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Department of General Surgery and Transplantation, Charite Campus Virchow Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany [G. S.]; First Department of Surgery, Okayama University Medical School, Okayama 700-0914, Japan [M. K.]; and Section of Thoracic Molecular Oncology, Departments of Thoracic and Cardiovascular Surgery [J. A. R., T. M.] and Tumor Biology [J. A. R.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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Introduction |
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Apoptosis, or programmed cell death, is an active, gene-directed form of cell death that is different from cell necrosis with respect to its morphology, biochemistry, pharmacology and biological significance. Many types of mammalian cells undergo apoptosis during normal development or in response to a variety of stimuli, including DNA damage, growth factor deprivation, and abnormal expression of oncogene or tumor suppressor genes (5, 6, 7) . Apoptosis is a widely accepted, important mechanism that contributes to cell growth reduction. 2-ME has been shown to induce apoptosis in a number of different cell types (4 , 8 , 9) .
The present study was undertaken to explore whether 2-ME induces apoptosis in pancreatic cancer cell lines, which exhibit aggressive clinical behavior. We studied the effect of 2-ME on the growth in culture of four pancreatic cancer cell lines and its effect on the metastatic behavior of MIA PaCa-2 in mice. Most of the pancreatic cancer cell lines appeared to be highly sensitive to 2-ME-mediated growth inhibition and underwent apoptotic cell death. Although the molecular mechanism of 2-ME action is not yet clearly understood, it appears to have potential as a therapeutic agent.
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Materials and Methods |
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Cell Proliferation Assay.
Cells were seeded at a density of 2 x 104 cells/well in 24-well plates. On the next day, cells were treated with different concentrations of either 2-ME or its inactive analogue 16-epiestriol (ranging from 0.01 to 10 µm concentrations). Cells were fed every 2 days; treated cells were replenished with fresh drug. Cells were harvested every other day. Cell growth was monitored first by trypsinizing the cells and then by staining them with crystal violet and counting stained cells using a hemocytometer. All experiments were done in triplicate.
Cell Cycle Analysis.
Cells were treated with different concentrations of 16-epiestriol (MEC) or 2-ME for 24 h. The concentration of 2-ME that inhibited growth by 50% (IC50) in the proliferation assay was determined (1.5 µM for MIA PaCa-2, 2 µM for PaTu 8988t and PaTu 8902, and 10 µM for PaTu 8988s). After washing with PBS, subconfluent cultures were harvested from 100-mm diameter dishes after 24 h of treatment with 2-ME or 16-epiestriol (used as inactive control of 2-ME used in Table 1
and Fig. 3
), fixed in 70% ethanol, and stored at 4°C until use. Fixed cells were incubated with 50 µg/ml of propidium iodide and 10 µg/ml of RNase A at 37°C for 30 min. Cell cycle analysis was done using an EPICS Profile II flow cytometer (Coulter Corp., Hialeah, FL). Data were analyzed using the Coulter Cytological program. All experiments were repeated at least two times.
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In Vivo Experiments.
Twenty female athymic mice, 4–6 weeks of age, were used for our studies. To reduce immune function further, animals were irradiated with 350 rads from a 137Cs source. The next day, 70–80% confluent MIA PaCa-2 cells were trypsinized, washed once in HBSS, and resuspended in PBS. One hundred µl containing 3 x 106 MIA PaCa-2 cells were injected into the mice, through the tail veins, to induce lung metastases. The animals were randomly grouped into two groups of 10. 2-ME was dissolved in 2% DMSO of olive oil to obtain a solution containing 1 mg of 2-ME in 50 µl. 2-ME was administered p.o. using an intubation needle. The animals were fed daily with 50 µl of the above-mentioned solution with or without 2-ME. Blinded treatment was performed from day 3 through day 21 after tumor cell injection. After treatment, the mice were killed, their lungs were harvested and fixed with Fekete’s solution (60% ethanol, 3% formaldehyde, and 4% glacial acetic acid) after intratracheal injection of a 15% India ink solution. The total number of unstained colonies on the lung surface was counted under a stereoscopic microscope by two investigators without knowledge of the treatment groups.
Immunohistochemical Staining for Angiogenesis Using a CD31 Antibody.
Two extra groups of five animals each (2-ME treated and nontreated) were used for the immunohistochemical staining. After the mice were fed daily for 3 weeks with 2-ME at the above-mentioned concentrations, the mice were killed, and the lungs were harvested and embedded in OCT compound (Miles, Inc., Elkhart, IN) for frozen sections. After being blocked with 5% goat serum and 1% horse serum, the slides were incubated with rat anti-mouse CD31 (PECAM-1; PharMingen, San Diego, CA) at 4°C overnight. Because primary antibody was rat anti-mouse, a secondary antibody, peroxidase-conjugated anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used. CD31 was visualized with diaminobenzidine. The number of vessels of a total of 30 lung colonies was counted in each group. The average number of vessels of each group was compared.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The rate of proliferation was significantly slower for these cell lines after treatment with 2-ME. An initial dose-response study indicated that all cell lines, except one, showed similar kinetics of growth inhibition. The PaTu 8902, PaTu 8988t, and MIA PaCa-2 cell lines showed marked growth inhibition (50–90%) after 5 days of treatment with 2 µM 2-ME in culture. The IC50 of 2-ME ranged from 2 to 10 µM among the cell lines. The PaTu 8988s cell line was less sensitive to 2-ME than the others. After 48 h of 2-ME treatment, the cells were examined under a phase contrast microscope. The cells had shrunken and retracted from the neighboring cells, and the nuclei were fragmented, a characteristic feature of apoptosis. However, no morphological changes were observed when these cell lines were treated with 16-epiestriol, an inactive metabolic byproduct of estrogen with no effect on cell growth (data not shown). These results suggest that 2-ME action on these cell lines was specific, and it inhibited the pancreatic cancer cell growth by inducing apoptosis in a time- and dose-dependent fashion. Moreover, it is important to note that although 2-ME can inhibit the growth of pancreatic cell lines, it had no effect on normal bronchial cells, even when treated with much higher concentrations (4)
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. Treatment with 2-ME that resulted in growth inhibition elicited a prominent, prolonged accumulation of cells at S phase in the three cell lines that were more sensitive to the treatment. However, in the PaTu 8988s cell line, which was relatively less sensitive, no significant effect on the cell cycle was seen.
The 2-ME-induced apoptotic cell population was examined using flow cytometry (Fig. 2)
. A considerable fraction of the 2-ME-treated cells appeared as sub-G1, compared with control, untreated cells or 16-epiestriol-treated cells. Thus, the profile of DNA content in the three sensitive cell lines, PaTu 8902, PaTu 8988t, and MIA PaCa2, indicated that a significant fraction had undergone apoptotic cell death after 2-ME treatment. In contrast, the PaTu8988s cell line, which was relatively resistant to 2-ME-mediated growth inhibition, showed little apoptotic cell death on flow cytometric analysis. These results indicate that 2-ME inhibits the growth in the three sensitive pancreatic cancer cell lines by inducing apoptotic cell death.
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. The characteristic TUNEL staining was observed when the PaTu 8902, PaTu8988t, and MIA PaCa-2 cell lines were exposed to 2 µM 2-ME for 48 h, and a high percentage of apoptotic cells was detected. However, control, mock-treated cells or 16-epiestriol-treated cells showed little DNA fragmentation, but 2-ME-treated cells displayed many apoptotic bodies. 2-ME treatment resulted in 30–90% apoptotic cell death in these cell lines (
90% in the MIA PaCa-2 cell line).
To study the antitumor effect of 2-ME in vivo, we compared the growth and number of metastatic lung colonies in treated and untreated control nude mice 21 days after the MIA PaCa-2 cells were injected into the tail veins (Fig. 4, a and b)
. There were 10 animals in each group, as described in "Materials and Methods." Animals were given oral 2-ME, and results showed that 2-ME-treated mice had a 60% lower incidence of lung colonies than control untreated animals (Fig. 5)
; this result was statistically significant (P < 0.0005 by t test analysis). This experiment was repeated three times and showed similar results (Table 2)
. The animals did not have any signs of toxicity, such as weight loss or modified behavior, during 2-ME treatment. All animals injected with MIA PaCa-2 cells developed lung colonies. To examine the effect on angiogenesis in the lung colonies, immunohistochemical staining for CD31, a specific marker for endothelium, was performed. However, no significant difference in the number of blood vessels inside the colonies was seen (Fig. 5)
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Discussion |
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1%). However, a significant number of apoptotic cells were detected 24 h after treatment with 2-ME. Apoptosis induced by various other agents appears to be mediated by a common set of downstream elements that act as regulators and effectors of apoptotic cell death. In many cases, p53 is required for apoptosis in a number of models, and stabilization of p53 leads cells to undergo apoptosis (4 , 13 , 14) . We have shown previously that in lung cancer cell lines, 2-ME induces endogenous wild-type p53 protein posttranscriptionally with associated bypass of the G1-S checkpoint and causes p53-dependent apoptosis (4) . Overexpression of p53 in response to several death stimuli induces apoptosis. We, therefore, examined the p53 gene status in our four pancreatic cancer cell lines. Western blot analysis of the p53 protein using an anti-p53 monoclonal antibody, PCR-single strand conformational polymorphism analysis of exons 4–9, and DNA sequencing indicated that all of these lines harbor mutated p53. PaTu 8988t and PaTu 8988s, both deriving from the same tumor, have a C-to-T mutation in codon 282, whereas PaTu 8902 harbors a T-to-A mutation in codon 176. MIA PaCa-2 cells revealed a C-to-T mutation in codon 248 (data not shown). Therefore, apoptosis induction by 2-ME contrasted with the previous results, indicating that 2-ME elicited p53-independent apoptotic cell death in these pancreatic cancer cell lines.
17ß-Estradiol has been reported to have a 
1000-fold higher binding affinity to the cytosolic estrogen receptor than that of 2-ME (15)
. On the other hand, 2-ME showed much more potent activity for apoptosis induction in the cells compared with 17ß-estradiol and 16-epiestriol (2
, 4
, 8)
. Moreover, the effect of 2-ME action is independent of estrogen receptor expression because blocking the estrogen receptors by tamoxifen does not abrogate 2-ME-induced apoptosis.4
These data therefore suggest that 2-ME acts through a separate pathway. Thus, our work and other studies (3)
suggest that 2-ME has a pleiotropic effect on cell growth and apoptosis of cells that depends on the tissue origin and genetic makeup. These results further suggest an association between apoptosis induction and inhibition of cell proliferation by 2-ME and that 2-ME inhibited pancreatic cancer cell proliferation through an apoptotic mechanism.
Angiogenesis, the generation of new capillaries from preexisting vessels, is critical to cancer progression (16 , 17) . Angiogenesis requires enzymatic degradation of the basement membrane, then vascular endothelial cell migration into the perivascular space, proliferation and alignment to form tubular structures, and finally, new vessel formation (16 , 17) . However, the molecular mechanism leading to pathological angiogenesis remains to be elucidated. Tumor angiogenesis has long been thought to be important for the growth of solid tumors (18) , and this view is strengthened by the fact that 2-ME inhibits both the neovascularization and growth of s.c. melanoma and sarcoma tumors (2) . However, we find no significant difference in vessel density among lung colonies in untreated versus 2-ME-treated mice. Nevertheless, this does not completely eliminate antiangiogenesis as a mechanism of growth inhibition of tumor colonies in our model because inhibition of neovascularization could limit the size of the lung colonies.
An earlier report indicated that 2-ME treatment of the estrogen-dependent cell line MCF-7 caused G2-M arrest associated with the depolymerization of tubulin (19) . However, we observed no such effect of 2-ME on the tubulin structure in immunofluorescence studies of our lung cancer cell lines (data not shown) nor in A431 cells, even at concentrations of 20 µM(20) . It has been further suggested that 2-ME caused mitotic arrest in a leukemia cell line by inhibiting the calmodulin pathway (20) . However, when pancreatic cancer cell lines were treated with 2 µM of 2-ME, there is an effect on the distribution of tumor cells in the different phases of the cell cycle (4) . A significant proportion of the cells are blocked at S phase. The molecular mechanism that blocks the cells to S phase has yet to be identified. Using Western blot analysis, we examined the E2F1 and Rb gene product, which have been implicated in the progression of the cells to S phase, but noticed no significant difference in the E2F1 expression or in Rb phosphorylation state (data not shown). It is interesting to note that PaTu8988s cells, which were not blocked at S phase after 2 µM 2-ME treatment, could avoid the 2-ME-mediated apoptotic cell death. Possibly, conflicting signals that caused premature entry of the cells to S phase and to retain the cells at S phase for a long time could result in the decision of a cell to undergo programmed cell death.
2-ME appears to have several unique features: (a) it is a nontoxic metabolic byproduct of estrogen present in normal human urine; (b) 2-ME treatment results in the extended, selective induction and stabilization of wild-type p53 protein necessary for apoptosis; (c) the effect of such treatment is restricted specifically to cancer cells and has no effect on normal bronchial epithelial cells, thus increasing the therapeutic index; and (d) 2-ME is effective in vivo, even when administered p.o. Interestingly, 2-ME has been reported to induce apoptosis in transformed cells but not in normal cells (21) . Thus, it provides a potential means for inducing programmed cell death in aggressive pancreatic cancer cells. This might be of clinical interest because there are few therapeutic options for pancreatic cancer, besides surgery. 2-ME induction of apoptosis in human pancreatic cancer cells, therefore, holds promise as a therapy for pancreatic cancer.
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FOOTNOTES |
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1 This study was partially supported by Grant P50-CA70907 from the National Cancer Institute (NCI) for a Specialized Program of Research Excellence (SPORE) in Lung Cancer; by a Development Grant from NCI for The University of Texas M. D. Anderson Cancer Center SPORE in Lung Cancer (to T. M.); by gifts to the Division of Surgery and Anesthesiology from Tenneco and Exxon for the Core Laboratory Facility; by NCI Support Core Grant CA16672 to The University of Texas M. D. Anderson Cancer Center; by a grant from the Mathers Foundation; and by a sponsored research agreement with Introgen Therapeutics, Inc. 
2 To whom requests for reprints should be addressed, at The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 109, Houston, TX 77030, Phone: (713) 792-6932; Fax: (713) 794-4901.
3 The abbreviations used are: 2-ME, 2-methoxyestradiol; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP biotin nick-end labeling.
4 T. Mukhopadhyay and J. A. Roth, unpublished observations.
Received 7/30/98; revised 11/16/98; accepted 11/19/98.
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