In vitro und in vivo Analyse der Wirkung von Vitamin C und Gemcitabine auf eine humane Pankreaskarzinom- Zelllinie (BxPC-3) sowie HUVEC’s und die Tumorinhibition im Pankreaskarzinom SCID-Maus-Modell

Abstract

Introduction: The standard adjuvant therapy of patients with pancreatic carcinoma is gemcitabine. The role of vitamin C in prevention and therapy of different tumours was controversially discussed in the past. High serum levels of vitamin C are responsible for the protective effect in gastric carcinoma and also MMP-Inhibition was observed in the human pancreatic carcinoma cell line (MIAPaCa-2). This MMP-Inhibition leads to anti tumour effects. Aim of the study was to analyse the effect of gemcitabine and vitamin C in vitro with proliferation assays on BxPC-3 cells and HUVEC’s. Furthermore this was also analysed in vivo in a human pancreatic cancer mouse model (SCID). Methods: Human pancreatic cancer cell line (BxPC-3) was injected subcutaneously (s. c.) into the dorsa of male, immunodeficient (SCID) mice. When tumour volume was 100 mm³, mice were randomized into each six groups (n = 5–7/group) receiving gemcitabine twice a week 125 mg/kgKG i. p. (group 1). Vitamin C mono therapy every other day 23 mg/mouse i. p. (group 2), 2,3 mg/mouse i. p. (group 3) 0,23 mg/mouse i. p. (group 4). The combination group (group 5) was treated twice a week with gemcitabine 125 mg/kgKG i. p. and additionally thrice a week vitamin C 2,3 mg/kgKG i. p. The control group (group 6) was treated with placebo solution i. p. Tumour volume was measured every third day with a digital calliper. At the end of the experiment tumour sections were analysed according to the microvessel density (CD31), apoptosis (TUNEL) and proliferation (Ki-67). Furthermore in vitro the IC₅₀ value of each compound and of the combination therapy was analysed on BxPC-3 cells and HUVEC’s. Non-parametric statistical analysis was performed. Results: Tumour inhibition rate (%) for BxPC-3 (slow growing tumour) was 48 % for group 1, 0 % for group 2, 8 % for group 3, 27 % for group 4 and 62 % for group 5. The inhibition of tumour growth was statistically significant in group 2 and group 5 compared with group 6 (p = 0,006). All mice gained weight throughout the study.

The immunohistological results for MVD were as follows: group 1: 5,68± 0,39; group 5: 6,49 ± 0,70; group 4: 4,75 ± 1,29 and group 6: 5,73 ± 1,27 (Mean ± SD). There was no significant difference between group 1 and group 6, but there was a significant increase of MVD between group 5 and 6 (p = 0,037).

We could achieve the following IC₅₀ values in the proliferation assays on HUVEC’s: 1,45 nM with gemcitabine and 4,94 mM with vitamin C. The IC₅₀ value in the proliferation assay on BxPC-3 cells were 9,47 nM with gemcitabine and 1,4 mM with vitamin C. Discussion: We could demonstrate a significant inhibition of the tumour growth in the combination therapy group. The MVD analysis is a critical feature to compare the success of tumour inhibition in vivo. Our in vitro data could demonstrate IC₅₀ values for the two compounds which are available in vivo. Therefore we suppose that a combination therapy of gemcitabine and vitamin C could be of interest in a multimodal therapy schedule of patients with pancreatic cancer.