The induction of myeloma cell death and DNA damage by tetrac, a thyroid hormone derivative

1. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 1

   Tetrac inhibits MM cell proliferation and viability and induces apoptosis. Four MM cell lines were treated with increasing concentrations of tetrac (10–100 µM) for 24–96 h and evaluated for (A) Cell proliferation (WST-1, ELISA), (B) cell number (flow-cytometry), (C and D) apoptosis (Annexin-V/PI) and (E) cell cycle, by flow-cytometry. Experiments were repeated three times in triplicate. *P ≤ 0.05, **P ≤ 0.01.
2. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 2

   Tetrac activates caspases and PARP-1 cleavage, but not AIF in MM. RPMI-8226 cells were incubated with tetrac (1–50 µM) for 1–8 h, total protein was extracted and Western blots were carried out for (A) caspase-9, caspase-3, PARP-1 and (B) AIF. Shown is a representative experiment of two performed (C) Representative quantification of band intensity of the corresponding Western blots is presented as ratio of cleaved target protein over total protein intensity. Values are means ± s.d. Densitometry is expressed as percentage compared with the vehicle-treated group (considered as 100%, marked by a dashed line). Experiments were repeated twice. *P ≤ 0.05, **P ≤ 0.01.
3. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 3

   Involvement of p53 and caspases in action of tetrac on MM cells. Temperature-sensitive RPMI-8226 cells were treated with tetrac ± Z-VAD-FMK and grown at 32°C or 37°C and evaluated for (A) cell proliferation (WST-1), (B) cell number (flow-cytometry), (C) apoptosis (Annexin-V/PI) and (D and E) cell cycle, flow-cytometry. Experiments were repeated three times at triplicate. *P ≤ 0.05, **P ≤ 0.01. Significance between tetrac’s effect under 32°C and 37°C is marked by #.
4. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 4

   Tetrac increases DNA damage response markers in MM cells. RPMI-8226 cells were treated with tetrac (1–50 µM) for 1–8 h and were evaluated for pATM, PARP-1 and pγH2AX level and activation by Western blot. Experiments were repeated twice. Representative quantification of band intensity of the corresponding Western blots is presented as ratio of target protein to tubulin intensity. Values are means ± s.d. Densitometry is expressed as percentage compared with the vehicle-treated group (considered as 100%, marked by a dashed line). *P ≤ 0.05, **P ≤ 0.01.
5. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 5

   Induction of cell death in MM cells is blocked by RGD. CAG cells were treated for an overnight with various concentrations of tetrac and examined for cell viability (WST-1) in combination with (A) RGD or (B) RGE. Experiments were repeated five times in triplicate. (C) Surviving cells (An−/PI−) and (D) apoptotic cells (An+/PI− and An+/PI+) were evaluated by flow-cytometry. Experiments were repeated three times in triplicate. (E) pγH2AX protein level and activation (Western blots). The experiment was repeated twice. Representative quantification of band intensity of the corresponding Western blots is presented as ratio of target protein to tubulin intensity. Values are means ± s.d. Densitometry is expressed as percentage compared with the vehicle-treated group (considered as 100%, marked by a dashed line). *P ≤ 0.05, **P ≤ 0.01.
6. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 6

   Tetrac induces apoptosis in primary BM cells from MM patients. (A) Mononuclear cells from a MM BM sample (BM#1) were separated by Ficoll gradient, treated in triplicates with tetrac (100 nM–1 µM) for 96 h and then analyzed for cell death (Annexin-V/PI, flow-cytometry). (A) Representative results for whole BM cells and (B) CD138+ cells. Samples of the same subject treated with vehicle served as controls. A full color version of this figure is available at https://doi.org/10.1530/ERC-17-0246.
7. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 7

   Tetrac sensitizes MM cell lines and primary cells to bortezomib. RPMI-8226 and CAG cells were treated with bortezomib (25 nM) with/without tetrac (100 µM) and were analyzed for (A) cell proliferation (WST-1, ELISA) and (B) survival (annexin-V/PI, flow-cytometry). Experiments were repeated at least 3 times, in triplicate. Primary BM cells from MM patients were treated with bortezomib (25 nM) with/without tetrac (100 nM–1 µM) and were analyzed for (C and D) apoptosis (annexin-V/PI, flow-cytometry). (E) Representative annexin/PI results from a BM sample (BM#1) in whole BM cells (upper panel) and gated CD138+ plasma cells (lower panel). *P ≤ 0.05, **P ≤ 0.01. A full color version of this figure is available at https://doi.org/10.1530/ERC-17-0246.
8. View larger version:

   • In this window
   • In a new window

   • Download as PowerPoint Slide

   Figure 8

   Primary bone marrow cells from MM patients highly express αvβ3 integrin. Flow-cytometry analysis of cells incubated with (A) αvβ3 antibody and (B) CD138-APC and CD45-FITC antibodies. Malignant plasma cell clone is depicted in pink; mononuclear cells in green. (C) Analysis of expression of integrin in CD138+ (pink) vs CD138-cells (green). (D) Forward scatter (FSC) and side scatter (SSC) plot of the various cell populations: plasma cells (blue), promyelocytes (purple), monocytes (yellow) and lymphocytes (green). (E) Relative αvβ3 expression in the various cell populations. Isotype control is depicted in black.

• © 2018 Society for Endocrinology