Low-dose radiation enhances cytotoxicity of pirarubicin, chemotherapeutic agent, in K562 and K562/adr leukemia cancer cells

1. Low-dose radiation enhances cytotoxicity of
Pirarubicin, chemotherapeutic agent, in K562
and K562/adr leukemic cancer cells
Khin The Nu Aye
Ph.D. Degree Program in Biomedical Sciences, Faculty of Associated Medical Sciences,
Chiang Mai University, under the CMU Presidential Scholarship, Chiang Mai, Thailand,
1

2. Introduction
2
Cancer – the second leading cause of global death
Different cancer treatments
 Surgery
 Radiation therapy
 Bone marrow transplant
 Immunotherapy/Hormone therapy
 Targeted drug therapy
9.6
million
deaths

3. 3
 Cancer Treatments may be used as:
(1) Primary Treatment
(2) Adjuvant Treatment
(3) Palliative Treatment
 Multidrug Resistant Effect : Main reason for treatment
failure , One of multidrug resistant cancers(MDR) – Leukemia
Introduction

4. 4
Adjuvant therapy
the combination of the low- dose
radiation and chemotherapy
Low- dose radiation
Effect of radio hypersensitivity, radiation
hormesis
Introduction

5. Objective
To determine effect of low-dose radiation on
cytotoxicity of Pirarubicin in K562 and K562/adr
leukemic cancer cells.
5

6. 6
K 562 K 562 /ADR
Complete Media
(RPMI 1640 + 10% FBS + Penicillin-Streptomycin 1%)
Cell lines and culture media
Cell lines
K562 = myelogenous leukemia cell line
K562/adr = Adriamycin resistant derivative of K562 cell line

7. 7
Control0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
1. Cell Cytotoxicity Experiment :
1.1 Radiation only
Cell Viability Assay
Perkin Elmer (LS 55)
Luminescence Spectrometer
5x104 cells/ml
Resazurin
48-hour incubation
4-hour incubation

8. 8
Control0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
1. Cell Cytotoxicity Experiment :
1.2 Radiation and Drug Treatment
Cell Viability Assay
Perkin Elmer (LS 55)
Luminescence Spectrometer
5x104 cells/ml
Resazurin
24-hour
incubation
4-hour incubation
24-hour
incubation
Pirarubicin
(10nM/100 nM)

9. 9
Control0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
2. Mitochondria Activity Experiment :
2.1 Radiation only
5x105 cells/ml
MTT
3-hour incubation
20-minute
incubation
Optical density(OD)
Shimadzu (UV 2700i)
Spectrophotometer

10. 10
Control0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
2.2 Radiation and Drug Treatment
5x105 cells/ml
MTT
90-min
incubation
20-min
incubation
90-min
incubation
Optical density(OD)
Shimadzu (UV 2700i)
Spectrophotometer
Pirarubicin
(10nM)

11. 11
Control 0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
3. Mitochondria Membrane Potential Experiment :
3.1 Radiation only
1x106 cells/ml
Rhodamine-B
MTT
3-hour incubation
Perkin Elmer (LS 55)
Luminescence Spectrometer
∆Ψm

12. 12
Control0.02Gy 0.05Gy 0.1Gy
K 562 K 562/ADR
X-Radiation
0.02Gy, 0.05Gy, 0.1Gy
3.2 Radiation and Drug Treatment
1x106 cells/ml
90-min
incubation
Pirarubicin(10nM)
90-min incubation
Rhodamine-B
MTT
Perkin Elmer (LS 55)
Luminescence Spectrometer
∆Ψm

13. 13
3. Statistical Analysis
One-way analysis of variance (ANOVA)
Between groups, Unpaired Student’s t-test
P-value ≤ 0.05

14. 14
Radiation only Radiation and Drug
K562/ADR
K562
0.1Gy
Control
Results: Cell morphology
THP10 uM
+ 0.1Gy
Control

15. Results : Cell viability
70
80
90
100
110
120
*p < 0.01
*p < 0.01
%
Cell
viability
X-ray, Gy
K562 : 48 hs post-irradiation
0 0.02 0.05 0.10
p = 0.06
70
80
90
100
110
120
0 0.02 0.05 0.10
%
Cell
viability
X-ray, Gy
K562/adr : 48 hs post-irradiation
*p < 0.01
*p < 0.05
*p < 0.01
15

16. Results : Enhance cytotoxicity
16
70
80
90
100
110
120
*p < 0.05
%
Cell
viability K562 : 24 hs Rad + 24 hs Pira 10 M
*p < 0.05
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
10 nM
Pira nM
70
80
90
100
110
120
K562 : 24 hs Rad + 24 hs Pira 100 M
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 100 100 100 100
%
Cell
viability
*p < 0.05
*p < 0.05
100 nM
Pira nM
70
80
90
100
110
120
K562/adr : 24 hs Rad + 24 hs Pira 10 M
%
Cell
viability
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
10 nM
Pira nM
70
80
90
100
110
120
K562/adr : 24 hs Rad + 24 hs Pira 100 M
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 100 100 100 100
%
Cell
viability
100 nM
Pira nM

17. Results : Mitochondrial activity
17
0 Gy 0.02 Gy 0.05 Gy 0.1 Gy
0.00
0.02
0.04
0.06
K562 : 3 hs Rad
*p < 0.01
*p < 0.01
O.D.
at
560
nm
X-Ray dose
*p < 0.01
0 Gy 0.02 Gy 0.05 Gy 0.1 Gy
0.00
0.02
0.04
0.06
O.D.
at
560
nm
K562/adr : 3 hs Rad
X-Ray dose
*p < 0.01
*p < 0.01
*p < 0.01
0.00
0.02
0.04
0.06
K562 : 1.5 hs Rad + 1.5 hs Pira 10 M
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
O.D.
at
560
nm
*p < 0.05
*p < 0.05
*p < 0.05
Pira nM
0.00
0.02
0.04
0.06
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
K562/adr : 1.5 hs Rad + 1.5 hs Pira 10 M
O.D.
at
560
nm
*p < 0.05
*p < 0.05
*p < 0.05
Pira nM

18. Results : MMP
18
0 Gy 0.02 Gy 0.05 Gy 0.1 Gy
100
120
140
160
180
200
220
m
X-Ray dose
K562 : 3 hs Rad
*p < 0.01
*p < 0.01
*p < 0.01
0 Gy 0.02 Gy 0.05 Gy 0.1 Gy
100
120
140
160
180
200
220
K562/adr : 3 hs Rad
m
X-Ray dose
*p < 0.05
*p < 0.05
*p < 0.05
100
120
140
160
180
200
220
240
K562 : 1.5 hs Rad + 1.5 hs Pira 10 M
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
m
*p < 0.05
*p < 0.05
*p < 0.05
Pira nM
100
120
140
160
180
200
220
240
K562/adr : 1.5 hs Rad + 1.5 hs Pira 10 M
Rad, Gy - - 0.02 0.05 0.1
Pira,M - 10 10 10 10
m
Pira nM

19. Conclusion
 This finding suggested that low-dose radiation could
enhance cytotoxicity of Pirarubicin in K562 and
K562/adr leukemic cancer cells.
 The mechanism should be further investigation.
19

20. Acknowledgments
Scholarship :
Ph.D. Degree Program in Biomedical Sciences, Faculty of Associated
Medical Sciences, Chiang Mai University, under the CMU
Presidential Scholarship, Chiang Mai, Thailand
Advisor :
Assoc. Prof. Dr. Montree Tungjai : E-mail mtungjai@gmail.com
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