Biological and Molecular Targeted Therapy for HHCSheng-Long Ye, MD, PhDLiver Cancer InstituteZhongshan HospitalFudan UniversityShanghai, China

Problem in HCCSevere cirrhosisIntrahepatic spread and distant metastasisLow resectabilityHigh postoperative recurrence

Non-Surgical Treatment Regional therapy TACEAblation therapy PEI, RFA, MWCT, Laser, HIFU, CryoablationRadiotherapyChemotherapyBiological & Molecular targeted therapyTraditional Chinese medicine

Biotherapy for HCCCytokinesIFNT1IL-2Adoptive immunotherapyCIKCTLRadioimmunotherapyI131 MetuximabTumor vaccinesDC vaccineGene therapyClinical investigation

Median survival time IFN 63.8m vs Control 38.8mIFNprevents recurrence and prolongs survival in postresective HCC patientsRandomized control clinical trial of 236 HCC cases with 6-year follow-up

Survival and miR-26a Expression in HCCN Engl J Med 2009; 361: 1437-1447

Prevention of Postresective Recurrence in HCC by LAK/IL-2

Combination Treatment of p53 & TACE in HCC******P

Strategy for Molecular Targeted therapyGrowth factor & receptor inhibitorsAngiogenesis inhibitorsSignal transduction pathway inhibitorsMonoclonal antibodiesCell-cycle modulation & gene therapy

Targets & DrugsEGFR: TKI: Erlotinib, LapatinibGefitinib Ab: CetuximabVEGF TKI: Sorafenib Sunitinib Ab: BevacizumabRAF TKI: SorafenibmTOR Rapamycin EverolimusProtease Inhibitor BortezomibTargeted Therapy for HCC

2. Llovet JM et al. NEJM 2008, 359: 378-390. 1. Cheng A et al. Lancet Oncol 2009; 10: 25-34Comparison of Oriental and SHARP Study

EndpointOriental1SHARP2Hazard Ratio (95% CI)P-valueHazard Ratio (95% CI)P-valueOS0.68 (+47%) 0.0140.69 (+44%)

Llovet et al NEJM 2008;359:378-390

Cheng AL, et al. Lancet Oncol, 2009; 10: 25-34

Development of HCC Therapy with Sorafenib Doxorubicin+SorafenibSorafenib+5-FU /UFT /Xeloda Sorafenib+Erlotinib (SEARCH)TACE+Sorafenib (SPACE)Radical cureResection, RFA, PEI+Sorafenib (STORM)Long-term follow-up (GIDEON)

Molecular targeted therapies assessed in clinical trials in HCCSorafenib RAF, VEGF, PDGFR III- positiveBrivanib FGF, VEGF III-designSunitinib PDGFR, VEGFR, KIT III-designErlotinib EGFR II-closedCetuximab EGFR II-ongoingLapatinib EGFR, Her2/nu II-ongoingLinifanib VEGF, PDGF II-ongoingTSU-68 VEGFR,PDGFR, FGFR II-ongoingBevacizumab VEGF II-closedEvelorimus+bevacizumab mTOR, VEGF II-ongoing Erlotinib+bevacizumab EGFR, VEGF II-ongoingRamucirumab VEGFR-2 II-ongoingLenalidomide FGF, VEGF II-ongoingARQ-197 c-Met (HGF) II-ongoingBortezomib Proteasome II-negativeAcyclic retinoids Differentiation III-designNolatrexed Thymidylate synthase III-negativeT138067 Tubulin III-negative Treatment Target Clinical phase

Molecular Approaches to Targeting Metastasis in HCCPotential targets for organ-specific metastasisViral-gene therapy targeting AFP-expression HCCAntisense gene therapy targeting HCC metastasis

Stat3 ASO Inhibits Proliferation of Human HCC CellsLipo.Control24h 250nM BRC 24h Stat3 ASO24h Concentration dependence of Stat3 ASO Inhibition in HCCLM3 CellsInhibitory Effects of Stat3 ASO with Different Time in HCCLM3 Cells Stat3 ASO inhibits proliferation in HCCLM3 cells with concentration dependenceInhibition of proliferation in HCC cells reaches highest at 48h

Chart11

10097.393.7003.13.12.12.1

98.394.7752.12.11.51.533

97.392.3512.52.50.60.63.63.6

968332.744223.13.1

95.37018.22.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

ec

HUVECMTT ASSAY2003.12.10.10 pm 2003.12.13 6pm

MTTBRCBRCBRCSTAT3STAT3STAT3VR2AVR2AVR2AVR2BVR2BVR2B

200NM

400NM

600NM

800NM

BLANKBLANKBLANKBLANKBLANKBLANKBLANK

HUVEC3X10^6CELLS/WELL.03.12.10 10PM

18248090%03.12.11 12PMin fact 03.12.12 1:42am

6-8100UL03.12.12 10AM8-10

2448MTT2003.12.12.12PM24

in fact,2003.12.13 6pm (42)no operation

96-well platetotal volume per well is 100 mililiter.

BRC for 3 parallel wellsanti-stat3 for 3 parallel wellsanti-VR2A for 3 parallel wellsanti-VR2B for 3 parallel wellslipofectamine 2000 for 12 wells in the same concentration with the ratio of 1:1

200nm0.45 ug/75ul serum-free DMEMthe same with left columnthe samethe same0.15ul x 12 =1.8ul/300ul medium

400nm0.9ug/75ulsamesamesame0.3ul x 12 = 3.6ul/300ulmedium

600nm1.35ug/75ulsamesamesame0.45ul x 12 =5.4ul/300ul medium

800nm1.8ug/75ulsamesamesame0.6ul x 12 = 7.2 ul/300ul medium

oligo

for example200nm

2003.12.14 9amchang the medium 100ul/well56 hours after incubation with oligo

2003.12.15 921am10ulMTT/well80hours after incubation with oligo

2003.12.15 5pm4 storage

2003.12.15 7pm100ul570nm(490nm)630nm

MTTBRCBRCBRCSTAT3STAT3STAT3VR2AVR2AVR2AVR2BVR2BVR2B

200NM0.7370.570.4670.3970.4510.4790.5050.4680.3410.5030.6030.607

400NM0.3780.3960.1820.290.3210.2920.3230.3330.3010.3940.4310.493

600NM0.40.3330.2970.2580.2740.2560.2670.3710.3370.3590.3950.452

800NM0.2860.3560.3040.2690.2140.240.2430.4860.1910.3860.3680.324

PBS0.6930.5320.50.4320.4760.4540.5370.8250.853

BLANK CONTROL0.1430.3530.281

averagebrcstat3vr2avr2bpbsblank control

200nm0.5910.44233333330.4380.5710.590.259

400nm0.31866666670.3010.3190.43933333330.59

600nm0.34333333330.26266666670.3250.4020.59

800nm0.31533333330.2410.30666666670.35933333330.59

ec

BRC

anti-stat3 oligo

anti-VEGFR2 oligo

anti-VEGFR2B oligo

different dose

MTT OD value

dose-effect curve

-ok

brc

stat3

vr2a

vr2b

pbs

stat-modi-spss

for ppt version

stat-modi-spss

10097.393.7003.13.12.12.1

98.394.7752.12.11.51.533

97.392.3512.52.50.60.63.63.6

968332.744223.13.1

95.37018.22.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

-dose-not spss

100100100000000

99.396.378.71.15470053841.15470053841.51.53.53.5

98.892.751.31.3650396821.3650396823.83.82.52.5

9888.329.7221.81.82.12.1

101.790.427.73.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

10097.393.7003.13.12.12.1

98.394.7752.12.11.51.533

97.392.3512.52.50.60.63.63.6

968332.744223.13.1

95.37018.22.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

cell number

graph-dose

spss11.1 statistic

lip50lip150lip250lip350lip500

1009910010098

10096959695

100100979293

mean100.098.397.396.095.3

sd0.02.12.54.02.5

meansd

meanLipo.BRC oligon.2'-MOE Stat3 ASOLipo.BRC oligon.2'-MOE Stat3 ASO

5010097.393.703.12.1

15098.394.7752.11.53

25097.392.3512.50.63.6

350968332.7423.1

50095.37018.22.56.22.4

brc50brc150brc250brc350brc500sta50sta150sta250sta350sta500

100969285639678523219

98959381759272473020

94939283729375543615.5

mean97.394.792.383.070.093.775.051.032.718.2

sd3.11.50.62.06.22.13.03.63.12.4

sd

Lipo.BRC oligon.2'-MOE Stat3 ASO

5010097.393.7

15098.394.775

25097.392.351

350968332.7

50095.37018.2

correct

verified by spss

cell number reference

lip250brc250sta250

1009252

959347

979254

graph time250nM

Lipo.BRC oligon.2'-MOE Stat3 ASO97.392.351.0

normalized01001001002.50.63.6

1299.396.378.7

2498.892.751.3lip0012244872brc12244872sta12244872

489888.329.71009897.398100989090.392.382512925.4

72101.790.427.71001001001001069691.1888975542827.6

100100999699959786.79079493230

mean100.099.398.898.0101.796.392.788.390.478.751.329.727.7

sd0.01.21.42.03.81.53.81.81.73.52.52.12.3

00

1.53.5

3.82.5

1.82.1

1.72.3

cell number

003.13.12.12.1

2.12.11.51.533

2.52.50.60.63.63.6

44223.13.1

2.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

7721

000000

1.15470053841.15470053841.51.53.53.5

1.3650396821.3650396823.83.82.52.5

221.81.82.12.1

3.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

assume

1.hcclm3 mtt value

dose-effect050nM150nM250nM350nM500nM

irrational graph

BRC odnAS STAT3 odnmodifiedLipo.BRC oligon.2'-MOE Stat3 ASO

average

0100100brc0

100nM9689100nM9894963

200nM9375200nM949192.52

400nM8048400nM8276794

600nM7521600nM797275.55

800nM6016800nM6359613

1000nM52111000nM5648526

stat30

100nM9086883

200nM7872754

400nM5246494

600nM231820.54

800nM191215.55

1000nM16912.55

lipo0

3

5

4

3

2

2.hcclm3 mtt value

time-effect

assume

1001000000

96892.82842712472.82842712472.82842712472.8284271247

93752.12132034362.12132034364.24264068714.2426406871

80484.24264068714.24264068714.24264068714.2426406871

75214.94974746834.94974746833.53553390593.5355339059

60162.82842712472.82842712474.94974746834.9497474683

52115.65685424955.65685424954.94974746834.9497474683

BRC odn

AS STAT3 odn

MTT value (%control)

brcstalippbs

100nM0.720.670.750.780.960.8933333333

200nM0.700.560.750.93333333330.7466666667

400nM0.600.360.750.80.48

600nM0.560.160.750.74666666670.2133333333

800nM0.450.120.750.60.16

1000nM0.390.080.750.520.1066666667

ratio

brcstalippbsBRC odnAS STAT3 odn

0100100

0.78100nM9689

200nm0.460.35200nM9375

250nm0.400.07400nM8048

400nm0.410.01600nM7521

500nm0.220.030.75800nM6016

800nm0.120.060.751000nM5211

averagep value

brc0

100nM98949630.0028958122

200nM949192.52

400nM82767940.0198039412

600nM797275.55

800nM63596130.1256659164

1000nM56485260

stat309890

100nM90868839486

200nM78727549381

400nM52464949587

600nM231820.54

800nM191215.55

1000nM16912.55

0.0028958122

brc200s200

9478

brc400s4009172

82529076

76469574

85540.0015030722

7342

0.0000018692

0244872

lipo.32.83.240

BRC odn 400nM32.51.82.124

AS STAT3 odn 400nM31.60.90.848

72

lipo24lip48lip72

2.83.24.2

2.63.43.9

3.12.94.6

2.92.63.5

avrage2.853.0254.05

00.20816659990.350.4654746681

brc24brc48brc72

2.51.82.1

2.31.62.3

02448722.621.6

lipo.32.853.034.052.22.11.9

BRC odn 400nM32.41.881.98a2.41.8751.975

AS STAT3 odn 400nM31.51.050.6500.18257418580.22173557830.2986078811

stat324s48s72

1.60.90.7

1.81.20.8

1.20.70.5

1.41.40.6

a1.51.050.65

00.25819888970.31091263510.1290994449

000000

0.20816659990.20816659990.18257418580.18257418580.25819888970.2581988897

0.350.350.22173557830.22173557830.31091263510.3109126351

0.46547466810.46547466810.29860788110.29860788110.12909944490.1290994449

lipo.

BRC odn 400nM

AS STAT3 odn 400nM

Hours

cell number(x104)

smmc7721

MTTMTTBRCBRCBRCMDM2MDM2MDM2HRASHRASHRASSTAT3STAT3STAT3

200NM(1:0.5)1.4381.9342.1821.4861.5441.4751.6231.5861.61.5421.5920.977

200NM(1:1)1.2731.4471.8481.4071.4471.4381.4871.4311.4281.4441.5041.28

250NM(1:1)0.7851.0331.7791.3921.4261.3751.3791.5371.5140.9741.4011.032

400NM(1:1)1.1821.7581.4631.2581.4061.391.4851.281.2741.220.9611.047

500NM(1:1)0.6691.5651.6191.0641.3461.3061.2831.2061.0670.6771.10.996

800NM(1:1)0.7630.7991.2741.0841.3311.1231.2521.261.0281.2311.191.247

1000NM(1:1)1.1711.3571.4071.7721.1270.8951.2661.2421.1171.2031.1461.112

lip 200nm1.3281.206

brcmdm2hrasstalippbs

lip 500nm1.7411.885200a1.85133333331.50166666671.6031.37033333331.2671.8413333333

200b1.52266666671.43066666671.44866666671.40933333331.267

lip 1000nm1.1281.513250nm1.1991.39766666671.47666666671.1356666667

400nm1.46766666671.35133333331.34633333331.076

pbs1.7391.6852.1500nm1.28433333331.23866666671.18533333330.92433333331.813

800nm0.94533333331.17933333331.181.2226666667

blank control1.0551.0551.0761000nm1.31166666671.26466666671.20833333331.1291.3205

blank1.0621.1

procedure2003.07.30 9amaverage-blank

brcmdm2hrasstalippbs

2003.07.31 9am200a0.000.000.000.000.000.00

200b0.000.000.000.000.000.78

2003.08.01 10amMTT 37250nm0.000.000.000.000.78

400nm0.000.000.000.000.78

2003.08.01 2pm 570nm 500nm0.000.000.000.000.000.78

800nm0.000.000.000.000.78

1000nm0.000.000.000.000.000.78

MTTBRCBRCBRCMDM2MDM2MDM2HRASHRASHRASSTAT3STAT3STAT3

200NM(1:0.5)0.340.831.080.390.440.380.520.490.500.440.490.88

200NM(1:1)0.170.350.750.310.350.340.390.330.330.340.400.18

250NM(1:1)0.69-0.070.680.290.330.280.280.440.41-0.030.30-0.07

400NM(1:1)0.080.660.360.160.310.290.390.180.170.12-0.04-0.05

500NM(1:1)-0.430.470.52-0.040.250.210.180.11-0.03-0.420.00-0.10

800NM(1:1)-0.34-0.300.17-0.020.230.020.150.16-0.070.130.090.15

1000NM(1:1)0.070.260.310.670.03-0.100.170.140.020.100.050.01

1000nm0.24966666670.20266666670.14633333330.0670.2585

blank

mtt

1 DMSO

2

3 MTT

4

5 mtt,

brcmdm2hrasstalippbs

200a0.790.440.540.310.210.78

200b0.460.370.390.350.210.78

250nm0.140.340.410.070.78

400nm0.410.290.280.010.78

500nm0.220.180.12(0.14)0.750.78

800nm(0.12)0.120.120.160.78

1000nm0.250.200.150.070.260.78

brcmdm2hrasstalippbs

200a0.790.440.540.310.78

200b0.460.370.390.350.78

250nm0.400.340.410.070.78

400nm0.410.290.280.010.78

500nm0.220.180.120.030.750.78

800nm0.120.120.120.060.78

1000nm0.250.200.150.070.750.78

brcmdm2hrasstalippbs

200a1.010.560.690.401.00

200b0.590.590.500.450.78

250nm0.510.440.530.090.78

400nm0.530.370.360.010.78

500nm0.280.150.150.040.750.78

800nm0.150.150.150.080.78

1000nm0.320.260.190.090.750.78

brc

mdm2

hras

sta

lip

pbs

brc

mdm2

hras

sta

lip

pbs

brc

mdm2

hras

sta

lm3 mtt

48hrsdose-effect curve

pbspbspbslipliplipbrcbrcbrcstastasta

100

200

400

600

400nmtime-effect curve

pbspbspbslip400lip400lip400brc400brc400brc400sta400sta400sta400

6hrs

12hrs

24hrs

36hrs

48hrs

72hrs

Chart5

100100100000000

99.396.378.71.15470053841.15470053841.51.53.53.5

98.892.751.31.3650396821.3650396823.83.82.52.5

9888.329.7221.81.82.12.1

101.790.427.73.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

ec

HUVECMTT ASSAY2003.12.10.10 pm 2003.12.13 6pm

MTTBRCBRCBRCSTAT3STAT3STAT3VR2AVR2AVR2AVR2BVR2BVR2B

200NM

400NM

600NM

800NM

BLANKBLANKBLANKBLANKBLANKBLANKBLANK

HUVEC3X10^6CELLS/WELL.03.12.10 10PM

18248090%03.12.11 12PMin fact 03.12.12 1:42am

6-8100UL03.12.12 10AM8-10

2448MTT2003.12.12.12PM24

in fact,2003.12.13 6pm (42)no operation

96-well platetotal volume per well is 100 mililiter.

BRC for 3 parallel wellsanti-stat3 for 3 parallel wellsanti-VR2A for 3 parallel wellsanti-VR2B for 3 parallel wellslipofectamine 2000 for 12 wells in the same concentration with the ratio of 1:1

200nm0.45 ug/75ul serum-free DMEMthe same with left columnthe samethe same0.15ul x 12 =1.8ul/300ul medium

400nm0.9ug/75ulsamesamesame0.3ul x 12 = 3.6ul/300ulmedium

600nm1.35ug/75ulsamesamesame0.45ul x 12 =5.4ul/300ul medium

800nm1.8ug/75ulsamesamesame0.6ul x 12 = 7.2 ul/300ul medium

oligo

for example200nm

2003.12.14 9amchang the medium 100ul/well56 hours after incubation with oligo

2003.12.15 921am10ulMTT/well80hours after incubation with oligo

2003.12.15 5pm4 storage

2003.12.15 7pm100ul570nm(490nm)630nm

MTTBRCBRCBRCSTAT3STAT3STAT3VR2AVR2AVR2AVR2BVR2BVR2B

200NM0.7370.570.4670.3970.4510.4790.5050.4680.3410.5030.6030.607

400NM0.3780.3960.1820.290.3210.2920.3230.3330.3010.3940.4310.493

600NM0.40.3330.2970.2580.2740.2560.2670.3710.3370.3590.3950.452

800NM0.2860.3560.3040.2690.2140.240.2430.4860.1910.3860.3680.324

PBS0.6930.5320.50.4320.4760.4540.5370.8250.853

BLANK CONTROL0.1430.3530.281

averagebrcstat3vr2avr2bpbsblank control

200nm0.5910.44233333330.4380.5710.590.259

400nm0.31866666670.3010.3190.43933333330.59

600nm0.34333333330.26266666670.3250.4020.59

800nm0.31533333330.2410.30666666670.35933333330.59

ec

0000

0000

0000

0000

BRC

anti-stat3 oligo

anti-VEGFR2 oligo

anti-VEGFR2B oligo

different dose

MTT OD value

dose-effect curve

-ok

0000000

0000000

0000000

0000000

0000000

brc

stat3

vr2a

vr2b

pbs

stat-modi-spss

for ppt version

stat-modi-spss

10097.393.7003.13.12.12.1

98.394.7752.12.11.51.533

97.392.3512.52.50.60.63.63.6

968332.744223.13.1

95.37018.22.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

-dose-not spss

100100100000000

99.396.378.71.15470053841.15470053841.51.53.53.5

98.892.751.31.3650396821.3650396823.83.82.52.5

9888.329.7221.81.82.12.1

101.790.427.73.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

10097.393.7003.13.12.12.1

98.394.7752.12.11.51.533

97.392.3512.52.50.60.63.63.6

968332.744223.13.1

95.37018.22.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

cell number

100100100000000

99.396.378.71.15470053841.15470053841.51.53.53.5

98.892.751.31.3650396821.3650396823.83.82.52.5

9888.329.7221.81.82.12.1

101.790.427.73.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

7721

graph-dose

spss11.1 statistic

lip50lip150lip250lip350lip500

1009910010098

10096959695

100100979293

mean100.098.397.396.095.3

sd0.02.12.54.02.5

meansd

meanLipo.BRC oligon.2'-MOE Stat3 ASOLipo.BRC oligon.2'-MOE Stat3 ASO

5010097.393.703.12.1

15098.394.7752.11.53

25097.392.3512.50.63.6

350968332.7423.1

50095.37018.22.56.22.4

brc50brc150brc250brc350brc500sta50sta150sta250sta350sta500

100969285639678523219

98959381759272473020

94939283729375543615.5

mean97.394.792.383.070.093.775.051.032.718.2

sd3.11.50.62.06.22.13.03.63.12.4

sd

Lipo.BRC oligon.2'-MOE Stat3 ASO

5010097.393.7

15098.394.775

25097.392.351

350968332.7

50095.37018.2

correct

verified by spss

cell number reference

lip250brc250sta250

1009252

959347

979254

graph time250nM

Lipo.BRC oligon.2'-MOE Stat3 ASO97.392.351.0

normalized01001001002.50.63.6

1299.396.378.7

2498.892.751.3lip0012244872brc12244872sta12244872

489888.329.71009897.398100989090.392.382512925.4

72101.790.427.71001001001001069691.1888975542827.6

100100999699959786.79079493230

mean100.099.398.898.0101.796.392.788.390.478.751.329.727.7

sd0.01.21.42.03.81.53.81.81.73.52.52.12.3

00

1.53.5

3.82.5

1.82.1

1.72.3

7721

000003.13.12.12.1

0002.12.11.51.533

0002.52.50.60.63.63.6

00044223.13.1

0002.52.56.26.22.42.4

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Concentration (nM)

MTT Value (% of control)

assume

000000

1.15470053841.15470053841.51.53.53.5

1.3650396821.3650396823.83.82.52.5

221.81.82.12.1

3.78593889723.78593889721.71.72.32.3

Lipo.

BRC oligon.

2'-MOE Stat3 ASO

Hours

MTT Value (% of control)

1.hcclm3 mtt value

dose-effect050nM150nM250nM350nM500nM

irrational graph

BRC odnAS STAT3 odnmodifiedLipo.BRC oligon.2'-MOE Stat3 ASO

average

0100100brc0

100nM9689100nM9894963

200nM9375200nM949192.52

400nM8048400nM8276794

600nM7521600nM797275.55

800nM6016800nM6359613

1000nM52111000nM5648526

stat30

100nM9086883

200nM7872754

400nM5246494

600nM231820.54

800nM191215.55

1000nM16912.55

lipo0

3

5

4

3

2

2.hcclm3 mtt value

time-effect

1001000000

96892.82842712472.82842712472.82842712472.8284271247

93752.12132034362.12132034364.24264068714.2426406871

80484.24264068714.24264068714.24264068714.2426406871

75214.94974746834.94974746833.53553390593.5355339059

60162.82842712472.82842712474.94974746834.9497474683

52115.65685424955.65685424954.94974746834.9497474683

BRC odn

AS STAT3 odn

MTT value (%control)

brcstalippbs

100nM0.720.670.750.780.960.8933333333

200nM0.700.560.750.93333333330.7466666667

400nM0.600.360.750.80.48

600nM0.560.160.750.74666666670.2133333333

800nM0.450.120.750.60.16

1000nM0.390.080.750.520.1066666667

ratio

brcstalippbsBRC odnAS STAT3 odn

0100100

0.78100nM9689

200nm0.460.35200nM9375

250nm0.400.07400nM8048

400nm0.410.01600nM7521

500nm0.220.030.75800nM6016

800nm0.120.060.751000nM5211

averagep value

brc0

100nM98949630.0028958122

200nM949192.52

400nM82767940.0198039412

600nM797275.55

800nM63596130.1256659164

1000nM56485260

stat309890

100nM90868839486

200nM78727549381

400nM52464949587

600nM231820.54

800nM191215.55

1000nM16912.55

0.0028958122

brc200s200

9478

brc400s4009172

82529076

76469574

85540.0015030722

7342

0.0000018692

0244872

lipo.32.83.240

BRC odn 400nM32.51.82.124

AS STAT3 odn 400nM31.60.90.848

72

lipo24lip48lip72

2.83.24.2

2.63.43.9

3.12.94.6

2.92.63.5

avrage2.853.0254.05

00.20816659990.350.4654746681

brc24brc48brc72

2.51.82.1

2.31.62.3

02448722.621.6

lipo.32.853.034.052.22.11.9

BRC odn 400nM32.41.881.98a2.41.8751.975

AS STAT3 odn 400nM31.51.050.6500.18257418580.22173557830.2986078811

stat324s48s72

1.60.90.7

1.81.20.8

1.20.70.5

1.41.40.6

a1.51.050.65

00.25819888970.31091263510.1290994449

000000000

0000.20816659990.20816659990.18257418580.18257418580.25819888970.2581988897

0000.350.350.22173557830.22173557830.31091263510.3109126351

0000.46547466810.46547466810.29860788110.29860788110.12909944490.1290994449

lipo.

BRC odn 400nM

AS STAT3 odn 400nM

Hours

cell number(x104)

smmc7721

MTTMTTBRCBRCBRCMDM2MDM2MDM2HRASHRASHRASSTAT3STAT3STAT3

200NM(1:0.5)1.4381.9342.1821.4861.5441.4751.6231.5861.61.5421.5920.977

200NM(1:1)1.2731.4471.8481.4071.4471.4381.4871.4311.4281.4441.5041.28

250NM(1:1)0.7851.0331.7791.3921.4261.3751.3791.5371.5140.9741.4011.032

400NM(1:1)1.1821.7581.4631.2581.4061.391.4851.281.2741.220.9611.047

500NM(1:1)0.6691.5651.6191.0641.3461.3061.2831.2061.0670.6771.10.996

800NM(1:1)0.7630.7991.2741.0841.3311.1231.2521.261.0281.2311.191.247

1000NM(1:1)1.1711.3571.4071.7721.1270.8951.2661.2421.1171.2031.1461.112

lip 200nm1.3281.206

brcmdm2hrasstalippbs

lip 500nm1.7411.885200a1.85133333331.50166666671.6031.37033333331.2671.8413333333

200b1.52266666671.43066666671.44866666671.40933333331.267

lip 1000nm1.1281.513250nm1.1991.39766666671.47666666671.1356666667

400nm1.46766666671.35133333331.34633333331.076

pbs1.7391.6852.1500nm1.28433333331.23866666671.18533333330.92433333331.813

800nm0.94533333331.17933333331.181.2226666667

blank control1.0551.0551.0761000nm1.31166666671.26466666671.20833333331.1291.3205

blank1.0621.1

procedure2003.07.30 9amaverage-blank

brcmdm2hrasstalippbs

2003.07.31 9am200a0.000.000.000.000.000.00

200b0.000.000.000.000.000.78

2003.08.01 10amMTT 37250nm0.000.000.000.000.78

400nm0.000.000.000.000.78

2003.08.01 2pm 570nm 500nm0.000.000.000.000.000.78

800nm0.000.000.000.000.78

1000nm0.000.000.000.000.000.78

MTTBRCBRCBRCMDM2MDM2MDM2HRASHRASHRASSTAT3STAT3STAT3

200NM(1:0.5)0.340.831.080.390.440.380.520.490.500.440.490.88

200NM(1:1)0.170.350.750.310.350.340.390.330.330.340.400.18

250NM(1:1)0.69-0.070.680.290.330.280.280.440.41-0.030.30-0.07

400NM(1:1)0.080.660.360.160.310.290.390.180.170.12-0.04-0.05

500NM(1:1)-0.430.470.52-0.040.250.210.180.11-0.03-0.420.00-0.10

800NM(1:1)-0.34-0.300.17-0.020.230.020.150.16-0.070.130.090.15

1000NM(1:1)0.070.260.310.670.03-0.100.170.140.020.100.050.01

1000nm0.24966666670.20266666670.14633333330.0670.2585

blank

mtt

1 DMSO

2

3 MTT

4

5 mtt,

brcmdm2hrasstalippbs

200a0.790.440.540.310.210.78

200b0.460.370.390.350.210.78

250nm0.140.340.410.070.78

400nm0.410.290.280.010.78

500nm0.220.180.12(0.14)0.750.78

800nm(0.12)0.120.120.160.78

1000nm0.250.200.150.070.260.78

brcmdm2hrasstalippbs

200a0.790.440.540.310.78

200b0.460.370.390.350.78

250nm0.400.340.410.070.78

400nm0.410.290.280.010.78

500nm0.220.180.120.030.750.78

800nm0.120.120.120.060.78

1000nm0.250.200.150.070.750.78

brcmdm2hrasstalippbs

200a1.010.560.690.401.00

200b0.590.590.500.450.78

250nm0.510.440.530.090.78

400nm0.530.370.360.010.78

500nm0.280.150.150.040.750.78

800nm0.150.150.150.080.78

1000nm0.320.260.190.090.750.78

000000

000000

000000

000000

000000

000000

000000

brc

mdm2

hras

sta

lip

pbs

000000

000000

000000

000000

000000

000000

000000

brc

mdm2

hras

sta

lip

pbs

0000

0000

0000

0000

0000

0000

0000

brc

mdm2

hras

sta

lm3 mtt

48hrsdose-effect curve

pbspbspbslipliplipbrcbrcbrcstastasta

100

200

400

600

400nmtime-effect curve

pbspbspbslip400lip400lip400brc400brc400brc400sta400sta400sta400

6hrs

12hrs

24hrs

36hrs

48hrs

72hrs

Stat3 ASO vs LIPOP=0.005; Stat3 ASO vs BRC oligon.P=0.002Antisense stat3 Oligodexynucleotides Inhibits Metastatic Potential in Human HCCLM3 HCC cells

Stat3 ASO Improves Survival of Nude Mice Bearing Human HCCLM3 CellsDay 2 Administration Day 10 AdministrationNS49.04.9 dBRC52.76.7 dStat3 ASO 10114.6 dNS46.24.3dBRC50.26.2dStat3 ASO 82.311.1dStat3 ASO vs ControlsP

ThanksEXPO 2010, Shanghai, China

**Shanghai