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Objectives
Aim:
Show that genetic depletion/ pharmacological inhibtion of GSK-3 results in decreased renal cancer cell proliferation and survival Show aberrant GSK-3b nuclear over expression in RCC cell lines and most human renal carcinomas Show a synergistic anti-cancer effect of GSK-3 inhibitor and Docetaxel in RCC.
Introduction
Renal cell carcinoma (RCC) is highly resistant to
molecules)
Introduction
Glycogen synthase kinase-3 Pluripotent serine/threonine kinase Important for epithelial cell homeostasis oncogene activation increase proliferation 2 types: a and b Inhibition of GSK-3 apoptosis due to decreased expression of NF-kb target genes Bcl-2, XIAP (chronic lymphocytic leukaemia and pancreatic cancer cells)
Method
Immunohistochemistry Nuclearcytosolic fractionation
RNA interference
Q- RTPCR
MTS
BrDU assay
lines compared with normal renal cell Increased phosphorylation of GS more inactive GS Higher levels of phosphorylation of glycogen synthase( GS) Lesser conversion of glucose to glycogen
Figure 1A
Protein lysates from the indicated RCC cell lines were used and
have higher intensity bands in RCC cell lines compared to normal kidney A ratio of pGS to total GS would show how much protein are active/inactive
Figure 1A
Kidney ACHN KRC/Y Caki1 Caki2 A704 A498
GSK-3b
pGS
Total GS
b-Actin
Loading control
Figure 1B
Kidney
C Nu
ACHN
C
NFkb p65
H3
SOD
Patient 2 N T
cytoplasm
C Nu C Nu
C Nu C Nu
GSK-3
GSK-3
pGS
H3
bActin
If GSK-3b activated enters nucleus If GSK-3b inactivated found in cytoplasm
SOD
Immunohisotchemical analysis
Weak cytoplasmic expression of gsk-3b in a fraction of glomerular and tubular epithelial cells in normal kidney
GSK-3b is accumulated in the nucleus of renal cancer cells
1
2 3
53
7 14
50
6 12
52
7 10
Pharmacological inhibition and genetic depletion of GSK-3 decrease proliferation and survival of renal cancer cells
Studies have been done to suggest that GSK-3 is important
factor of NF-kb-mediated cancer cell survival and proliferation in pancreatic and chronic lymphocytic leukaemia (CLL) To determine if GSK-3 is essential for RCC cell survival and proliferation, 3 small molecule inhibitors were used: AR-A014418 (ATP-competitive) SB-216763 (ATP-competitive) TDZD8 ( non ATP competitive)
Pharmacological inhibition and genetic depletion of GSK-3 decrease proliferation and survival of renal cancer cells
All 3 inhibitors can decrease viability of ACHN renal cancer
cell Anti-cancer effect of AR-A014418 was tested on 6 other renal cancer cell lines: KH39,KU19-20,Caki1,Caki2,KRC/Y and A498 Ar-A014418 is a potent and specific GSK-3 inhibitor Inhibition of GSK-3 decreased renal cancer cell viability in a dose and time dependent manner
Pharmacological inhibition and genetic depletion of GSK-3 decrease proliferation and survival of renal cancer cells
Using BrDU incorporation assay:
Found that pharmacological inhibition of GSK-3 suppresses
cancer cells Results suggest that GSK-3 is a positive regulator of renal cancer cell proliferation and survival
surivival, accompanying apoptotic morphological changes observed by Hoest staining GSK-3a does not affect cancer cells
Scrambled siRNA
No apoptosis
Figure 4A
24 h 0 25
ACHN 48 h 25 50 0
24 h
Caki1
48 h AR-A014418 M
50
25
50
25
50
pGS
Total GS
PARP
-Actin
Figure 4B,C
Figure 4D,E,F
Input DMSO AR-A Ig DMSO AR-A p65 DMSO AR-A
XIAP
Bcl-2
-Actin
kidney cancer is intrinsically chemoresistant Increased expression of Bcl-2 and XIAP is important reason Ho: whether inhibition of GSK-3 could be useful in combination with conventional chemotherapeutic agent in treatment Docetaxel is well establised drug with limited cytotoxic effect in clinical RCC Result: inhibition of GSK-3sensitised ACHN and Caki1 cells to Docetaxeldecrease cell survivability
Figure 5
In a nutshell.
GSK-3b has important function in pathogenesis of human
cancer GSK-3 is a positive regulator of RCC cell survival, proliferation and chemoresistance GSK-3b aberrant nuclear accumulation in most renal cell Inactive GSK-3b is able to translocate to nucleus from cytoplasm but rapidly degraded by proteosomal pathway within nucleus of cancer cell
In a nutshell
Factors leading to progression of RCC: Increased activity of NF-kb Increased expression of anti apoptotic molecules (Bcl-2 and XIAP) GSK-3 inhibitors inactivation of NF-k b sensitised cancer cells to chemotherapy conventional chemotherapy drugs can be more effective Thus, work has identified GSK-3 b as a novel potential therapeutic target in RCC
Reference:
Bilim.V.,Ougolkov.A.,Yuuki.K., Naito.S., Kawazoe.H.,, Muto.A.,
Oya.M., Biladeau.D., Motoyama.T. and Tomita.Y. (2009). Glycogen synthase kinase-3: a new therapeutic target in renal cell carcinoma. British Journal of Cancer (101) 2005-2014 Fang.X.,Yu.S.X.,Lu.Y.,Bast,R.C.Jr.,James R. Woodgett, and Mills.G.B. Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A.(2000)PNAS.(97) 11960-11965 Mishra.R.(2010).Glycogen synthase kinase 3 beta: can it be target for oral cancer.molecular cancer.(9) Narayan.G, Prasad. S.B.(2012). A NEW INSIGHT OF GSK3b REGULATION:IMPLICATIONS IN CANCER THERAPY. Journal of Scientific Research. (56) 25-34
Thank you
Presented by:
Farhath Jabien
BBSD1 1012A
UOB: 09074657