Cancer Stem Cells

AntiAnti--Cancer Stem Cell ( C S C )Cancer Stem Cell ( C S C )

Hoechst 33342 & Hoechst 33342 & Side Population (SP) CellsSide Population (SP) Cells

Hoechst 33342: dsDNA-binding dye

UV Excitation Emission350 nm 460 nm

FilterBlue Red

450/20 nm 674 nm(Goodell MA et al_1996)

SPSP

Do selected TCM herbs as components of the clinically used anti-cancer formula, with reported anti-tumour bioactivity, specifically target cancer stem cells(CSC)?

1. Examination of the crude extracts of selected anti-cancer herbal medicines

2. Subsequent fractionation to obtain pure compounds that specifically target CSC

CSC in vitro system => Changes in the

proportion of the Side Population as surrogate CSC

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

61.5

Solvent control (80% EtOH) for HM1 to HM5. Dissolved in DMSO.

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

44.3

3.78

10.9

3.32

10.9%

Standard 3: Negative controls: solvent control

10-1 100 101 102 103

FL3 LOG: PI

10-1

100

101

102

103

F

L

1

L

O

G

:

F

I

T

C

0.47 2.37

73.124

a b

c

a: DMSO 0.1% control

b: Chemotherpeutics Taxol 0.5 ug/ml

C: Thalidomide 20 ug/ml

16% 73%

35%

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

14.6

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

55.6

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

14.6

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

55.6

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

55.6

PI

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

20.2

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

70.1

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

20.2

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

70.1

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

70.1

PI

a. 0.2% DMSO,

b. 0.0002% DMSO

14.6%

20.2%

Positive vsnegative controls

Chemo Taxol

0.0001 ug/ml

Chemo Taxol

0.001 ug/ml

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

19.8

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

62.2

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

19.8

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

62.2

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

62.2

PI

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

20

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

59.7

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

20

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

59.7

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

59.7

PI

19.8%

20.0%

Neg control

Chemo Taxol

0.01 ug/ml

Chemo Taxol

0.1 ug/ml

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

18.2

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

54.2

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

18.2

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

54.2

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000F

S

C

-

A

54.2

PI

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

21.1

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

8.57

PI0 1000 2000 3000 4000

UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

21.1

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

8.57

PI0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

8.57

PI

18.2%

21.1%

Neg control

Propolis from different geographic areas have different chemical profiles

1620

2428

32

900720

540360

180

Mass (a.m.u.)

0.0

3.0

1.5

Intensity (log)

A

b

s

o

r

b

a

n

c

e

(

%

)

0

50

100

Retention Time (mi

n)

P1 P2 P5 P4 STD P3

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

6.36

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

43.8

1.82

16.5

1.82

HM2 and HM4

at 100 ug/mL,

had high killing effect on the cancer cells,

but did not preferentially killed the SP.

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

1.95

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

43.1

3.08

18.3

1.91

HM2

HM crude extracts that killed the cancer cells with no killing preference for the SP

HM4

16.5%

18.3%

Herbal Medicine (HM) crude extract

that selectively targets and kills SP

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

42.5

HM 1 at 100 ug/mL, selectively reduced SP%

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

21.9

8.96

0.83

0.11

0.83%

POSITIVE control

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

2.74

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

33.2

3.17

0.49

1.38

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

25.1

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

14.5

10.5

0.43

0.062

b.) Fractions from HM1 that selectively killed SP

Fraction 6 (F6) and fraction 7(F7) at 100 ug/mL, selectively killed the SP

F6

F7

0.49%

0.43%

POSITIVE control

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

59.4

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

39.1

4.02

12.3

2.07

C2: Solvent control for fraction 1

0 5000 104 50000105

B575 D-A

0

1000

2000

3000

4000

F

S

C

-

A

53.6

0 1000 2000 3000 4000UV670 A-A

0

1000

2000

3000

4000

U

V

4

4

0

_

B

-

A

34.5

3.74

15.3

2.32

C3: Solvent controls for fractions 4, 6 & 7

C2

C3

Solvent controls for fractions obtained from HM. Dissolved in DMSO.

12.3%

15.3%

SOLVENTcontrols

Peer-reviewed RESEARCH FUNDINGS

Anthony Rothe Memorial Trust (Australia)

University of Sydney Cancer Research Fund

International Myeloma Foundation (USA)

Multiple Myeloma Research Fund (USA)

Cancer Institute NSW Career Development Fund (Australia)

NSW Cancer Council Project Fund (Australia)

Australia-China Special Fund

Australian National Institute of Complementary Medicine

George Li, Kei Fan, Iyad Ahmed, Nantiga Virgona, Bhagwant Sekhon, Tetsuo Yamagishi, Rebecca Roubin

Gouyi Mo, Suilin Mo, Daniel Sze, Shihong Yang, Celine Loh, Kristin Muller

Cancer Immunology Group,

Faculty of Pharmacy,

University of Sydney