Multiple antitumor effects of picropodophyllin in colon carcinoma cell lines: clinical implications

Abstract

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.

Figure 1

RT-PCR analysis of IGF-1R, IGF-2, IGF-1 and MMP-7 mRNA expression in tissues from colorectal cancer patients. Forty-eight matched samples from tumor (T) and normal control (C) tissue were analyzed for target gene expression by semi-quantitative RT-PCR analysis, where expression of β-actin mRNA served as internal reference. Results from four representative patients are shown.

Figure 2

Effect of PPP on growth of colon cancer cell lines. Left panel, cell lines were treated with different concentrations of PPP in triplicates followed by analysis using the resazurin assay at indicated times. Right panel, in parallel experiments, cells were treated with 0.5 μM PPP and counted manually in quadruplicates using trypan blue exclusion resulting in total number of viable cells treated and non-treated with PPP. Three experiments were performed and one representative is shown exhibiting means ± SD.

Figure 3

Effect of PPP on cell viability. Left panel, cells were treated with 0.5 μM PPP and counted manually in quadruplicates using trypan blue exclusion resulting in number of viable adherent and suspension cells, respectively. Right panel, in parallel, the number of dead adherent and suspension cells was counted and viability calculated and expressed as mean % viable cells ± SD. All experiments were performed three times and one representative is shown.

Figure 4

Effects of IGF-1 and PPP on cell migration. Petri dishes with near-confluent monolayers of the cell lines were scratched before treatment with 50 ng/ml IGF-1 or indicated concentrations of PPP for 48 h (HT-29) or 24 h (HCT-116, DLD-1 and CaCO-2). Three experiments were performed and images show representative areas of quadruplicate scratches.

Figure 5

Effects of PPP and/or IGF-1 on expression of AKT, ERK and MMP-7, -9 and -2. The cell lines were pretreated for indicated times with 0.5 μM PPP in complete medium and then stimulated 5 min with 50 ng/ml IGF-1 before lysis. (A) The expression of phospho/total-IGF-1R, phospho/total-AKT and phospho/total-ERK in the HT-29 and HCT-116 cell lines and (B) MMP-7, -9 and -2 in the HT-29 cell line were analyzed by Western blotting where GAPDH served as loading control. All experiments were performed three times and one representative is shown.