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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

chemotherapy and progressive

High apoptotic threshold Activation of Nuclear Factor-kb(NF-kb) Increased expression of Bcl-2 and XIAP (anti-apoptotic

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

Small molecule inhibitor

Western blotting

RNA interference

Expression pattern of GSK-3b

Q- RTPCR

MTS

BrDU assay

Effect of GSK3 inactivation

GSK-3B is expressed and active in human renal cancer cells

Western blot:
Higher levels of GSK-3b expression in RCC cell

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

normal kidney is a control

separated by SDS-PAGE,
transferred to PVDF membrane probed with antibodies against GSK-3b, phospho-glycogen

synthase(pGS) and total glycogen synthase (GS)

pGS( inactive GS,phosphorylated by GSK-3) was found to

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

RCC cell lines

GSK-3b

pGS

Total GS

b-Actin
Loading control

Figure 1B
Kidney
C Nu

Using western blot with nuclear/cytosolic fractionation

Caki1 Caki2 KRC/Y A498 KH39 KU19-20
Nu C Nu C Nu C Nu C Nu C Nu C Nu GSK-3

ACHN
C

NFkb p65

H3

SOD

Figure 1C: Expression of GSK-3b

Patient 1
normal

Figure 1D: Nuclear/cytosolic fractionation was used

Patient 3 Patient 4 N T nucleus
tumor

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

pGS expression is seen

Table 2. Results of immunohistochemical study for GSK-3 and pGS

Total Histological type Oncocytoma Clear cell Other 2 64 10 0 60a 8 0 62b 7 GSK-3 nuclear pGS positive

pT stage (malignant tumours only)

1
2 3

53
7 14

50
6 12

52
7 10

Grade (malignant tumours only) 1 2 3 Total 27 43 4 74 RCCs and 2 oncocytomas 26 39 3 68 27 39 3 69

Abbreviations: pGS=phospho-glycogen synthase; RCC=renal cell carcinoma.

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

proliferation of renal cancer cells

Using Hoest staining

Found a dose-dependent induction of apoptosis in AR-A014418-treated

cancer cells Results suggest that GSK-3 is a positive regulator of renal cancer cell proliferation and survival

To determine if the inhibitory effect was specific to GSK-3b,

GSK-3a and 3b was depleted in ACHN using siRNA

Result: depletion of GSK-3b significant decrease in renal cancer cell

surivival, accompanying apoptotic morphological changes observed by Hoest staining GSK-3a does not affect cancer cells

Most sensitive cell line

Scrambled siRNA

Check uniqueness of drugs

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

Cleaved PARP XIAP

Bcl-2

-Actin

Figure 4B,C

Figure 4D,E,F
Input DMSO AR-A Ig DMSO AR-A p65 DMSO AR-A

XIAP

Bcl-2 WCL C Nu GSK-3 H3 SOD

PARP Cleaved PARP XIAP

Bcl-2
-Actin

AR-A014418 and Docetaxel synergistically suppress survival of renal cancer cells

Chemotherapeutic effect for RCC is very limited as

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