multikinase inhibitor, has shown promising survival results in ad-vanced HCC. Targeted therapies can be combined to interrupt sig-naling in related tumorigenic pathways. We aim to study RAS andmTOR pathway activation in human HCV-hepatocarcinogenesisand the effect of its abrogation with a multikinase inhibitor (Sor-afenib, Bayer) and an mTOR inhibitor (Rapamycin).

METHODS: We investigated the expression of 4 genes of the RASpathway in 77 human HCC samples by qRT-PCR and used immu-nohistochemistry to localize expression of phosphorylated mTOR.K-Ras was sequenced in 100 HCC cases. HCC cell lines were incu-bated with increasing concentrations of Sorafenib and Rapamycin toassess viability (MTT assay), proliferation (3[H] Thymidine Incor-poration), and apoptosis (flow cytometry). Immunoblotting was per-formed to demonstrate abrogation of downstream protein targets.

RESULTS: H-RAS was upregulated 4-fold in advanced HCC hu-man samples (p�0.01). No point mutations were detected. Mem-branous staining of phospho-mTOR was lost in HCC (p�0.01).There was an additive effect of Sorafenib and Rapamycin in vitro,leading to decreased viability and inhibition of proliferation(p�0.001). Flow cytometry confirmed apoptosis of HCC cell lineswith Sorafenib alone and in combination.

CONCLUSIONS: Ras and mTOR pathways are activated in HCC.Sorafenib and Rapamycin abrogated the pathways and have an ad-ditive effect. This provides the rationale for animal experiments andpotentially for future translation into clinical trials in the primaryand adjuvant setting in HCC.

Triptolide a potential therapeutic candidate forpancreatic cancerPhoebe Phillips, PhD, Daniel Borja-Cacho, MD, Vikas Dudeja, MD,Joshua McCarroll, PhD, Rajinder Dawra, PhD,William Grizzle, MD, PhD, Selwyn Vickers, MD, PhD, FACS,Ashok Saluja, PhDUniversity of Minnesota, Minneapolis, MN

INTRODUCTION: We have previously demonstrated that HSP70overexpression in pancreatic cancer cells confers resistance to apopto-sis and that HSP70 inhibition induces apoptosis in these cells. There-fore, the aims of this study were to examine the effect of triptolide (ananti-tumor diterpenoid triepoxide) on i) pancreatic cancer cells byassessing viability, apoptosis and HSP70 levels; and ii) pancreaticcancer growth and metastases in vivo.

METHODS: Pancreatic cancer cells (MiaPaCa-2/PANC-1) andnormal pancreatic duct cells (PDC) were incubated with triptolide orvehicle (control). We assessed the effect of triptolide on cell viability,apoptosis and HSP70 expression in these cells. We used an ortho-topic model of pancreatic cancer by injecting MiaPaCa-2 cells intothe pancreas; 6 days later we administered triptolide (0.2mg/kg/day)or vehicle subsequently for 60 days.

RESULTS: Triptolide decreased viability in MiaPaCa-2 andPANC-1, with no effect on PDC viability (Triptolide 200 nM � %of C: MiaPaCA-2 56�3, PANC-1 60�2, Pancreatic duct cells85�4). Triptolide increased apoptosis in cancer cells (measured by

Annexin V, caspase-3 [Table] and TUNEL stain). Triptolide signifi-cantly decreased pancreatic cancer growth in vivo (Tumor cm3: Con-trol 1.03�.43 vs triptolide 0.14�0.05, *p�0.05, n�7, Mean �SE). Triptolide decreased local-regional spread to multiple organscompared to control mice. Triptolide decreased HSP70 expression invitro and in vivo compared to controls.

Table. Effect of triptolide on caspase 3 and Annexin V in pancre-atic cancer cells.Treatment(24h)

Caspase 3 Activity(% of C) Annexin V (% of C)

MiaPaCa-2 PANC-1 MiaPaCa-2 PANC-1

Control (C) 100 � 0 100 � 0 100 � 0 100 � 0

Triptolide 100nM 1078 � 89� 507 � 87� 194 � 14� 198 � 15�

Triptolide 200nM 1282 � 144� 801 � 39� 247 � 26� 306 � 70�

�p�0.05 compared to Controls, n�3, Data Expressed as Mean � SE

CONCLUSIONS: Triptolide causes pancreatic cancer cell death invitro and in vivo by induction of apoptosis, this may be mediated viaHSP70 inhibition. Triptolide is a potential therapeutic agent to pre-vent the progression and metastases of pancreatic cancer.

A specific VEGF-R tyrosine kinase inhibitorenhances the antiproliferative effect oftrastuzumab in ErbB-2 overexpressing breastcancer cellsElizabeth Min Hui Kim, MD, Catherine Lobocki, MS,Linda Dubay, MD, Vijay Mittal, MDSt John-Providence Hospital and Medical Centers, Southfield, MI

INTRODUCTION: Trastuzumab (Herceptin) has been found tohave antiproliferative effects in ErbB-2 overexpressing breast cancerscells. VEGF-R activation has been shown to induce apoptosis incancer cells. This study’s purpose was to evaluate the antiproliferativeeffect of the novel combination of trastuzumab and a specificVEGF-R tyrosine kinase inhibitor in breast cancer cell lines.

METHODS: Increasing doses of trastuzumab (0.125-2 nM) and aVEGF-R tyrosine kinase inhibitor (1.25-20 mM) were tested aloneand in combination. Two ErbB-2 overexpressing cell lines (BT474and SKBR3) and ErbB-2 negative cell line(MCF7) were studied.Inhibition of growth was assessed after 5 and 7 days. Apoptosis wasdetermined after 48 hrs of treatment.

RESULTS: Time and dose-dependent growth inhibition was dem-onstrated in all three cell lines tested with VEGF-R inhibitor, whiletrastuzumab was only effective in the ErbB-2 positive cells. After 5days, trastuzumab (0.5 nM) inhibited cell growth by 35.6% and31.9%, and the VEGF-R inhibitor (5 mM) by 37.4% and 54.3% inBT474 and SKBR3, respectively. The combination (trastuzumaband VEGF-R inhibitor), significantly inhibited growth in BT474and SKBR3 by 60.4% and 75.8 % (p � 0.001, oneway ANOVA)compared to either agent alone. In MCF7, no increase in inhibitionwas seen with the combination compared to VEGF-R inhibitionalone. Apoptosis was increased by 1.9 to 10.6-fold using the combi-nation in the BT474 and SKBR3.

CONCLUSIONS: Trastuzumab plus VEGF-R inhibition decreasedproliferation and increased apoptosis in ErbB-2 overexpressing breast

S94 Surgical Forum Abstracts J Am Coll Surg