1 / 19

PMX 290 induces a G2/M cell cycle block

. . . . . .  p < 0.05  p < 0.001 Student’s T Test. PMX 290 induces a G2/M cell cycle block. Cell cycle analysis: HCT116 cells, 24h exposure Accumulation of cells in the G2/M phase of the cell cycle Population with sub G1 DNA content apparent.

york
Télécharger la présentation

PMX 290 induces a G2/M cell cycle block

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1.       p < 0.05  p < 0.001 Student’s T Test PMX 290 induces a G2/M cell cycle block Cell cycle analysis: HCT116 cells, 24h exposure Accumulation of cells in the G2/M phase of the cell cycle Population with sub G1 DNA content apparent DMSO 1 mM PMX 290 1 mM PMX 2058 Strictly confidential

  2. 13.7% 12.4% 38.2% 19.5% DMSO 1 mM PMX 290 1 mM PMX 2058 3.2% 1.8% PMX 290 induces apoptosis AnnexinV / propidium iodide analysis: HCT116: 24h, PMX 290 Caspase 3 and PARP cleavage ← PARP113 kDa ← cleaved PARP 89 kDa ← cleaved PARP24 kDa ← cleaved caspase 317/19 kDa ← D 0.1 0.5 1 5 0.1 0.5 1 5 0.5 PMX 290 PMX 2058 CPT Strictly confidential

  3. Induction of Reactive Oxygen Species (ROS) by PMX 290 7h     Induction of ROS detected with H2DCF-DA by flow cytometry 16h  p < 0.05  p <0.001 Strictly confidential

  4. Glutathione (GSH) depletion sensitises cells to PMX 290 HCT116 GSH depleted using buthionine L-sulfoximine Strictly confidential

  5. PMX 290 is a mammalian thioredoxin reductase (TrxR) inhibitor PMX 290 has been shown to be an irreversible inhibitor of NADPH-reduced mammalian thioredoxin reductase (DTNB assay). IC50 = 2.7 mM Chew et al: FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  6. TrxR Actin 0 5 3 1 0.5 PMX 290 (µM) PMX 290 inhibits TrxR in cells Thioredoxin reductase but not thioredoxin activity inhibited in cell lysates of HCT116 treated with PMX 290 Time dependent inhibition of BIAM labelling of selenocysteine residue of thioredoxin reductase DMSO 0.5 1 3 5 PMX 290 (µM) Chew et al: FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  7. PMX 290 (mM) DMSO Time (min) 60 3 15 30 60 HRP- Streptavidin -ratTrxR PMX 290 targets selenium residue of TrxR IC50 (mM) 7.9 8.3 > 75 > 75 > 75 PMX 290 does not inhibit E. coli TrxR/Trx system or yeast glutathione reductase (GR). PMX 290 prevents binding of BIAM that targets selenocysteine residue Chew et al: FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  8. Proposed model of TrxR inhibition Modified from Chew et al; FASEB Journal (2008): 22(6), 2072-83. Strictly confidential

  9. Whole Cell Lysates IP: Trx1 S—S Trx1 SH—SH Trx1 P ASK1  ASK1 ASK1 ASK1 ASK1 P SH—SH Trx1  Trx1 Trx1 S—S Inactive Active DMSO DMSO 5 M DMSO DMSO 5 M -Ab +Ab PMX -Ab +Ab PMX 290 290 H2O2, TNF-, cisplatin  p-p38 MEKK4/7 MEKK3/6  total p38 JNK p38  p-JNK   total-JNK  Apoptosis 0 1 2 3 4 5 6 7 24 h 0.5 mM PMX 290 Apoptosis Signal Regulating Kinase 1 (ASK1) inhibition by Trx1 Strictly confidential

  10. Dominant negative ASK1 (dnASK) does not prevent apoptosis induced by PMX 290 ← dnASK ← actin K709R mutation leads to catalytically inactive ASK1 mock transfection dnASK1 Strictly confidential

  11. Difference in Trx/TrxR inhibition data Trx/TrxR/Insulin reduction assay Different concentrations/conditions to Chew paper but fundamentally similar assay Strictly confidential

  12. PMX 290 inhibits VEGF secretion under hypoxic and normoxic conditions VEGF secretion inhibited at sub-toxic concentrations of PMX 290. Jones et al; Clin. Cancer Research (2006): 12 (18), 5384 – 5394. Strictly confidential

  13. PMX 290 inhibits Hif-1 - HRE DNA binding and decreases mRNA level of Hif target genes Jones et al; Clin. Cancer Research (2006): 12 (18), 5384 – 5394. Strictly confidential

  14. PMX 290 MDA 468 PMX 290 increase Hif protein levels in cytoplasm MDA468 cells Unexpected increase in Hif-1 and Hif-2 seen with PMX 290 treatment under both normoxic and hypoxic conditions. VHL independent, possibly due to increase in HSP70/90 expression Thioredoxin overexpression has been shown to increase Hif protein levels and transactivation (Welsh, Cancer Research, 2002) Jones et al; Clin. Cancer Research (2006): 12 (18), 5384 – 5394. Strictly confidential

  15. Normoxic control Hypoxic control Hypoxic + PMX 290 • Hif driven EGFP expression is inhibited by 0.25 mM PMX 290. • No increase in Hif-1 protein expression in response to PMX 290. • Potentiation of 5-FU induced apoptosis under hypoxia PMX 290 inhibits Hif-1 transcriptional activity with no change in protein levels in HCT116 Ravizza R et al., Eur J Cancer (2009) Strictly confidential

  16. spheroids monolayer Control 2.5 mM PMX 290 Ravizza R et al., Eur J Cancer (2009) Subtoxic concentrations of PMX290 reduce HIF-1 activity and enhance the response to 5-FU • 3D HCT116 spheroid model Strictly confidential

  17. 0.5 mM PMX 290, 24h, HCT116 cells • Some indication of oxidative stress (e.g. HO-1) • CHOP (DDIT3), Trib3, GRP78 (HSPA5/BiP), ATF3 all upregulated and involved in ER stress Induction of endoplasmic reticulum stress related mRNAs Strictly confidential

  18. PMX 290 in vitro effects • Potent in vitro growth inhibition of cancer cell lines • G2/M cell cycle arrest and apoptosis induction Mechanism of action • PMX 290 induces ROS in sensitive cells • GSH depletion sensitises cells to PMX 290 • Evidence for TrxR inhibition through targeting of selenium residue • Only 40% inhibition of TR in cell lysates at lethal doses • Apoptosis induction independent of Trx/ASK pathway • Conflicting evidence for TR inhibition • Possible other targets, e.g. ER stress and Hif Summary Strictly confidential

  19. PMX 290 Hif effects • Conflicting cell type-specific alterations of Hif protein levels but… • Consistent inhibition of Hif transcriptional activity at sub toxic doses in monolayer and 3D cell models • Inhibition of Hif transactivation activity • Inhibition of VEGF secretion by hypoxic cells • Potentiation of 5-FU induced apoptosis in hypoxic monolayers and 3D spheroids Summary Strictly confidential

More Related