Page 17 - D. Cancer biology

Anti-cancer Effects of β-apopicropodophyllin
Against Colorectal Cancer Cells.

,
,
Na-Gyeong Lee¹², A-Ram Kang², Jin-Hee Kwon², Hong-Duck Um², Joon Kim¹*, Jong Kuk Park²*
,
¹Department of Life Sciences, Korea University, Seoul 02841, Korea
²Division of Applied Radiation Bioscience, Korea Institute of Radiological & Medical Sciences, Seoul 01812, Korea
ABSTRACT INTRODUCTION
In previously study, we have isolated podophyllotoxin acetate (PA), a naturally β-Apopicropodophyllin, APP is a synthesized derivative of podophyllotoxin (PPT) which is a
occurring derivative of podophyllotoxin (PPT), from Natural Product Library. PA had anti- natural product that has been used as a traditional drug to decrease immunosuppression. It
cancer activities against one of the non-small cell lung cancer (NSCLC) cell lines, but is was shown to reversibly bind tubulin and disrupt its polymerization to prevent mitotic spindle
physiologically unstable for therapeutic drug and contains highly toxicity. For that, we formation, induce cell cycle arrest, and inhibit cell proliferation. [1] Previously study showed
synthesized several new derivatives of PPT and identified β-apopicropodophyllin (APP) that treatment of lung cancer cells with APP altered microtubule polymerization and triggered
Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase that regulates multiple cellular functions such DNA damage to induce cell cycle arrest, while also stimulating pro-apoptotic ER stress. [2] So
as a potential cell death inducer. In this study, we tested whether APP has anti-cancer
as cell cycle, cell survival, drug resistance. Aberrant elevation of Pim-1 kinase is associated with numerous types of cancer. Pim-1 plays an
drug effect against colorectal cancer. Colorectal cancer (CRC) is the second most we examined whether APP could act as a radiosensitizer in colorectal cancer cells as like in
important role in cancer cell survival. Ent-7beta-hydroxy-15-oxokaur-16-en-18-yl acetate (HOA) is a natural compound, which has been isolated
from the leaves of Croton tonkinensis Gagnep. In the present study, we investigated degradation of Pim-1 by HOA. Treating HOA with time, cell
death increased and the expression of Pim-1 was decreaed. PARP, cleaved PARP and cleaved Caspase-3 of PC-3 cell was identified through ined NSCLC cells. Colorectal cancer (CRC) is very common disease in the world, the second most
common cancer in women and the third most common in men. First, we exam
whether APP could act as a radiosensitizer in CRC cells. APP additionally showed common cancer in women and the third most common in men. Also some cases are
Western Blot and showed apoptosis. The degree of apoptosis was confirmed by FACS. Pim-1 didn't show any change in RNA expression level anti-
detected through RT-PCR. Our results demonstrated that Pim-1 was degraded by proteasome, confirmed by treating Lactacystin and Chloro- in
PC-3 cell. activity against HCT116 and DLD-1 cell lines with IC50 values of 7.877nM and
cancer associated with people who are less than 65 years old. [3]
8.219nM, respectively. Annexin V-Propidium iodide assay indicated a combination of Radiation therapy is widely used for CRC treatment, but high dose of radiation can damage
APP and γ-ionizing radiation (IR) that increased apoptotic cell death. We also performed surrounding normal tissues and low dose of radiation may lead to resistance to radiation
H2DCFDA-based assay, and then observed APP and IR co-treatment increased ROS therapy [4]. To overcome these problem of radiation therapy, we have developed novel
levels in CRC cells. Taken together, co-treatment of APP and IR might promote increase radiosensitizer. The purpose of this experiment also is an improvement of the radiation
of cell death followed by ROS induction in CRC cells. treatment efficiency with radiation therapy by developing novel radiosensitizer drug.
RESULTS
HCT116 B
A *** A HCT116 DLD-1 3500
120 100 140 180 169.71 con
con APP IR IR+APP 100 80 ** 120 100 100 114.06 93.78 160 140 3000 APP only
Cell viability (%) 60 40 59.73 53.06 25.62 ROS (%) 80 60 67.28 ROS 120 100 80 100 88.06 111.29 Tumor size(mm 3 ) 2500 IR IR+APP
2000
0 20 con APP 7.5nM IR 3Gy IR+APP 40 20 60 40 20 1500
b-Apopicropodophyllin 0 con NAC IR+APPIR+APP+NAC 0 con NAC IR+APPIR+APP+NAC 1000
(APP) B DLD-1 500
Figure 1. Chemical structure of β-apopicropodophyllin 120 *** C con 0
(APP). 100 NAC 5mM 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
con APP IR IR+APP 100 80 67.30 63.90 *
Cell viability (%) 60 40 45.69 20 HCT116 18.27 Day
20 15 13.73
A HCT116 0 con APP 7.5nM IR 3Gy IR+APP IR+APP IR+APP+NAC Apoptosis (%) 10 con
48h C con APP 7.5nM 5 3.76 5.79
100
72h
Cell viability (%) 50 30 HCT116 * 22.36 0 con NAC IR+APPIR+APP+NAC APP only
IR 3Gy IR+APP 20 * 12.31 D
0 Apoptosis (%) con NAC 1mM
0 10 20 30 40 50 60 70 80 90 100 110 10 8.43 IR only
Drug concentration (nM) 4.3 DLD-1
15 13.19
B DLD-1 0 con APP 7.5nM IR 3Gy IR+APP 10.70
Apoptosis (%)
48h 10 IR+APP
Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a serine/threonine kinase that regulates multiple cellular functions such as cell cycle, cell survival, drug resistance. Aberrant elevation of Pim-1 kinase is associated with numerous types of cancer. Pim-1 plays an important role in cancer cell
IR+APP
72h
Cell viability (%) 50 con APP 7.5nM DLD-1 0 Figure 6. Combination of APP and IR enhances
100
5.66
IR+APP+NAC
survival. Ent-7beta-hydroxy-15-oxokaur-16-en-18-yl acetate (HOA) is a natural compound, which has been isolated from the leaves of 5 4.47
D
30 ** con NAC IR+APPIR+APP+NAC apoptosis in vivo: ‘Control’, mock control; ‘APP only’,
5mg/kg APP only; ‘Radiation only’, 3Gy IR only;
0 23.71 ‘IR+APP’, combination of 3Gy IR and 5mg/kg APP; Mice
0 10 20 30 40 50 60 70 80 90 100 110 *
Drug concentration (nM) 20 16.58 were injected with HCT116 cells and divided into the
IR 3Gy IR+APP Apoptosis (%) 11.91 E HCT116 DLD-1 indicated treatment groups.
Figure 2. IC50 values of APP for colorectal cancer cell 10 8.03 non-IR IR(3Gy) non-IR IR(3Gy)
lines. APP (7.5nM) - + - - + + - + - - + +
(A and B) MTT assay for detection cell viability and IC50. 0 con APP 7.5nM IR 3Gy IR+APP NAC - - + - - + - - + - - + APP IR
PARP
c-PARP
A B E cas3
HCT116 DLD-1 HCT116 DLD-1 ROS
250 APP - + - + - + - + c-cas3
192.53 150 146.09 150.28 IR (3Gy) - - + + - - + + cas9
200
169.25 114.80 Bcl-xl
ROS (%) 150 100 100 98.28 ROS (%) 100 100 c-cas3 c-cas9 Caspase
b-actin
50 50 b-actin
0 0 Figure 5. Refraining ROS can restore cell viability suppressed by
con APP 7.5nM IR 3Gy IR+APP con APP 7.5nM IR 3Gy IR+APP APP and IR in HCT116 and DLD-1 cell lines.
Figure 4. Combination of APP and IR enhances apoptosis in (A and B) H2DCFDA-based assay for detection of ROS. (C and D)
CRC cells. (A and B) Cell counting for detection cell viability. Annexin V-PI assay for detection of apoptosis. (E) Immunoblot
Figure 3. Combination of APP and IR induces ROS (C and D) Annexin V-PI assay for detection of apoptosis. (E) assay for detecting PARP, caspase-3 and caspase-9 activation in Apoptosis
accumulation in CRC cell lines. (A and B) H2DCFDA- Immunoblot assay for detecting Bcl-xl and caspase-3 cells treated with NAC, APP and IR. For PARP, ‘c-PARP’ indicates
based assay for detection of ROS. The cells were stained activation in cells treated with APP and IR. For caspases3 ‘c- the cleaved form. For caspases-3 and caspase-9, ‘c-cas3’ and ‘c-
with 25μM H2DCFDA for 5min. Fig. 7. Scheme of radiosensitizing effect by APP: co-
cas3’ indicates the cleaved form. cas9’ indicate the cleaved form. treatment of APP and IR could induce apoptotic cell
death via ROS accumulation.
CONCLUSION REFERENCES CONTACT INFORMATION
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