Robert Newman, Ph.D. and Peiying Yang, Ph.D.

Transcription

1 Robert Newman, Ph.D. and Peiying Yang, Ph.D. Department of Experimental Therapeutics and General Oncology The University of Texas, M.D. Anderson Cancer Center 1

2 Cardiac glycoside extracts from plants and animals as therapeutic agents Digitalis purpuria: Source of cardiac glycosides used for Rx of congestive heart failure 2

3 Cardiac glycoside extracts from plants and animals as therapeutic agents But what about use of cardiac glycosides for treatment of cancer? Digitalis purpuria: Source of cardiac glycosides used for Rx of congestive heart failure Nerium oleander: Source of the oral product PBI from Phoenix Biotechnology, Inc. and Anvirzel from Nerium Biotechnology, Inc. Bufo Toad Source of Chinese Medicine Huachansu from Anhui Jianchan Biochemical Inc. 3

4 Cardiac glycosides and Cancer Stenkvist et al. Cardiac glycosides and breast cancer. Lancet 10, Stenkvist,B., et al. Cardiac glycosides and breast cancer, revisited. N. Eng. J. Med. 306, Haux, J. Digitoxin is a potential anticancer agent for several types of cancer. Med. Hypoth. 53(6), Stenkvist B. Cardenolides and cancer. Anticancer drugs 12,

5 Cardiac glycosides and Cancer In the last 15 years there have been over 1100 articles cited in PubMed that refer to cardiac glycosides and cancer. In addition in the past three years alone there have been four review articles regarding cardiac glycosides for cancer management: Mijatovic et al., Biochem. Biophysi. Acta, 2007 Prassas et al., Nature Review, 2008 Alghoul, et al., Ther. Drug Monit, 2008 Newman et al., Mol. Interv.,

6 Oleandrin, bufalin and lipid soluble cardiac glycosides Na, K-ATPase MAPK PI3k ROS Glycolysis ERk1/2 AP-1 Akt HIF1α NF-Kb P21 cp1 Cell cycle arrest Autophagic cell death Apoptosis Red cross Down-regulation; Blue arrow Up-regulation; Dashed arrow hypothesized down regulation 6

7 Background: Rationale Na, K-ATPase Transmembrane protein Four isoforms of subunits binding site for Na +, ATP, and cardiac glycosides Three isoforms of subunits One subunit Binding affinity of subunits to cardiac glycosides: 2 and 3 >> 1 (250 fold greater binding to CGs) Na, K-ATPase 3 subunit is over-expressed in colon cancer cells compared to normal colon cells, while 1 expression was reduced (Sakai et al., FESEB Letter, 2004) 7

8 August,

9 Relative Na,K-ATPase 3 subunit expression is associated with cell sensitivity to oleandrin Mouse 120 Percent growth of control cells nm 15.6 nm 210 nm Panc-02 BxPC3 MiaPaca PANC-1 IC Actin Concentration (nm) 1000 Human Cell lines 3/β-actin IC50 value (nm) Panc-1 (HUMAN) 71 MiaPaca (HUMAN) 32 BxPC3 (HUMAN) 4 Panc-02 (MOUSE) alpha3/ actin ratio Yang, P., et al., Mol. Cancer Ther

10 The expression of Na,K-ATPase 3 subunit is localized in or near the plasma membrane in normal colon tissues but has a distinct perinuclear position in colon cancer tissues Normal 3 isoform Normal A B Cancer Cancer C D 10

11 Staining Scores Tumor Normal Nuclear Cytoplasmic Nuclear Cytoplasmic Tumor tissue: Increased 3 expression overall; Shift in expression to a perinuclear/nuclear location 11

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13 The distribution of 3 (fluorescence staining) depends on CaCo-2 cell phenotype: Wild type/undifferentiated vs. differentiated Undifferentiated Differentiated 3 isoform Green staining = 3; blue staining = nuclei. Note decreased 3 subunit presence near nuclei in differentiated cells. 13

14 0 hr 6 hr 16 hr 24 hr 14

15 Undifferentiated CaCO-2 cells Differentiated CaCO-2 cells A B E F C D G H Vacuoles and autophagozomes Normal cellular architecture 15

16 Percent growth of control cells mmllllllllllllllllllllllllllllllllllllllm Wildtype CaCo2 mmmmmmmmmmmmm Differentiated CaCo2 m Normal phenotype Malignant phenotype Concentration of oleandrin (nm) 16

17 Undifferentiated CaCO-2 cells ERK perk -Actin Differentiated CaCO-2 cells ERK perk -Actin Oleandrin (nm) PBI-05204(nM)

18 Molecular Cancer Therapeutics, 9: ,

19 Na, K-ATPase plays a significant role in the formation and maintenance of epithelial phenotype in mammalian cells. Molecular Cancer Therapeutics, 9: ,

20 Normal cell architecture: Limited 3 expression Location is near cell membrane TGF-B1 Oleandrin: Anvirzel, PBI Malignant /abnormal cell architecture: Abundant 3 expression- Location is perinuclear 20

21 Na, K-ATPase 3 subunit is located primarily on the plasma membrane of cells in normal human colon and lung tissues, but translocates to an intracellular compartment surrounding the nucleus in colon or lung tumor tissues. Cellular distribution of the Na,K-ATPase 3 subunit in CaCo-2 cells is altered when the phenotype is changed from an undifferentiated to a differentiated state. A perinuclear location is associated with adverse cell events such as cancer. The sensitivity of undifferentiated and differentiated CaCO-2 cells to oleandrin appears to be associated with the relative cellular expression and distribution of Na,K-ATPase 3 subunit. Among molecular events oleandrin enhances ERK phosphorylation in undifferentiated CaCO-2 cells, while no such change is observed in oleandrin treated differentiated CaCO-2 cells. 21

22 Na,K-ATPase must now be recognized as more than a simple membrane bound ion pump responsible for Na and K exchange. The 3 subunit and perhaps other components of the enzyme show a differential distribution in normal as opposed to malignant cells Because lipid soluble cardiac glycoside compounds such as oleandrin bind selectively to the 3 subunit and such binding is correlated with antiproliferative activity, the relative expression of this subunit might serve as a marker of tumor sensitivity to therapeutic products containing oleandrin such as Anvirzel and PBI

23 Univ. Texas M. D. Anderson Cancer Center Dr David Menter Carrie Cartwright Jibin Ding Dr. Imad Shureiqi Phoenix Biotechnology Inc. is gratefully acknowledged for funding support 23