A very low toxic agent induces apoptosis and reduces growth of human hepatocellular carcinoma cells
Journal of Gastroenterology and Hepatology
Volume 21 Page 1207 - July 2006
Guido Schumacher,* Sylvia Scheunert,* Anne Rueggeberg, Max G. Bachem, Andreas K. Nussler,* Antonino Spinelli,* Tapas Mukhopadhyay,Â§,1 Johann Pratschke* and Peter Neuhaus*
*Department of General, Visceral, and Transplantation Surgery, andDepartment of Anesthesiology, Humboldt University, CharitÃ© Campus Virchow Klinikum, Berlin, andDepartment of Clinical Chemistry, University of Ulm, Ulm, Germany; andÂ§Departments of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
Aim: To examine the efficacy on growth inhibition of 2-methoxyestradiol (2-ME) on human hepatocellular carcinoma in vitro.
Methods: Hep3B, SK-Hep1, and PLC/PRF/5 cells were used. Proliferation assays using 2-ME should show a dose-dependent reduction of cell number. Different staining methods in cells derived from human hepatocellular carcinoma and normal human hepatocytes were performed to demonstrate possible tumor specific induction of apoptosis. FACS-analysis was done to confirm the induction of apoptosis after treatment with 2-ME.
Results: A reduction of the cell number of 90-98% was observed in all cancer cells after treatment with 2 Âµmol 2-ME. The mechanism of action appeared to be induction of apoptosis. Normal human hepatocytes were unaffected by 2-ME. The most sensitive cell line to 2-ME, SK-Hep1, showed an up-regulation of the p53 and p21 proteins.
Conclusions: 2-Methoxyestradiol appears to be highly effective in reducing tumor growth in vitro in human hepatocellular carcinoma. It may be tumor specific and applicable for clinical trials.
Hepatocellular carcinoma (HCC) is one of the most frequent primary cancers worldwide with the highest incidence in the East Asian countries. Apart from surgery, no cure of this disease has been described. Progress made in the understanding of the molecular mechanisms of tumor growth encourages studies of new therapeutic compounds and their mechanisms. We were interested in examining a possible growth inhibitory effect of 2-methoxyestradiol (2-ME) on human HCC cells. 2-ME is a physiological metabolite of estrogen, excreted in the urine. It appears to be independent of the presence of estrogen receptors.1,2 Several studies have shown that 2-ME inhibits tumor growth in different types of cancer. In vitro and in vivo experiments showed significant effects on tumor growth inhibition of breast cancer,3 lung cancer,4,5 pancreatic cancer,6 neuroblastoma,7 hepatoma,8,9 prostate cancer,9 and angiosarcoma.10,11 Several different mechanisms of growth inhibition seem to be involved. Inhibition of the tubulin fibers in the mitotic fuse was observed in breast cancer,3 but not in lung or pancreatic cancer.
2-Methoxyestradiol is a strong inhibitor of angiogenesis.12 Induction of apoptosis after treatment with 2-ME was observed in lung cancer cells. 2-ME stabilized the p53 protein as a crucial cell cycle regulator, which led to induction of apoptosis in these wt-p53 expressing cells. In contrast, induction of apoptosis independent of the p53 mutation status occurred in pancreatic cancer cells mutated for p53.6 Another mechanism of apoptosis induced by 2-ME has been shown in epithelial carcinomas such as breast, liver and colorectal cancer cells. A phosphorylation of c-jun N-terminal kinase (JNK) was found, which appeared to be correlated with phosphorylation of Bcl-2.9 Leukemia cells underwent apoptosis through a reactive oxygen species and serine-threonine kinase Akt dependent process.13
Herein we used HCC cells with varying p53 status and non-cancer human hepatocytes to analyze their comparative sensitivity to 2-ME.
Preparation of the drugs
2-Methoxyestradiol was kindly provided by EntreMed (Rockville, MD, USA). It was dissolved in absolute ethanol to give a 20 mmol/L solution, which was subsequently diluted with saline to obtain a stock solution of 660 Âµmol/L. The solution was aliquoted and stored at -20Â°C.
16-Epiestriol (MEC), an estrogen metabolite without known growth inhibitory activity, was purchased from Sigma (Deisenhofen, Germany) and dissolved, diluted and stored in the same manner as 2-ME.
For each experiment, a new aliquot was thawed and used.
Cells and tissue culture
Three cell lines derived from human HCC were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were used for all experiments. They were chosen according to the mutational status of p53, a crucial protein for cell cycle regulation and apoptosis. SK-Hep1 cells express wild-type p53. Hep3B cells harbor a deletion of the p53 protein, and PLC/PRF/5 cells express a mutation for p53. All cancer cell lines were maintained in modified Eagles's medium (MEM) supplemented with 10% fetal bovine serum, 1% glutamine, 1% nonessential amino acids, 1% sodium pyruvate, antibiotics, and antimycotics. Culture medium was refreshed twice per week and a 1:5 subculture split done once per week.
Cultures of normal human hepatocytes were obtained according to a protocol described previously.14 Briefly, perfusion of normal human liver, which was freshly resected mostly due to metastatic disease, was performed under sterile conditions using a solution with an enzymatic mixture containing collagenase and DNase. Normal human hepatocytes subsequently purified could be kept in culture for approximately 10 days in Williams Medium E supplemented with 10% fetal bovine serum, glutamine, sodium pyruvate, and nonessential amino acids. The p53 status in the normal human hepatocytes was assumed to be wild-type.
Cell proliferation assay
Cells were seeded from a subconfluent monolayer in 24-well plates at a density of 1-2 Ã- 104 cells per well depending on the doubling time of each cell line. After 48 h, cells were incubated with 2-ME or MEC. The doses ranged from 0.5 to 2 Âµmol in steps of 0.5 Âµmol. Cell counts were done in triplicate on day 1, 3, and 5 after incubation using a hemocytometer. Trypan blue exclusion was used to identify dead cells.
Western blot analysis
The detection of different protein expression patterns was performed using Western blot analysis according to a standard protocol as described elsewhere.15 The antibodies used were p53 (p53 Ab-3, NeoMarkers, Union City, CA, USA) with a 1:500 dilution, p21WAF1 (Ab-1, Oncogene Research Products; Calbiochem, Darmstadt, Germany) with a dilution of 1:300, and Î²-Actin (monoclonal anti-Î²-actin; Sigma-Aldrich, Deideshofen, Germany) with a dilution of 1:700. As secondary antibody we used the horseradish peroxidase-conjugated antibody (ImmunoPure, antigoat, mouse IgG; Pierce, Rockford, IL, USA) with a dilution of 1:5000. The loading quantity of the proteins was 50 Âµg/well for p53, 100 Âµg/well for p21WAF1 and 15 Âµg/well for Î²-actin detection.
Demonstration of apoptosis by immunofluorescence microscopy
Immunofluorescence microscopy was performed to demonstrate Apo2.7 expression, Annexin V binding and transferase deoxytidyl uridine end labeling (TUNEL) reaction. To assess the presence of apoptosis, HCC cell lines and hepatocytes were grown as monolayer on glass culture slides. 2-ME or MEC was added for two days. Thereafter monolayers were fixed for 1 h using buffered formaldehyde (6%), washed and air-dried. Staining was performed in one run. The procedures for staining are described in detail elsewhere.16 Fluorescence of Apo2.7 (yellow-green), Annexin-binding (red) and TUNEL-reaction (yellow) were observed and photographed using a fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with epilumination. To compare different staining intensities exposure time was always the same in each apoptosis test.
Cell cycle analysis
Cells were seeded in 100-mm diameter dishes at 1 Ã- 106 cells per dish and incubated with 2-ME or MEC at a dose that reduces growth by 50%. The doses used were 2.5 and 5.0 Âµmol for Hep3B and PLC/PRF/5, and 1.5 and 3.0 Âµmol for SK-Hep1 cells. After 3 days of incubation, cells were trypsinized, washed in phosphate-buffered saline (PBS), and fixed in 70% ice-cold ethanol for 60 min and stored at 4Â°C until used. The procedure for fluorescent activated cell sorting (FACS) analysis was performed according to a protocol previously described.17 Briefly, fixed cells were incubated with 1 mg/mL of RNase (Sigma) for 15 min at room temperature. Thereafter, 0.5 mL propidium iodide (PI) solution (Sigma; 100 Âµg/mL PBS) was added for 15 min at room temperature in the dark. Cells were washed once in PBS and kept at 4Â°C in the dark until measurement. 10 000 cells were analyzed using a FACS scan flow cytometer (Becton-Dickinson).
Preparation of cultured normal human hepatocytes
Six-well plates were covered with 3 mL 2% collagen-precoat in PBS per well. After 30 min of incubation, the solution was removed. 1 Ã- 106 normal human hepatocytes per well, which were harvested from freshly resected livers, were seeded. After 48 h of incubation at 37Â°C the cells were treated with 2-ME (2.5, 5 and 10 Âµmol) or with MEC (5 and 10 Âµmol), followed by incubation for 48 h at 37Â°C. Staining for apoptosis was performed according to the procedure described above. Photographs from viable hepatocytes were taken under a phase contrast microscope.
Potent growth inhibition after treatment with 2-ME
Proliferation assays are shown as absolute numbers of cells. There was no difference in cell growth inhibition after treatment with MEC when compared to control cells after a period of 5 days. There was a dose-dependent reduction of cell number after treatment with 2-ME (Fig. 1). 1.5 and 2 Âµmol 2-ME for 5 days reduced cell growth by 80-99%. SK-Hep1 cells revealed the highest degree of reduction of cell number. The low dose of 0.5 Âµmol 2-ME reduced the cell number by 71% after 5 days in this cell line. Normal human hepatocytes showed neither proliferation in the control groups nor reduction of the cell number, even at doses of up to 10 Âµmol 2-ME (data not shown).
p53 Protein is up-regulated in wild-type p53 expressing SK-Hep 1 cells
Western blotting showed that after treatment with 1.5 and 3.0 Âµmol 2-ME the wild-type p53 could be up-regulated in the wild-type p53 expressing cell line SK-Hep 1. p53 protein showed a fourfold increase after 2-ME treatment. No change in expression was observed after treatment with MEC. As a consequence, p21WAF1 (a direct downstream effector of p53) was up-regulated by six-fold in these cells, indicating that there is a functionally active p53 (Fig. 2a). The cell line PLC/PRF/5 harbors a mutated p53 protein, which is highly expressed and functionally inactive. The p21WAF1 protein did not show any increase in expression after treatment with 2-ME (Fig. 2b). The Hep3B cells, which are deleted for the p53 gene, expressed no p53 protein. The p21WAF1 protein was not changed in expression pattern after 2-ME treatment (Fig. 2c).
Strong induction of apoptosis
Specific staining for Apo 2.7, Annexin V, and TUNEL showed the induction of apoptosis in all 2-ME treated cancer cells (Fig. 3). All three staining techniques were strongly positive for apoptosis. In contrast, no induction of apoptosis was seen in MEC treated cells and in normal human hepatocytes. FACS analysis confirmed the presence of apoptotic cells in 2-ME treated cells as shown by an increase of the sub-G1 phase of the cell cycle, which corresponds to small size DNA derived from DNA fragmentation during the process of apoptotic cell death (Fig. 4). The counts of cells in the sub-G1 fraction allowed a quantification of the percentage of apoptotic cells. No apoptosis was seen after treatment with equivalent doses of MEC or control cells. There was no induction of apoptosis in normal human hepatocytes.
No changes of cell cycle phases
The number of cells in G1 phase decreased in groups treated with 2-ME by 50-70%. In these groups there was a high fraction of apoptotic cells (Fig. 4). G2 and S phase remained unchanged in all cell lines after both 2-ME and MEC treatment.
We have shown that 2-ME potently reduces the number of human HCC cells in vitro in a dose-dependent manner. This reduction of cell number of human HCC cells is of particular significance, because only a very small effect on survival can be achieved in patients with HCC, besides via surgery. High levels of apoptosis were induced after low doses of 2-ME. Previous studies have shown a high impact of 2-ME on other cancer types in vitro and in vivo.36,11,1820 Hepatoma cells have been also investigated recently. An induction of apoptosis in Hep3B cells after 2-ME treatment was also observed in vitro in combination with other cytostatic agents.8,9 It further appears to inhibit mammary carcinogenesis.21 All experiments showed a strong dose-dependent reduction of cell number of cancer and endothelial cells.
Variations were seen in the mechanism of action in different tumor types. An antiangiogenic effect and reduction of endothelial cell growth has been frequently described.3,7,1012,22,23 Other mechanisms are inhibition of the tubulin polymerization,24 stabilization of the p53 and enhanced induction of p53 dependent apoptosis,4,18 or p53 independent apoptosis.6 In our system, staining of the 2-ME treated cells showed that all cell lines underwent high levels of apoptosis. To examine both p53 dependent and independent pathways, we used SK-Hep1 cells expressing wild-type p53 and two cell lines expressing mutated p53 (Hep3B and PLC/PRF/5). Apoptosis was induced in all three cell lines, suggesting a p53 independent pathway in the p53 mutated cell lines. However, the wild-type p53 expressing cell line SK-Hep1 seems to be more sensitive to 2-ME rather than Hep3B and PLC/PRF/5 cells showing a higher percentage of apoptotic cells, which could be confirmed and quantified by FACS analysis. We found an increase in the sub-G1 region, which is represented by small DNA fragments, a characteristic feature of apoptosis. Cell cycle phases changed in G1, when apoptotic cell death occurred. There was a 50-70% reduction of the G1 phase. This may be caused by cells undergoing apoptosis from the G1 status, as S and G2/M phases remained unchanged. 2-ME has been described to arrest cells in mitosis with or without tubulin polymerization,24,25 causing G2-arrest. The absence of changes of the G2 phase suggests that there is no effect on cellular microtubules in the present system. These data confirm results shown in pancreatic cancer cells where no changes in cell cycle phases could be observed.6 These analyses suggest that the reduction of cell number by 2-ME may be caused only by induction of apoptosis instead of arresting the cell cycle.
McCormick et al. performed a large toxicity study on rats and dogs using 2-ME doses of up to 160 mg/kg bodyweight. No significant increase of liver-related blood values and a slight reduction of white blood cells and platelets as indicator for bone marrow suppression was observed in rats, but not in dogs. Moderately reduced size of testes and uterus was observed in rats and dogs after three weeks of treatment.26 Normal skin fibroblasts were unaffected by 2-ME.27 We also saw normal cells unaffected by 2-ME. Treatment of normal human hepatocytes did not show any signs of apoptosis or cell damage even at doses of up to 10 Âµmol 2-ME. As human cultured hepatocytes do not proliferate, in contrast to cancer cells with high proliferation index, a growth inhibition dependent on the proliferation index could be suggested. However, toxicity studies in vivo show very little signs of cell damage in normal cells, even in rapidly proliferating tissues such as intestine or bone marrow. No diarrhea and only marginal bone marrow suppression have been observed. A tumor-specific effect of 2-ME on cell growth is therefore suggested. An inhibition of superoxide dismutases, essential enzymes for the elimination of superoxide radicals by 2-ME, may explain a certain tumor specificity, because cancer cells are highly dependent on superoxide dismutases.28 As a possible risk of clinical application, one report presented a potential carcinogenicity of 2-ME observed in Syrian hamster embryo fibroblasts.29
Overall, 2-ME appears to be an effective and tumor-specific compound for reduction of cell number of human HCC. Low levels of toxicity in vivo and oral administration make it a potential and promising agent for clinical application.
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