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Medical Research Service, Stratton Veterans Affairs Medical Center (A.S., F.B.D., H.-Y.L., P.J.D.), the Clinical Research Institute, Albany Medical College (F.B.D., P.J.D.), and the Wadsworth Center, New York State Department of Health (P.J.D.), Albany, New York 12208
Address all correspondence to: Hung-Yun Lin, Ph.D., Clinical Research Institute MC-16, Albany Medical College, Albany, New York 12208. E-mail: . Address all requests for reprints to: Dr. Paul J. Davis, Clinical Research Institute, MC-16, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208. linhungyun{at}hotmail.com
Abstract
Two papillary thyroid carcinoma (PTC) and two follicular thyroid carcinoma (FTC) cell lines treated with resveratrol (RV), 1–10 µM, showed activation and nuclear translocation of MAPK (extracellular signal-regulated kinase 1/2). Cellular abundance of the oncogene suppressor protein p53, serine phosphorylation of p53, and abundance of c-fos, c-jun, and p21 mRNAs were also increased by RV. Inhibition of the MAPK pathway by either H-ras antisense transfection or PD 98059, an MAPK kinase inhibitor, blocked these RV-induced effects. Addition of pifithrin-
, a specific inhibitor of p53, or transfection of p53 antisense oligonucleotides caused decreased RV-induced p53 and p21 expression in PTC and FTC cells. Studies of nucleosome levels estimated by ELISA and of DNA fragmentation showed that RV induced apoptosis in both papillary and follicular thyroid cancer cell lines; these effects were inhibited by pifithrin- and by p53 antisense oligonucleotide transfection. PD 98059 and H-ras antisense transfection also blocked induction of apoptosis by RV. Thus, RV acts via a Ras-MAPK kinase-MAPK signal transduction pathway to increase p53 expression, serine phosphorylation of p53, and p53-dependent apoptosis in PTC and FTC cell lines.
RESVERATROL IS A phytoalexin that occurs naturally in grapes and several medicinal plants (1, 2). It has chemopreventive activity in mouse models of mammary gland and skin cancer (2) and has been reported to have other antitumor effects (2, 3, 4). The mechanism of the antitumor effects of resveratrol (RV) is not well understood, but in some tumor cell models may involve induction of apoptosis (3, 5, 6). Apoptosis is an intrinsic protective mechanism (7, 8) by which genetically damaged cells or excessive numbers of cells inappropriately induced by a mitotic stimulus may be eliminated (9). A potential strategy of cancer management is the pharmacologic induction of apoptosis in established cancer cells.
p53 is an oncogene suppressor protein present at low levels in the normal cell (10). In response to stresses such as DNA damage (10), levels of cellular p53 protein rise; this increase in p53 appears to result from a posttranslational mechanism that stabilizes the protein (11). When specifically phosphorylated at several serines, p53 promotes apoptosis (12). p53 is a substrate for serine kinases such as Jun N-terminal kinase (JNK) (13), p38 kinase, and extracellular signal-regulated kinases 1 and 2 (ERK1/2) (14). p53 alone, or in conjunction with other proteins such as c-Jun (15), is involved in induction of apoptosis by anticancer drugs directed against cancers of the prostate (15), lung (15), breast (16), and thyroid gland (17).
MAPK (ERK1/2), an inducible component of normal cellular signal transduction processes, has been shown to be constitutively activated (phosphorylated) in several cancer cell lines (18, 19, 20). In contrast to such constitutive activity of ERK1/2, transient activation of the kinases may contribute to induction of apoptosis (21, 22). ERK1/2 is also an upstream regulator of the p53 response to DNA damage caused by the apoptosis-inducing drug cisplatin (23).
In the present studies of established human papillary (PTC) and follicular thyroid carcinoma (FTC) cell lines, we found that exposure of cells to RV revealed activation of MAPK. MAPK activation was associated with a subsequent increase in abundance of nuclear p53 protein that was shown to be phosphorylated at serines 6 and 15 in FTC and serine 15 in PTC cells. These changes in cellular MAPK and p53 were associated with induction of apoptosis. We have recently reported that p53 is a substrate for activated MAPK (24).
Materials and Methods
Cell lines
Two human PTC cell lines (BHP 2–7 and BHP 18–21), generously provided by Dr. J. M. Hershman (West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA) were studied. Two FTC cell lines (FTC 236 and FTC 238) were kindly provided by Dr. Orlo Clark (University of California at San Francisco-Mt. Zion Medical Center, San Francisco, CA) with permission of Dr. Peter Goretski. PTC cells were cultured in Roswell Park Memorial Institute 1640 medium, and FTC cells in 50% DMEM/50% Ham’s F-12 plus 10 mU/ml of TSH (Sigma, St. Louis, MO); media were supplemented with 10% FBS, and cells were maintained in 5% CO2 at 37 C. All cell cultures were incubated with fresh media containing 0.25% hormone-depleted FBS for 2 d before study.
Cell fractionation
After treatment with RV and/or other agents for indicated times, the cells were washed with ice-cold PBS and lysed in hypotonic buffer (20 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 3 µg aprotinin/ml, 1 mg pepstatin/ml, 20 mM NaF, and 1 mM DTT), with 0.2% NP-40. Lysis occurred over 10 min with cells on ice. After centrifugation at 4 C for 1 min at 13,000 rpm, the supernatants were collected as cytosol. Nuclear extracts were prepared by resuspension of the crude nuclei in high salt buffer (hypotonic buffer to which was added 20% glycerol and 420 mM NaCl) at 4 C, with rocking for 1 h. The nucleoprotein-containing supernatants were collected after centrifugation at 4 C for 10 min at 13,000 rpm.
Immunoblotting
Proteins were separated by discontinuous SDS-PAGE (9%) and transferred by electroblotting to Immobilon membranes (Millipore Corp., Bedford, MA). After blocking with 5% milk in Tris-buffered saline containing 0.1% Tween, membranes were incubated overnight with one of various antibodies including rabbit polyclonal antiphosphorylated MAPK (ERK1 and ERK2) (New England Biolabs, Inc. [NEB], Beverly, MA), anti-serine-15- or anti-serine-6-phosphorylated p53 (NEB), anti-p21 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-c-Fos (Santa Cruz Biotechnology, Inc.), or mouse monoclonal anti-c-Jun or anti-p53 (Santa Cruz Biotechnology, Inc.). The secondary antibody was either goat anti-rabbit IgG (1:1000) or rabbit anti-mouse IgG (1:1000) (DAKO Corp., Carpinteria, CA). Immunoblotted proteins were visualized by chemiluminescence and scanned for illustration (BioImage, Millipore Corp.).
Transfection of H-ras and p53 antisense oligonucleotides
Using a technique previously described by our laboratory (24), thyroid cancer cells were transfected with 7.5 µg of either H-ras antisense or scrambled oligonucleotide (25) or with 5.0 µg of either p53 antisense or scrambled oligonucleotide (26) (Operon Technologies, Alameda, CA) in the presence of LIPOFECTAMINE PLUS reagent (Life Technologies, Inc., Grand Island, NY) for 6 h. The medium was then replaced with fresh medium containing 0.25% thyroid hormone-depleted FBS (24) and cells were treated with or without 10 µM RV for 48 h.
Determination of c-fos, c-jun, p21, and GAPDH mRNAs
Total RNA was isolated as described previously (27). First strand cDNAs were synthesized from 1 µg of total RNA using oligodeoxythymidine and Avian Myeloblastosis Virus Reverse Transcriptase (Promega Corp., Madison, WI). First-strand cDNA templates were amplified for GAPDH, c-fos, c-jun, and p21 by PCR using a hot start (Ampliwas, Perkin-Elmer Corp., Foster City, CA). Primer sequences used were as described by Irving et al. (28). The PCR cycle consisted of an initial step of 95 C for 3 min, followed by 94 C for 1 min, 55 C for 1 min, 72 C for 1 min for 25 cycles, and a final cycle of 72 C for 8 min. PCR products normalized to give equal signals from GAPDH were subjected to electrophoresis in 2% agarose gel containing 0.5 µg/ml of ethidium bromide. Gels were visualized under UV light and photographed with Polaroid film (Polaroid Co. Cambridge, MA). Photographs were scanned for illustration.
Nucleosome ELISA assay for detection of apoptosis
Cells were harvested and washed with PBS. Nucleosome ELISAs were carried out according to the protocol provided by Oncogene Research Products (Cambridge, MA).
DNA fragmentation
Total genomic DNA extraction followed the protocol of the SUICIDE-TRACK DNA LADDER ISOLATION KIT (Oncogene). DNA electrophoresis was performed in 1.5% agarose gels containing 0.5 µg/ml of ethidium bromide. Gels were visualized under UV light and photographed with Polaroid film. Photographs were scanned as indicated above.
Using the methods described above, all experiments reported were carried out at least three times with comparable results.
Results
Activated MAPK in thyroid cancer cell lines
With the addition to BHP 18–21 and FTC 236 cells of RV, 0.1 to 100 µM, for 4 and 24 h, nuclear extracts revealed the presence of activated MAPK, detected with antiphospho-MAPK that recognized both ERK1 and ERK2 isoforms (Fig. 1A
). There was concentration-dependent activation of MAPK with 0.1–100 µM RV at 4 h and with 0.1–10 µM RV at 24 h. However, less activated MAPK was found after treatment with 100 µM RV for 24 h. This was attributed to induction by RV of apoptosis at 24 h (results not shown). The pattern of activation of MAPK induced by RV showed some variation among thyroid cancer cell lines during longer time periods. In the absence of RV, some activation of nuclear MAPK was detected after 3–5 d in cultures of papillary and follicular thyroid cancer cell lines (Fig. 1B). For example, there was a sustained activated MAPK response to RV for 5 d (1 or 10 µM) in BHP 2–7 and BHP 18–21 cells, but activated MAPK was decreased at 5 d in FTC 238 cells (RV, 1–10 µM). In FTC 236 cells, treatment with 10 µM RV, in contrast to 1 µM, resulted in almost complete disappearance of activated MAPK at 1 and 2 d. As indicated above, this finding is attributed to the susceptibility of the cells to RV-induced apoptosis.
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In BHP 2–7 cells treated with RV, there was increased abundance of p53 protein (Fig. 2A). RV at 10 µM was more effective than the phytoalexin at 1 µM. RV also caused accumulation of p21, an inhibitor of cyclin-dependent protein kinase activity (29), after 3–5 d of treatment, and c-Fos and c-Jun proteins were similarly increased (Fig. 2A). Using RT-PCR, we detected increased abundance of c-fos and c-jun mRNAs in BHP 2–7 cells treated with 1 µM RV (Fig. 2B).
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). p21 content was also increased in cells after 3–5 d of treatment with 10 µM RV, and c-Fos content increased with 1 and 10 µM RV (Fig. 3A). Increases in p53, c-fos, and p21 mRNAs in RV-treated cells were seen, particularly after 3 d, with both concentrations (Fig. 3B).
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). RV-induced increases in p53 and serine-15-phosphorylated p53 were seen in FTC 236 cells with 1 or 10 µM RV, whereas in FTC 238 cells increases in p53 and c-Fos were seen particularly with 1 µM RV (Fig. 4A). Treatment of cells with RV increased p53 and p21 mRNA levels in both FTC 236 and FTC 238 cells, and c-fos mRNA in FTC 236 cells (Fig. 4B).
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To study whether the increase in p21 mRNA in RV-treated thyroid cancer cells is p53-dependent, PTC and FTC cells were exposed to 10 µM RV in the presence or absence of 20 µM PFT-, a specific p53 inhibitor (30). Total p53 accumulation and serine 15 phosphorylation of p53 were reduced by PFT- in both PTC cell lines (Fig. 5A, lanes 4). The abundance of p21 mRNA decreased in cells treated with RV and PFT-, compared with levels in cells treated with RV alone, indicating a reduction in p53-dependent p21 gene expression in both PTC cell lines (Fig. 5A). Similar results also were observed in two FTC cell lines (Fig. 5B). We also used p53 antisense oligonucleotide transfection to decrease p53 content in cells. The abundance of p53 and expression of p21 mRNA induced by RV were diminished by antisense but not by scrambled oligonucleotide transfection in BHP18–21 cells (Fig. 6A, lanes 3). Similar results were also observed in FTC 236 cells (Fig. 6B). These results with PFT- and p53 antisense oligonucleotide transfection strongly suggest that the expression of p21 induced by RV is p53-dependent.
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To demonstrate that activated MAPK (ERK1/2) is essential for the action of p53, cells were treated with RV in the presence or absence of PD 98059 (PD), a specific MEK inhibitor (24). Results indicated that PD blocked the activation and nuclear translocation of ERKs 1 and 2 induced by RV in BHP 2–7 and BHP 18–21 thyroid cancer cell lines (Fig. 7A, lanes 4 compared with lanes 3). The phosphorylation of serine 15 on p53 induced by RV was inhibited by PD (Fig. 7A). There was no detectable serine 6 phosphorylation induced by RV (results not shown). c-Fos expression, stimulated by RV (lanes 3), was also inhibited by PD (lanes 4). p53 mRNA did not change significantly with RV treatment (2 d) in BHP 2–7 cells, but c-fos mRNA increased with RV, an effect inhibited by PD (Fig. 7B). On the other hand, p53 and c-fos mRNA increased with RV treatment in BHP 18–21 cells, an effect diminished by PD (Fig. 7B).
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). Phosphorylation of serines 6 and 15 on p53 was only minimally reduced by the MEK inhibitor in both follicular cancer cell lines (Fig. 8A). RV-induced p53 mRNA in FTC 236 and FTC 238 cells was not appreciably affected by PD (lanes 4, Fig. 8B). Induction of c-fos and c-jun mRNA by RV (lanes 3) was reduced by addition of PD to these FTC cells (lanes 4).
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, A and B, lanes 5 vs. 4). In cells transfected with a scrambled oligonucleotide (lanes 6), the response to RV was the same as in untransfected cells (lanes 4). The phosphorylation of serine 15 of p53 in BHP 2–7 and FTC 238 cells was also inhibited by H-ras antisense transfection (Fig. 9, A and B, lanes 5). FTC 238 cells displayed serine 6 phosphorylation of p53 in response to RV, which was inhibited by H-ras antisense transfection (Fig. 9B), but BHP 2–7 cells did not show serine 6 phosphorylation in response to RV. A similar lack of serine 6 phosphorylation was found with BHP 18–21 cells, whereas the response of FTC 236 cells resembled that of FTC 238 cells (results not shown). Not only the abundance and phosphorylation of p53 protein, but also the increase of p53 and p21 mRNA caused by RV (Fig. 9, C and D, lanes 4) were inhibited by H-ras antisense oligonucleotide transfection in BHP 2–7 and FTC 238 cells (lanes 5). These results demonstrate that activation of ERK1/2 by RV is Ras-dependent, and that a reduction in Ras affects serine phosphorylation of p53 and abundance of p53 and p21 mRNA in cells treated with RV.
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RV caused increased nucleosome content indicating apoptosis in two papillary thyroid carcinoma cell lines, BHP 2–7 and BHP 18–21, and two follicular thyroid cancer cell lines, FTC 236 and FTC 238 (Fig. 10). When cells were treated with RV in the presence of PD, apoptosis was inhibited.
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, apoptosis was inhibited in BHP 2–7 and BHP 18–21 cells, as well as in FTC 236 and FTC 238 cells (Fig. 11). With the use of p53 antisense oligonucleotide transfection, we further confirmed the p53-dependence of RV-induced apoptosis in the same thyroid cancer cell lines (Fig. 12).
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, A and B). These results again support an essential role for the H-Ras-MEK-MAPK signaling pathway in p53-dependent apoptosis induced by RV.
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In the present studies, treatment of human thyroid cancer cells with the naturally occurring polyphenol, RV, disclosed the presence of inducible MAPK, and that such induction was associated with subsequent apoptosis. RV in low concentrations (1 pM-10 µM) has been reported by others to activate MAPK in human neuroblastoma SH-SY5Y cells (31), although higher concentrations (50–100 µM) of the stilbene inhibited MAPK activity in this cell line (31) and in other cells (32). In the studies described above, some activation of ERK1/2 was apparent in papillary and follicular thyroid cancer cells cultured for 3–5 d in the absence of RV, and exposure of these cells to RV resulted in further accumulation of these activated kinases. The RV effect was time- and concentration dependent. Activation of ERK1/2 by RV was blocked by either H-ras antisense oligonucleotide transfection or the MEK inhibitor, PD 98059. Thus, a Ras- and MEK-dependent signal transduction cascade is implicated in biologically-relevant activation of MAPK in these cells.
Other laboratories have proposed that constitutively active MAPK is required for maintenance of the malignant state, but that short-term activation of MAPK may direct cells to apoptosis (33). However, constitutive activation of MAPK in such studies may be a response of cell exposure to culture conditions for several days, as Fig. 1
in the present report suggests. What is clear, however, is that the modest activation of MAPK in control cells in our studies after 3–5 d in culture did not lead to activation of p53 and to apoptosis. It has been suggested by others that short-term activation of MAPK may be required for induction of apoptosis in normal cells (21).
Normal p53 function is essential to the induction of apoptosis in human and murine cells subjected to DNA damage (10). In some cancer and leukemic cells, high levels of p53 are found (16) but reflect the presence of dysfunctional, usually mutated, protein. Apoptosis does not occur in such cells in response to the constitutively expressed p53. In the untreated follicular and papillary thyroid carcinoma cell lines we studied, low-to-moderate amounts of p53 were present constitutively, but apoptosis was not detected unless the cells were exposed to RV. The p53 that was transiently induced by RV appeared to retain function of the wild-type protein, in that its appearance was associated with immediate-early gene product accumulation and with apoptosis. By using an inhibitor of p53-dependent transcriptional activation, PFT-, and p53 antisense oligonucleotide transfection, to block both RV-induced increase in abundance of p53 (Figs. 5 and 6) and RV-induced apoptosis (Figs. 11 and 12), we substantiated a role for p53 in promotion by RV of apoptosis in these thyroid cancer cells.
RV (10–100 µM) induces apoptosis in several cancer cell models, including prostate cancer (4), breast cancer (34, 35), lymphoblasts (3), and leukemia (5). The present studies show for the first time that papillary and follicular thyroid cancer cell lines have an apoptotic response to RV. Huang et al. (3) have described RV-induced apoptosis in embryonic fibroblasts that express wild-type p53, but not in p53-deficient cells. Basolo and co-workers (17) have reported that expression of (presumptively wild-type) p53 is correlated with diversion of cells to apoptosis in papillary thyroid cancer. The present studies involving RV are consistent with these observations. Because both the H-ras antisense and MEK inhibitor paradigms decreased activation of p53 and the induction of apoptosis, it is clear that the latter two steps are linked to the MAPK pathway. In addition, because both the MEK inhibitor (Figs. 7 and 8) and H-ras antisense paradigms (Fig. 9) decreased activation of p53 and the induction of apoptosis (Figs. 10 and 13), it is clear that the latter two steps are linked to the MAPK pathway and provide a Ras- and MAPK-related mechanism by which the stilbene can increase cellular content of p53.
Overexpression of p53 has been shown to induce c-fos expression in a manner that differs from p53 expression in response to potentially oncogenic growth factors, such as epidermal growth factor (36). p53-induced c-fos expression is relevant to apoptosis. As Figs. 2–4 in this report show, RV not only promoted accumulation of p53, but also of c-Fos and c-Jun, in thyroid cancer cells. The abundance of respective mRNAs of c-fos and c-jun was also increased in RV-treated cells.
Studies by Hsieh et al. (37) have also demonstrated that RV increases accumulation of cellular p53 and the cyclin-dependent protein kinase inhibitor, p21, in cultured pulmonary artery endothelial cells. p21 was originally identified as a transcriptional target of p53 (38). It is now known that this protein is a component of a complex containing cyclins, cyclin-dependent kinases and proliferating cell nuclear antigen (39). An increased amount of p21 in the quaternary complex leads to inhibition of DNA synthesis and cell cycle arrest (40) that permits DNA repair to proceed. In both papillary and follicular thyroid carcinoma cells, we also found that RV increased cellular abundance of p21 protein and mRNA (Figs. 2–6 and 9). This stilbene-dependent increase in p21 mRNA was reduced by cell treatment with the p53-specific inhibitor, PFT- (Fig. 5) and p53 antisense oligonucleotide transfection (Fig. 6), suggesting that RV-induced p21 expression is p53-dependent. These results are consistent with a role for p21 in p53-dependent apoptosis that is caused by RV. RV-induced p53-dependent p21 expression was inhibited by H-ras antisense oligonucleotide treatment (Fig. 9) and corresponded to a reduction in RV-induced apoptosis (Fig. 13), further solidifying the connection between MAPK and p53 signals.
Phosphorylation of p53 determines the biological activity of the protein. At several serine residues, including serine 6 and serine 15, phosphorylation has been shown to be relevant to DNA damage (10) and apoptosis (41). In our studies, there were differences between papillary and follicular thyroid cancer cells in the serine phosphorylation patterns caused by RV. That is, both serine 6 and serine 15 were phosphorylated in follicular cancer cells treated with RV, but serine 6 was not phosphorylated in papillary cancer cells. However, serine phosphorylation-dependent apoptosis was induced by RV in both cell types. Stilbene-related serine 15 phosphorylation in papillary thyroid cancer cells was inhibited by PD 98059 to a greater extent than in follicular thyroid cancer cells, but the H-ras antisense oligonucleotide paradigm was highly effective in all cancer cell lines in blocking serine 15 phosphorylation of p53. RV-induced serine 6 phosphorylation in follicular thyroid cancer cells was minimally affected by PD 98059 but was completely inhibited by antisense H-ras transfection under conditions of these experiments. This suggests that the mechanism of RV-induced phosphorylation of p53 involves MAPK at serine 15, but another serine kinase at residue 6. As noted above, p53 is also a substrate for JNK (13) and p38 kinase (14). It is not clear how relevant serine 6 phosphorylation is to apoptosis (41), and in the present studies we show that serine 6 phosphorylation by RV is not required in papillary thyroid cancer cells for apoptosis to occur.
Acknowledgments
Drs. J. Hershman and O. Clark kindly provided thyroid cancer cell lines for study.
Footnotes
This work was supported in part by the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P.J.D.), and grants from the Candace King Weir Foundation and the Charitable Leadership Foundation.
Abbreviations: AS, Antisense oligonucleotide; DTT, dithiothreitol; ERK1 and ERK2, extracellular signal-regulated kinases 1 and 2; FTC, follicular thyroid carcinoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, Jun N-terminal kinase; MEK, MAPK kinase; PD, PD98059; PFT-, pifithrin-; pMAPK, phosphorylated MAPK; PTC, papillary thyroid carcinoma; RV, resveratrol; Scr, scrambled oligonucleotide.
Received September 10, 2001.
Accepted December 10, 2001.
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