Apoptosis of Carcinoma Human ColonColo 205 and Breast SKBr3 Cells by AntiCancer Drugs

1
(No Transcript)
2
Apoptosis of Carcinoma (Human Colon-Colo 205 and
Breast -SKBr3) Cells by Anti-Cancer Drugs Subhash
Basu

• Dept. of Chem. and Biochem, Univ. of Notre Dame,
  IN.
• Dept. of Chem., Kent State Univ, OH.
• Cleveland Clinic, Cleveland,OH.

3
NORMAL AND CANCER CELL GROWTH REGULATION
Normal Cells
Tumor Cells
CANCER CELLS
SAME AS TUMOR CELLS BUT 1) APOPTOTIC PROGRAM IS
READY AND REMAIN INHIBITED 2)APOPTOSIS IS
INDUCABLE
24hrs
24hrs
24hrs
24hrs
24hrs
Not Programmed for Cell Death (Non Apoptotic)
Undifferentiated Dividing Cells
Differentiated Cells
n1
2n
APOPTOTIC (Programmed) CELL DEATH
4
(No Transcript)
5
HOW DO WE DETECT APOPTOSIS ?

• 1. Light microscopic examination of morphology of
  cultured cells.
• 2. Using Fluorescence Dye (PSS1)which binds to
  the outer layer Phosphatidyl-Serine (early
  stage)
• 3. Using Propidium Iodide which enters through
  permeable plasma membrane and binds to DNA (late
  Stage)..
• 4. Determining activation of specific Caspase(s)
  by Western Blots (early-late stages)
• 5. DNA-laddering visualization by gel
  electrophoresis.(late stage)

6
WHAT PROVOKES APOPTOSIS ?

• Inhibition of chromosomal DNA replication. (
  cis-platin in Colo-205)
• Ceramide-generated from inhibition of the first
  committed step in the biosynthesis of
  glycosphingolipids (L/D-PPMP L/D-PDMP
  in Colo-205/SKBr3)
• Ceramide-generated from breakdown of
  glycosphingolipids.(GD3 GD1b Gangliosides in
  SKBr3)
• Ceramide-generated from breakdown of
  sphingomyelin.
• Deregulation of the transcription factors.

7
Structure of Apoptotic Drugs
Betulinic Acid
8
Apoptotic Colo-205 by Drugs
Cis-Platin 150 µM 48 hrs
L-PPMP 15 µM 48 hrs
Control Cells were synchronized with 0.5 mM
HU(24 hrs) X 2
Melphalan 16 µM 48 hrs
Tamoxifen 16 µM 48 hrs
9
DNA Laddering in Apoptotic Colo-205 (cis-Platin
Treated)
10
APOPTOSIS AND DNA METABOLISM BY ANTI- CANCER
DRUGS(cis-Platin/L(D)-PPMP/GD3)
APOPTOSIS
DNA-REPLICATION
APOPTOSIS
DNA-REPLICATION
cis-Platin ?
UNREGULATED
INHIBITED
cis-Platin ?
REGULATED
?X
Y?
CASPASES
cis-Platin ?
Pol-a
Cyto-C
Cyto-C
NUCLEARDNA
NUCLEARDNA
MITOCHONDRIA
MITOCHONDRIA
DIFFERENTIATED NOMAL CELLS (n1)
UNDIFFERENTIATED TUMOR OR CANCER CELLS (2n)
(COLO-205/SKBR-3/PA-3/ALL)
11
ANTI CANCER DRUGS
Biologically Active Platinum Complexes
cis-DDP (cis - Platin)
cis Pt (NH3)2Cl4
Biologically Inactive Platinum Complexes
Trans-DDP
Pt (dien) Cl
12
Kelly, T., Moghaggas, S., Bose, R., and Basu,
S.(1993) Cancer Biochem. And Biophys., 13,
135-146.
13
(No Transcript)
14
Bose, R. N., Li, D., Yang, W. W., and Basu, S.
(1999) J. Biomol. Struct. Dynamics, 16,
1075-1085
15
Bose, R. N., Li, D., Yang, W. W., and Basu, S.
(1999) J. Biomol. Struct. Dynamics, 16,
1075-1085
16
APOPTOSIS AND DNA METABOLISM BY ANTI-CANCER DRUGS
(cis-PLATIN // L-/D-PPMP // GD3)
cis-PLATIN
DNA-REPLICATION
APOPTOSIS
DNA-REPLICATION
APOPTOSIS
Pol-?
?
?
REGULATED
Y ?
X?
INHIBITED
Cyto-c
CASPASES
NUCLEAR DNA
Cyto-c
NUCLEAR DNA
MITOCHONDRION
DNA-DEGRADATION
MITOCHONDRION
DIFFERENTIATED NORMAL CELLS (N1)
UNDIFFERENTIATED TUMOR OR CANCER CELLS (
2N ) (COLO-205 / SKBR-3 / PA-3 / ALL)
17
Model of a Replisome Complex
- DNA Polymerase-?ç
5'
- Primase
- Helicase
- DNA Polymerase-?
3'
5'
3'
18
APOPTOSIS AND DNA METABOLISM BY ANTI-CANCER DRUGS
(cis-PLATIN // L-/D-PPMP // GD3//Betulinic Acid)
Anti-CANCER DRUGS
DNA-REPLICATION
APOPTOSIS
DNA-REPLICATION
APOPTOSIS
Pol-?
?
?
REGULATED
Y ?
X?
INHIBITED
Cyto-c
CASPASES
NUCLEAR DNA
Cyto-c
NUCLEAR DNA
MITOCHONDRION
DNA-DEGRADATION
MITOCHONDRION
DIFFERENTIATED NORMAL CELLS (N1)
UNDIFFERENTIATED TUMOR OR CANCER CELLS (
2N ) (COLO-205 / SKBR-3 / PA-3 / ALL)
19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
Bio-Synthesis of Ceramide
23
Analysis of L-14C-Serine Incorporation in
Colo-205 Cells ( L-PPMP or cis-Platin)
2 ml
3 ml
4 x 0.5 ml filtered through GF/A disks
Extraction for 14C-GSLs
Filter Assay
Upper Layer (saved)
Dissolved in 50-100 ml C-M (11)
Radioactivity quantitated by toluene
scintillation system
Scraped and radioactive areas quantitated
24
Incorporation of L-14C-Serine in Colo-205
cells(L-PPMP Treated Cells - 20-22 hr. / Live
Cells)
CPM x 10-3 / 106 cells
25
Incorporation of L-14C-Serine in Colo-205
cells(L-PDMP Treated Cells - 20 hr. / Total
Cells)
CPM x 10-4 / 106 cells
26
(No Transcript)
27
ACTIVATION OF CASPASE-3 DURING APOPTOSIS
GSL (GD3/GD1b)
P-Choline
(Sphingomylin)
Fas/TNF
PI-3 Kinase
Glc-Gal
(GD3)
?
Sphase
Pro-Caspase-8
SA
CGase
Phospho Akt
SA
FADD
?
CERAMIDE
Active -Casp-8
pBad
PTP
Mitochondrion
Sphingosine
Pro-Caspase-3
Cyto-C
Apaf-1
Caspase-3Activation
Casp-3 (p19/12)
Pro-Caspase-9
Active-Casp-9
Active Casp-3(p17/12)
Sph-1-P
?
DFF/ICAD/PARP etc.
PKC
?
DNA Cleavage
28
(No Transcript)
29
(No Transcript)
30
STRUCTURES OF DISIALOSYL GANGLIOSIDES(APOPTOTIC)
31
GD3 or GD1b Gangliosides Induce PS Movement To
The Outer Leaflet of Plasma Membrane and Change
Mitochondrial Permeability Transition (PT)As
Observed by the Binding of a Fluorescent Dye
PSS-380(Newly Synthesized by Dr. Bradley Smiths
Group at Notre Dame Patent Pending)
32
IDENTIFICATION OF APOPTOTIC CANCER CELLS USING
FLUORESCENT DYES
33
Structure of Fluorescent DyesPSS-380 and
Propidium Iodide
PSS-380 B. Smith. et.al (in press)
Propidium Iodide (Commercial)
34
APOPTOSIS OF BREAST CANCER CARCINOMA (SKBR3)
CELLS(EFFECT OF GD3/6hrs)
Control Phase Contrast
Control Blue with PSS-380
Control Red with PPI
20uM GD3 Blue with PSS-380
20uM GD3 Phase Contrast
20uM GD3 Red with PPI
80uM GD3 Phase Contrast
80uM GD3 Red with PPI
80uM GD3 Blue with PSS-380
35
APOPTOSIS OF BREAST CANCER CARCINOMA (SKBR3)
CELLS(EFFECT OF GD3 / 24hrs)
Control Phase Contrast
Control Blue with PSS-380
Control Red with PI
20uM GD3 Phase Contrast
20uM GD3 Blue with PSS-380
20uM GD3 Red with PI
80uM GD3 Phase Contrast
80uM GD3 Blue with PSS-380
80uM GD3 Red with PI
36
STRUCTURES OF DISIALOSYL GANGLIOSIDES(APOPTOTIC)
37
APOPTOSIS OF BREAST CANCER CARCINOMA (SKBR3)
CELLS(EFFECT OF GD1b/6hrs)
Control Phase Contrast
Control Blue with PSS-380
Control Red with PPI
40uM GD1b Blue with PSS-380
40uM GD1b Phase Contrast
40uM GD1b Red with PPI
80uM GD1b Phase Contrast
80uM GD1b Blue with PSS-380
80uM GD1b Red with PPI
38
APOPTOSIS OF BREAST CANCER CARCINOMA (SKBR3)
CELLS(EFFECT OF GD1b / 24hrs)
Control Phase Contrast
Control Blue with PSS-380
Control Red with PI
40uM GD1b Phase Contrast
40uM GD1b Blue with PSS-380
40uM GD1b Red with PI
80uM GD1b Phase Contrast
80uM GD1b Blue with PSS-380
80uM GD1b Red with PI
39
GD3 or GD1b Gangliosides Induce PS Movement To
The Outer Leaflet of Plasma Membrane and Change
Mitochondrial Permeability Transition (PT)As
Observed by the Binding of a Fluorescent Dye
PSS-380(Newly Synthesized by Dr. Bradley Smiths
Group at Notre Dame Patent Pending)
40
Summary of Ceramide Induced Apoptotic Pathway
CD-95 cross linking ? Increased level of
ceramide by cleavage of sphingolipids in
membrane ? Synthesis of GD3 ? Induction of
PT ? Release of cytochrome-c Flipping PS on the
outer PM ? Activation of caspases ? Apoptosis
41
A,B, and C are images Taken before the addition
of gangliosides
A200uM GD3 B200uM GD3 Cyclosporin
A C200uM GD1a
Bernardi, P., et al.JBC Vol.274 22581-5 1999
42
ACTIVATION OF CASPASE-3 DURING APOPTOSIS
GSL (GD3/GD1b)
P-Choline
(Sphingomylin)
Fas/TNF
PI-3 Kinase
Glc-Gal
(GD3)
?
Sphase
Pro-Caspase-8
SA
CGase
Phospho Akt
SA
FADD
?
CERAMIDE
Active -Casp-8
pBad
PTP
Mitochondrion
Sphingosine
Pro-Caspase-3
Cyto-C
Apaf-1
Caspase-3Activation
Casp-3 (p19/12)
Pro-Caspase-9
Active-Casp-9
Active Casp-3(p17/12)
Sph-1-P
?
DFF/ICAD/PARP etc.
PKC
?
DNA Cleavage
43
FACTS ABOUT A CASPASE

• 1. Initiates disassembly in response to
  proapoptotic signal (initiators).
• 2. Contains three domainsNH2 sub- unit, large
  and small subunit.
• 3. Always cuts after aspartic acid (?)

44
Proposed Interaction Between Different
CaspasesDuring Apoptosis
45
ACTIVATION OF CASPASE-3 DURING APOPTOSIS(GD3 /
SKBR-3 CELLS)
46
ACTIVATION OF CASPASE-3 DURING APOPTOSISGD1aGD1
bGT1a (442) / SKBR-3 CELLS
47
(No Transcript)
48
(No Transcript)
49
CERAMIDE GLYCANASE CATALYTIC ACTIVITY
Annelids Leech, Earthworm,
Li, Y-T., et
al.Bacteria Rhodococcus species
Ito, I. et
al.Mollusc Mercenaria mercenaria
Basu, S. et
al.Mammalian Rabbit Rat Tissues
Basu, S. et
al.Avian Embr. Ch. Brains
Basu, S. et
al.Human Colon(Colo-205)
Breast(MCF7) Carcinoma Basu, M. et al.
50
(No Transcript)
51
Summary

• 1 Synchronized carcinoma cells
  (Colo-205/SKBR-3) showed apoptosis (programmed
  cell death) in the presence of either L/D-PDMP or
  -PPMP (0.5 to 8.0 mM)., GD3 or GD1b(10-160 mM)
  Agarose gel electrophoresis showed the
  characteristic DNA laddering of the apoptotic
  process.
• A New Fluorescence Dye(PSS-380) detected flipping
  of Phosphatidyl Serine from inside to outside of
  the plasma membrane during apoptosis.
• 3 Inhibitors of GSLs or GSLs (containing
  disialosyl gr.) added from outside showed
  increased activation of Caspase-3 and initiation
  of apoptotic process.
• 4 Apoptotic cells produced by PDMP PPMP
  (2.0- 16.0 mM) in the presence of L-14C/or
  3H-Serine (24-48 hrs) showed increased
  biosynthisis of radioactive CERAMIDE.
• Relevant Publications
• 1) M. Basu and S. Basu in Liposome Methods and
  Protocols (2002) Micelles and Liposomes in
  Metabolic Enzymes and Glycolipid
  Glycosyltransferase Assays(edited by S. Basu and
  M. Basu) , Humana Press, New York
  pages,107-130..
• 2) Basu,M et al. (1999) BioSci. Rep., 19,
  449-460. "Ceramide Glycanase Activities in Human
  Cancer Cells"

52
TARGETED DELIVERY OF DRUGS TO CANCER CELLS (By
LIPOSOME BULLETS)
Mab (CSLEX)
SA-Lex
Synthetic Liposomes Containing Anti-cancer
Drugs/ cis-Platin, Tamoxifen, Melphalan,
Betulinic Acid L/D-PPMP,
L/D-PDMP, GD3, Ceramides, GD1b, GT1b
Metastatic Cancer Cells (Colon, Breast,Neuronal,
Prostate, ALL)
53
ACKNOWLEDGEMENTS

• At Notre Dame Outside Collaboration
• Dr. Manju Basu 1.Dr. Jin-ichi Inokuchi
  (Hokkaido University)
• Rui Ma 2.Dr. Sipra Banerjee (Cleveland
  Clini
• Patrick Boyle 3. Dr. Rathin N. Bose (Kent
  State Univ.)
• Krastyu Ugrinov
• Chris Moulton
• Brett Campbell
• Blair Daus
• Shannon Bradley
• Brian Mikkula
• Patric Kelly
• Matthew Bradley
• Maria Miguel
• )

54
(No Transcript)
55
THANK YOU FOR YOUR PATIENCE CALM ENDURANCE !!!
56
The MUDD HALL Dream in 1969 by the New Chairman
(A Calm Endurance Between 1962-1970)
57
A ROSEMAN-LAB Party at Basu-Apartment in
Baltimore (Summer 1970 )
58
A Reception for the LYNCH LECTURER at Basu
Residence (April 10, 1989)
59
A Dinner at the Studebaker Palace---Tippecanoe
Place (April 11, 1989)
60
(No Transcript)
61
DNA-Laddering Process

• 1.Apoptosis signal activates specific proteases
  (Caspases ).
• 2.Leads to activation of endonuclease(s).
• 3.The specific cleavage of the DNA leaves pieces
  of DNA in increments of 200 b.p.

62
SUBSTRATES USED FOR CGASE ASSAYS
GgOse43-TCer Galb1-3GalNAcb1-4Galb1-4Gl
c-3-TCER nLcOse54,5-TCer
Gala1-3Galb1-4GlcNAcb1-3Galb1-4Glc-4,5-TCER
63
(No Transcript)
64
APOPTOSIS OF BREAST CANCER CARCINOMA (SKBR3)
CELLS(EFFECT OF GD3/2hrs)
Control Phase Contrast
Control Blue with PSS1
Control Red with PPI
10uM GD3 Phase Contrast
10uM GD3 Blue with PSS1
10uM GD3 Red with PPI
80uM GD3 Phase Contrast
80uM GD3 Blue with PSS1
80uM GD3 Red with PPI
65
Structure of Betulinic Acid

66
ACTIVATION OF CASPASE-3 DURING APOPTOSIS
GSL (GD3/GD1b)
P-Choline
(Sphingomyelin)
Fas/TNF
PI-3 Kinase
Glc-Gal
(GD3)
?
Sphmase
Pro-Caspase-8
SA
CGase
Phospho Akt
SA
FADD
?
CERAMIDE
Active -Casp-8
pBad
PTP
Mitochondrion
Sphingosine
Pro-Caspase-3
Cyto-C
Apaf-1
Caspase-3Activation
Casp-3 (p19/12)
Pro-Caspase-9
Active-Casp-9
Active Casp-3(p17/12)
Sph-1-P
?
DFF/ICAD/PARP etc.
PKC
?
DNA Cleavage
67
ACTIVATION OF CASPASE-3 DURING APOPTOSIS
GSL (GD3/GD1b)
P-Choline
(Sphingomylin)
Fas/TNF
PI-3 Kinase
Glc-Gal
(GD3)
?
Sphase
Pro-Caspase-8
SA
CGase
Phospho Akt
SA
FADD
?
CERAMIDE
Active -Casp-8
pBad
PTP
Mitochondrion
Sphingosine
Pro-Caspase-3
Cyto-C
Apaf-1
Caspase-3Activation
Casp-3 (p19/12)
Pro-Caspase-9
Active-Casp-9
Active Casp-3(p17/12)
Sph-1-P
?
DFF/ICAD/PARP etc.
PKC
?
DNA Cleavage
68
Apoptotic Colo-205 by Drugs
Cis-Platin 150 µM 48 hrs
L-PPMP 15 µM 48 hrs
Control Cells were synchronized with 0.5 mM
HU(24 hrs) X 2
Melphalan 16 µM 48 hrs
Tamoxifen 16 µM 48 hrs
69
STRUCTURES OF DISIALOSYL GANGLIOSIDES(APOPTOTIC)
70
(No Transcript)