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Downregulation of survivin by ginsenosdie CK increased anti-cancer effect
in human malignant neuroblastoma cells
Jung-Mi Oh , Sun Young Park 1,2 , Sungkun Chun 1,2
1,2
1 Department of Physiology, Jeonbuk National University Medical School, Jeonju, Korea
2 Brain Korea 21 Plus Program, Jeonbuk National University Medical School, Jeonju, Korea
Neuroblastoma (NB) is a solid tumor that mostly in children. Malignant NB have poor prognosis because conventional chemotherapeutic agents are hardly effective.
Survivin is a member of the inhibitor of apoptosis family proteins and is involved in tumor cell survival and invasion, and differentiation. Survivin is highly upregulated
in NB and correlated with poor prognosis. Malignant NB ( SK-N-BE(2) and SH-SY5Y) cells were highly expressed of survivin protein. In this study, we examined the
effects of survivin knockdown and ginsenoside CK on survival and differentiation of NB cells. We found that CK treatments inhibited cell mortality and down-regulated
survivin gene expression. Survivin knockdown using siRNA in NB cells induced morphological features of neuronal differentiation and apoptotic cell death. The
combination of survivin siRNA and CK induced increased expression of NSE and E-cadherin and decreased expression of Notch-1 and PCNA, thereby promoting
apoptosis and differentiation. Collectively, combination therapy with CK and inhibition of survivin may be a potentially effective agent for the treatment of NB.
Key words: Neuroblastoma, Ginsenosides, Survivin, EMT, Apoptosis
Introduction c
c
• Neuroblastoma is a solid tumor that mostly occurs in children.
• Survivin is an inhibitor of apoptosis protein and is
overexpressed in a wide spectrum of tumors, including
neuroblastoma.
• EMT is known to be essential for the initial and overall rate
limiting steps of neuroblastoma invasion and metastasis. d e
• In recent years, as the emphasis of EMT in the tumor
progression has been increased, anticancer strategies against
EMT have been widely investigated.
• Therefore, the inhibition of survivin by itself, or in
combination with other approach, has emerged as a promising
therapeutic strategy
Influence of survivin knockdown and CK treatment on
cell apoptosis
Objective
CK inhibits EMT in SK-N-BE(2) cells a
In this study, we examined the effect of survivin knock- a
down by siRNA and stimultaneous CK treatment in Control CK 5µM
neuroblastoma cells 0h (mm) 1 Control CK
A 0.8 **
Methods distance 0.6 ** **
24h 0.4 **
Separation and validation of effective ginsenosides Migratory 0.2
in ginseng using TLC & HPLC 0 0h 24h 48h
48h
Cell growth rate : MTT Time (post scratch)
assay c
b
Effects of apoptosis
Immnufluorescence
• Annexin/PI staining • Alexa-594
• Hoechst 33342 conjugate antibody
• Alexa-4884
Expression of survivin in neuroblastoma cell lines
conjugate antibody
Ginsenoside effects on migration & invasion b
a b
Real time-PCR, Western blot Evaluation of ginsenoside effects on genes
• Metastasis associated proteins : matrix metalloproteinase MMP-2, MMP-9 Knockdown of survivin in SK-N-BE(2) cells by siRNA
• Apoptosis associated proteins : BCL-2, Bcl-Xl, Survivin, PARP, Caspase-3 c
a
Results siRNA sequence
# 1: CUCAUCUAAGCUGCUUAUU(dTdT) / anti-sense : AAUAAGCAGCUUAGAUGAG(dTdT) (1012477)
# 2: AAGGAGAUCAACAUUUUCA(dTdT) / anti-sense : UGAAAAUGUUGAUCUCCUU(dTdT) (1012475)
CK effects on the SK-N-BE(2) cell proliferation # 3: GUGCAACCGCCUAGACUUU(dTdT) / anti-sense : AAAGUCUAGGCGGUUGCAC(dTdT) (1012479)
a b b
References Conclusion
* L. Zhang et al., J. Braz. J.Med. Biol. Res. 47(7), 548-553 (2014). * Hossain et al., Exp Xell Res. 318 (13) , 1597-1610 (2012)
Survivin silencing potentiated anti-cancer effects of CK in human
* Lee S.C et al., Oncotarget. 6(29) 27146-27159 (2015). * Li Y.D. et al., Int. J. Clin Exp Pathol., 8(2), 1731-1736 (2015) malignant neuroblastoma cells having survivin overexpression.
* H.M. Calderone et al. J Cancer Therapy , 5, 1289-1302 (2014).
