 ± 0.0017 (A*) NS NS NS Bands were quantified using Image Studio Lite (LI-COR Biosciences), where relative optical density values were obtained. The data were then normalized against β-actin. The lettering scheme denotes the following: lowercase letters (e.g., a, b, c, etc.) indicate analysis done by one-way ANOVA followed by the Newman–Keuls post hoc t test. Uppercase letters (e.g., A, B, C) indicate two-tailed t tests and symbols (*, **, and ***) indicate one-tailed t tests where significance was defined as P < 0.05. Significant values are displayed in the table and complete data sets along with their analyses are presented in Supplemental Tables 4–7. Letters and asterisks in parentheses below each treatment indicates the statistical comparison made with respect to that treatment group. The upper band indicates the protein of interest and the lower band indicates the β-actin band. NS, not significant.
- TABLE 6
In vitro metabolism and in vivo pharmacokinetic parameters of tamoxifen, C4, or C5 by MLM/human liver microsomal incubation and by subcutaneous/oral administration in female C57BL/6J mice, respectively
Data represent the mean ± S.D. percentage of drug conjugate remaining from microsomal incubations (n = 3) unless indicated otherwise.
Drug Route In Vitro Metabolism in Microsomes In Vivo Metabolism in Female C57BL/6J Mice 0 min 5 min 10 min 20 min Tmax Cmax Half-Life AUC0–24 Relative Bioavailability % h ng/ml h h × ng/ml % Tamoxifen MLM s.c. 100.67 ± 9.44 74.79 ± 10.13 60.36 ± 11.59 6 27.5 4.6 259.4 HLM 97.16 ± 7.84 80.03 ± 3.79 58.35 ± 24.55 C4 MLM s.c. 107.81 ± 16.14 88.36 ± 11.92 65.55 ± 14.60 6 40.9 6.8 289.1 HLM p.o. 95.20 ± 20.19 79.22 ± 8.43 65.78 ± 8.76 0.5 102.6 3.6 143.7 4.52 C5 MLM s.c. 112.11 ± 16.02 85.39 ± 12.22 69.45 ± 14.23 6 31.0 5.3 229.1 HLM p.o. 95.82 ± 10.62 75.32 ± 5.47 61.72 ± 4.96 24 8.9 6.9 1.6 0.11 Tamoxifen, C4, or C5 drug conjugates (10 μM) were incubated with MLMs or HLMs over time (10 minutes for MLMs and 20 minutes for HLMs) and the amount remaining at each timepoint was measured by HPLC-UV detection. Data were analyzed by one-way ANOVA followed by the Newman–Keuls post hoc t test where significance was defined as P < 0.05. Mice were administered a 1 mg/kg dose by the subcutaneous route or a 10 mg/kg dose by the oral route (conjugates only). HLM, human liver microsome.
Data Supplement
- Supplemental Data -
Supplementary Scheme 1
Supplementary Figures 1-7
Supplementary Tables 1-7
Supplementary Material
- Supplemental Data -
