Combined γ Tocotrienol and Met Inhibitor Treatment Suppresses Mammary Cancer Cell Prolifera;on, Epithelial to Mesenchymal Transi;on, and Migra;on Paul W. Sylvester, Ph.D. Pfizer Endowed Professor of Pharmacy College of Pharmacy University of Louisiana at Monroe, USA
Tocotrienols and Cancer Cancer is a group of more than 100 different diseases characterized by uncontrolled cellular growth, local tissue invasion, and distant metastases. Current strategies for treating cancer have focused on developing drugs directed against specific molecular targets associated with tumor cell growth and progression. Tocotrienols may have great potential in the prevention and treatment of cancer because of multiple mechanisms of action directed against tumor cell proliferation and viability.
Vitamin E and Cancer Antiproliferative Effects Tocotrienols inhibit growth factor receptor activation. Leading to the suppression of mitogenic signaling pathways. Resulting in the blockage of cell cycle progression and inhibition of cancer cell division.
EGFR (HER) Dimerization ATP ATP Gene Transcription Growth Proliferation Survival
Cells per Well (1X10 5 ) 3 2 1 0 0 0.75 1 2 2.5 3 3.5 4 γ Tocotrienol (µm) Day 1 Day 5 Cells per Well (1X10 5 ) 3 2 1 0 0 0.1 0.25 0.5 0.75 1 3 5 Erlo;nib (µm) Day 1 Day 5 3 3 Cells per Well (1X10 5 ) 2 1 Cells per Well (1X10 5 ) 2 1 0 0 0.1 0.25 0.5 1 3 5 Gefi;nib (µm) Day 1 Day 5 0 0 10 20 50 100 250 Trastuzumab (µg/ml) Day 1 Day 5
Effect of Combined Treatment on SA Cell Growth MTT Assay Ki67 Staining 4 Cells per Well (1X10 5 ) 3 2 1 C E T ET C T E G ET GT Treatment Groups G GT C Control; T γtocotrienol; E Erlotinib; G Gefitinib.
Effect of Combina;on Treatment on ErbB Receptor Levels ErbB3 (kda) C T E G ET GT ErbB4 C 185 ErbB3 185 ErbB4 T 185 phospho ErbB3 180 phospho ErbB4 60 αtubulin E ET
Effect of γ Tocotrienol on Met Tyrosine Kinase Receptor Ac;va;on
Met Receptor Tyrosine Kinase Met is the receptor for hepatocyte growth factor (HGF). HGF is a growth factor normally ac;ve in wound healing, liver regenera;on and organ morphogenesis. Met dysregula;on results in increase cellular prolifera;on, survival, decrease adhesion, cytoskeletal changes, and increased mo;lity. Cancers with aberrant Met ac;vity are highly aggressive, invasive and metasta;c and these pa;ents have a poor prognosis.
c Met Receptor Homodimeriza;on HGF ss ΩΩΩΩ ss ΩΩΩΩ P P P P P P P P
HGFInduced Met Activation A Cells per Well (1X10 5 ) 3.0 2.5 2.0 1.5 1.0 0.5 HGF 0 ng/ml HGF 1 ng/ml HGF 5 ng/ml HGF 10 ng/ml Day0 Day1 Day2 Day3 B Time (min) HGF 100ng/ml 0 5 30 60 PMet Met αtubulin
HGFInduced Met Activation MitogenFree HGF 10ng/ml 5 5 30 30 Red: Phosphorylated Met receptors Blue: DAPI
Time (5 min) HGF 100ng/ml EGF 100ng/ml EGFR PEGFR Met PMet αtubulin
Cell Number per Well (1x10 5 ) Cell Number per Well (1x10 4 ) Cell Number per Well (1x10 4 ) 5 4 3 2 1 5 4 3 2 1 3 2 1 SA 0 1 2 3 4 5 5.5 γtocotrienol (µm) 0 1 2 3 4 5 5.5 SU11274 (µm) MCF7 0 5 10 20 25 30 0 2 4 6 8 10 γtocotrienol (µm) SU11274 (µm) MDAMB231 0 5 10 15 20 25 0 4 6 8 10 12 γtocotrienol (µm) SU11274 (µm)
Effect of γ Tocotrienol on Met Phosphoryla;on HGF C γt 3 Red: PhosphoMet Blue: DAPI (nuclear stain)
Synergistic Antiproliferative Effects with Combined γtocotrienol and SU11274 Treatment MDAMB231 MDAMB231 Cell Number per Well (1x10 4 ) 3 2 1 # # # # γtocotrienol (µm) 20 15 10 5 MDAMB231 2 4 6 8 10 ND SU11274 (µm) 0 5 10 12.5 15 γtocotrienol (µm) 0 5 10 12.5 15 γtocotrienol (µm) SU11274 (4 µm)
A CLS1 MCF10A Cell Number per Well (1x10 4 ) 4 3 2 1 Cell Number per Well (1x10 4 ) 4 3 2 1 0 5 10 15 20 30 0 2.5 5 10 15 20 0 5 10 20 30 0 2.5 5 10 15 B γtocotrienol (µm) SU11274 (µm) γtocotrienol (µm) SU11274 (µm) CLS1 MCF10A Cell Number per Well (1x10 4 ) 5 4 3 2 1 Cell Number per Well (1x10 4 ) 5 4 3 2 1 0 1 2 3 3.5 0 1 2 3 3.5 0 5 10 15 20 0 5 10 15 20 γtocotrienol (µm) γtocotrienol (µm) γtocotrienol (µm) γtocotrienol (µm) SU11274 (3 µm) SU11274 (3 µm)
A Cells per Well (1X10 5 ) 3.0 2.5 2.0 1.5 1.0 0.5 0 3 4 4.5 5 5.5 SU11274 (µm) Cells per Well (1X10 5 ) 3.0 2.5 2.0 1.5 1.0 0.5 0 1 2 3 3.5 4 γtocotrienol (µm) B C D HGF 100ng/ml γtocotrienol (4µM) SU11274 (5.5µM) HGF 100ng/ml Time (hr) 0 0 24 48 72 Met PMet αtubulin Met αtubulin VehicleTreated SU11274 5.5 µm γtocotrienol 4 µm Red: Phosphorylated Met receptors Blue: DAPI
A Cells per Well (1X10 5 ) 2.5 2.0 1.5 1.0 0.5 B HGF 100ng/ml γtocotrienol (2µM) SU11274 (3µM) Control γtocotrienol 2µM SU11274 3µM SU11274 3µM γtocotrienol 2µM Met PMet αtubulin
Treatment Effects on Ki67 Labeling DAPI Ki67 Merge 2µM γt 3 3µM SU 3µM SU 2µM γt 3 Control
γtocotrienol and SU11274 Treatment Effects on Met Signaling Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU 2µM γt 3 Control 2µM γt 3 3µM SU 3µM SU MEK pmek MAPK pmapk STAT1 pstat1 STAT5 pstat5 αtubulin PI3K PDK1 ppdk1 Akt pakt PNFκB105 PTEN ppten αtubulin
A Following 24 hr Treatment 0 hr Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU MDAMB231 SA B SA MDAMB231 Wound Closure (%) 80 60 40 20 # # Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU
Effects of Subeffective Doses of γtocotrienol and SU11274 on Epithelial and Mesenchymal Cellular Markers Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU ECadherin βcatenin Cytokeratin8 Cytokeratin18 Vimentin αtubulin
Treatment Effects on Epithelial and Mesenchymal Cellular Markers ECadherin Control 2µM γt 3 3µM SU 2µM γt 3 3µM SU 2µM γt 3 3µM SU Mesenchymal Marker Epithelial Marker Vimentin Cytokeratin18 Cytokeratin8 βcatenin
Extracellular α HGF β Met Intracellular γtocotrienol SU11274 SOS Grb2 Src Shp2 GAB1 STAT1/5 Shc PI3K RAS RAF Rac/Rho FAC PDK PTEN MEK Akt IKKα/β MAPK PAC mtor NFκB Cell Proliferation Cell Adhesion, Motility Invasion, Cytoskeleton Cell Survival Transcription
Conclusions γtocotrienol inhibits both EGFreceptor and Metreceptor tyrosine kinase activity and mitogenic signaling. These results strongly suggest that γtocotrienol treatment may provide significant health benefit in the prevention and/or treatment of breast cancer in women with deregulated HGF/Met signaling either as monotherapy or in combination with Met Inhibitors in the treatment of highly invasive and metastatic forms of breast cancer.
Collaborators Sylvester El Sayed Kaddoumi Nazzal Karen P. Briski Ahmed Orabi Bilal Abuasal Hazem Ali Barry S. McIntyre Mudit Mudit Ala Abuznait Alaa Alayoubi Abdul Gapor Fathy Behery Hisham Qosa Sumit Shah Ganesh Samant Vikram Wali Sunitha Bachawal Amit Shirode Mohamed Akl Nehad Ayoub Abhita Malaviya Theja Suryatheja Parash Parajuli Roshan Tiwari Osama Alawin Acknowledgements Support American Institute for Cancer Research National Cancer Institute at NIH American Heart Association Malaysian Palm Oil Board Malaysian Palm Oil Council Carotech Hovid First Tech International Ltd.
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