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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 The Wistar Institute; 2 Department of Chemistry and 3 Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania; and 4 Department of Dermatology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan
Requests for reprints: Keiran S.M. Smalley, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-3951; E-mail: ksmalley{at}wistar.org.
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Abstract |
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Introduction |
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Unlike other solid tumors, melanomas typically lack p53 mutations and retain expression of the wild-type (WT) protein, often at high levels (6–9). This is surprising, given the highly malignant nature of melanoma and its great resistance to therapeutic intervention. Evidence suggests that melanoma cells can tolerate high levels of transcriptionally active p53 (9) and have an abnormal p53 response following DNA damage (6). It is therefore likely that p53 is not a functional tumor suppressor in melanoma. One of the major mechanisms of p53 inactivation in melanoma is through the increased expression of Mdm2 (and its human homologue Hdm2), a nuclear protein that binds to, promotes the nuclear export of, and subsequently degrades p53 through its intrinsic E3 ubiquitin ligase activity (10). In addition to its role in proteasomal degradation, Mdm2 also inhibits the transcriptional activity of p53 activity through specific binding at key residues (11). More than 50% of primary invasive and metastatic melanoma samples overexpress Hdm2 at the protein level, and it is likely that this contributes to functional inactivation of p53 in melanoma (12). p53 function can also be negatively regulated by another protein, which is structurally related to Mdm2 called Mdm4 (and its human homologue HdmX; ref. 13). Although there is evidence that some melanoma cell lines overexpress Mdm4 (14), the exact relationship between p53 function and Mdm2/Mdm4 expression remains to be unraveled (15).
p53 can be pharmacologically activated in cancer cell lines through a variety of mechanisms, leading to the induction of apoptosis (16–19). Of these, two groups have identified glycogen synthase kinase (GSK) 3ß as a major regulator of p53 localization and expression (20, 21). Although GSK3ß has received little attention as a possible therapeutic target from the cancer research community, several very recent studies have suggested that inhibiting GSK3ß may induce apoptosis in colorectal carcinoma cells through a p53-dependent pathway (18, 19). Classically, GSK3ß was described as a key regulator of glycogen metabolism and is also known to regulate other processes, such as apoptosis in neurons, protein synthesis, cell proliferation, microtubule dynamics, cell motility, and Wnt signaling (22–24).
In the current study, we explore whether tumors with p53-WT, such as melanoma, may benefit from strategies that pharmacologically activate the p53 pathway. Here, we describe for the first time the anticancer activity of organometallic GSK3ß inhibitors, represented by DW1/2 (25, 26). Treatment of melanoma cells with these compounds leads to the nuclear accumulation of p53, down-regulation of Mdm2 and Mdm4, increased NOXA expression, and apoptosis via the mitochondrial pathway. Similar results were seen when the melanoma cells were treated with Nutlin-3, an antagonist that disrupts the p53/Mdm2 binding interaction leading to enhanced p53 activation and apoptosis (27).
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Materials and Methods |
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Cell culture. Human melanocytes and melanoma cells were isolated and cultured as described by Smalley et al. (28). The lentiviral vector short hairpin RNA (shRNA) constructs for p53 were developed in the laboratory of Dr. Maria Soengas (University of Michigan, Ann Arbor, MI) based on published sequences (29, 30). The p53 adenovirus was from Dr. Wafik El-Deiry (University of Pennsylvania, Philadelphia, PA; ref. 6). Melanoma cells that were WT for p53 (WM793, 1205Lu, and WM115) or harbored p53 mutations (WM852, WM983A, WM983B, and WM164) have been sequenced previously (6, 7).
Adherent cell proliferation analysis. Cells were plated into a 96-well plate at a density of 2.5 x 104 per mL and left to grow overnight. Cells were treated with increasing concentrations of DW1, DW2, or DW1/2 in triplicate. In each instance, cells were left to grow for 72 h before being treated with 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 3 h (Sigma, St Louis, MO). After this time, medium was rapidly removed and the resulting crystals were solubilized in DMSO. Absorbance was read in a plate reader at 540 nm. Absorbance readings were subtracted from the value of blank wells, and the reduction in cell growth was calculated as a percentage of control absorbance in the absence of any drug. Data show the mean of at least three independent experiments ± SE.
Western blot analysis. Proteins were extracted and blotted as described by Smalley et al. (28). After analysis, Western blots were stripped once and reprobed for ß-actin to show even protein loading. Antibodies to phosphorylated GSK3ß, phosphorylated p53, cleaved caspase-9, cleaved caspase-3, Bcl-2, BAD, Akt, extracellular signal-regulated kinase (ERK), and Bax were from Cell Signaling Technology (Beverly, MA). The polyclonal antibody to Mdm4 (HdmX) was from Bethyl Laboratories (Montgomery, TX). The monoclonal antibody to ß-actin was from Sigma and the monoclonal antibody for p53 (DO-1) was from Calbiochem (San Diego, CA). Monoclonal antibodies to GSK3ß, ß-catenin, Mdm2, p21, p27, and NOXA were from BD Biosciences (Franklin Lakes, NJ).
Three-dimensional spheroid growth. Melanoma spheroids were prepared using the liquid overlay method and treated as described previously (31).
Cell cycle analysis. Cells were plated into 10-cm dishes at 60% confluency and left to grow overnight before being treated either with DW1/2 (100 nmol/L–1 µmol/L) for 24 h or with Nutlin-3 (5–20 µmol/L; Cayman Chemical Co. Ann Arbor, MI), LiCL (30 mmol/L), or SB216763 (20 µmol/L) for 24 to 72 h. Cells were then treated and analyzed as described previously (31).
Flow cytometric analysis of apoptosis and mitochondrial membrane potential (
m). After the treatment of the cells with DW1/2 (300 nmol/L or 1 µmol/L, 24 h), cells were washed once with Annexin binding buffer [10 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl, 5 mmol/L CaCl2], resuspended in 100 µL binding buffer containing 1 µL FITC-conjugated Annexin V (R&D Systems, Minneapolis, MN) and 25 nmol/L tetramethylrhodamine, methyl ester, perchlorate (TMRM; Molecular Probes, Eugene, OR), and incubated at 37°C for 15 min. Cells were then analyzed for Annexin V fluorescence (green) and TMRM retention (red fluorescence) using flow cytometry.
Immunofluorescence microscopy. Cells were seeded thinly onto ethanol-sterilized glass coverslips in six-well plates and left to grow overnight. After which they were treated with either DW1/2 (1 µmol/L), LiCl (30 mmol/L), SB216763 (20 µmol/L), or Nutlin-3 (15 µmol/L) for 24 h before being fixed in 4% paraformaldehyde and permeabilized with 0.2% (v/v) Triton X-100. Samples were then blocked in PBS containing 1% bovine serum albumin. Primary antibody incubations to p53, pH2AX, pATM (all at 1:100; Calbiochem), or cleaved poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology) were done at 37°C for 1 h under humidified conditions. Slides were prepared as described by Smalley et al. (28).
Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining. Cells were thinly seeded onto coverslips in six-well plates and left to adhere overnight. Cells were treated with DW1/2 (100 nmol/L–3 µmol/L) for 72 h before being fixed and permeabilized in 4% formaldehyde and 0.2% (w/v) Triton X-100. Cells were then treated using fluorescein/terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) reagents from an in situ cell death detection kit (Roche Diagnostics, Basel, Switzerland) for 1 h before being washed and treated with antifade. Cells were analyzed as for immunofluorescence.
Statistical analysis. Unless otherwise stated, all experiments show the mean ± SE of at least three independent experiments. Statistical significance was measured using the Student's t test, where P < 0.05 was judged to be significant.
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and GSK3ß, respectively, and the IC50 values for DW2 to be 9 and 15 nmol/L for GSK3 and GSK3ß, respectively, at an ATP concentration of 100 µmol/L. Treatment of 1205Lu melanoma cells with increasing concentrations (3 nmol/L–3 µmol/L) of DW1, DW2, and DW1/2 for 72 h (structures shown in Fig. 1A) led to concentration-dependent reduction in cell growth (Fig. 1B). In agreement with the in vitro kinase data, it was noted that DW2 was less potent than DW1. However, as the racemic mixture DW1/2 displayed similar potency to DW1 and did not require resolving, all subsequent experiments were done on this compound. DW1/2 had growth-inhibitory activity against a panel of five melanoma cell lines (WM793, WM115, WM164, WM852, and 1205Lu; Fig. 1C). Often the pharmacologic profile of drugs in two-dimensional cell culture is not predictive of the response in three-dimensional melanoma spheroid culture (31). To investigate this further, spheroids formed from 1205Lu cells were implanted into a three-dimensional collagen I gel and treated with DW1/2 (1–10 µmol/L) for 72 h. It was found that 1 µmol/L DW1/2 markedly reduced cell viability of the spheroid, as shown by the loss of green staining and increased red staining and lack of invasion into the surrounding collagen (Fig. 1D).
Treatment with DW1/2 induces apoptosis through the intrinsic mitochondrial pathway. Cell cycle analysis showed that treatment of melanoma cells (WM793 and 1205Lu) with DW1/2 for 24 h led to a concentration-dependent increase in the number of cells in the sub-G1 population, suggesting that DW1/2 was inducing apoptosis (Fig. 2A
). At the highest concentration (1 µmol/L), levels of apoptosis were 47% and 65% in the 1205Lu and WM793 cells, respectively. In other studies, cells were treated with drug for 24 h, fixed, stained, and labeled using a TUNEL kit. Again, like the cell cycle analysis, increasing concentrations of DW1/2 increased the level of TUNEL staining (data not shown). To investigate the mechanism of apoptosis, cells were treated with 1 µmol/L DW1/2 for 0, 2, 6, and 24 h, and protein was extracted and probed for expression of the cleaved caspase-3 and caspase-9 (Fig. 2B). Caspase-9 was cleaved rapidly (<6 h) and caspase-3 was cleaved after 6 h of drug treatment. Following DW1/2 treatment, there was little change in levels of Bcl-2, Bax, or BAD (Fig. 2B). However, there was a marked reduction in levels of phosphorylated BAD after 2 h of DW1/2 treatment. A prolonged treatment of the cells with DW1/2 (24 h) led to increased expression of NOXA (Fig. 2B). Further evidence for DW1/2-induced apoptosis through the intrinsic pathway was shown by an increase in Annexin V positivity and loss of mitochondrial membrane potential (m) as measured by loss of TMRM staining (Fig. 2C). These processes started as early as 3 h, with rapid progression during subsequent time points (6, 12, 24, 48, and 72 h; Fig. 2C). To confirm whether the DW1/2 had any selectivity for tumor cells over normal cells, primary human skin melanocytes derived from foreskins were treated with increasing concentrations of DW1/2 (30 nmol/L–1 µmol/L) for 24 h followed by Annexin V staining and flow cytometry (Fig. 2D). It was noted that only very little apoptosis was seen in the melanocytes (6%) following 1 µmol/L DW1/2 treatment (24 h) compared with 31% in the 1205Lu melanoma cells at the same drug concentration. In other studies, we looked at the ability of DW1/2 to reduce the growth of primary human skin fibroblasts in MTT assays and found a markedly weaker antiproliferative effect relative to that seen for WM793 melanoma cells (Supplementary Fig. S1A). To determine the cross-reactivity of DW1/2 on other major melanoma survival pathways, we also looked at levels of phosphorylated Akt and phosphorylated ERK. It was noted that there was a substantial decrease in phosphorylated Akt activity after 2 h of DW1/2 treatment but little effect on phosphorylated ERK levels (Supplementary Fig. S1B).
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p53-dependent apoptosis can also arise indirectly, as a consequence of DNA damage and through the generation of reactive oxygen species (ROS). As some metal ions, such as platinum, can induce p53 expression following DNA damage, we investigated whether the ruthenium-based DW1/2 compound also induced a DNA damage response. Treatment of 1205Lu cells for 0, 2, 6, or 24 h (1 µmol/L) did not lead to an increase in the nuclear staining of phosphorylated H2AX or phosphorylated ataxia-telangiectasia mutated (ATM), which are both markers of DNA damage (Supplementary Fig. S2A). In contrast, treatment of the same cells with cisplatin led to increased phosphorylation of both H2AX and ATM after 24 h of treatment. As further evidence of DW1/2 working through a non-DNA damage–mediated p53 induction mechanism, we showed the rapid up-regulation of the p53 target gene p21 after 6 h of DW12 treatment (Supplementary Fig. S2B). Finally, we investigated whether DW1/2 induced apoptosis through a ROS-dependent mechanism by pretreating the cells with the antioxidants TROLOX (200 µmol/L; Supplementary Fig. S3) or N-acetylcysteine (5 µmol/L) and found that there was no protection from DW1/2-induced apoptosis.
Knockdown of p53 reduces DW1/2-induced apoptosis in p53-WT melanoma cells. As melanoma cells have abnormalities in many apoptotic pathways, it is often difficult to compare between cell lines. To overcome this, we investigated the role of p53 in DW1/2-induced apoptosis within a defined, isogenic system where p53 was knocked down in WM793 cells using a stable shRNA lentiviral vector system (29, 30). After green fluorescent protein (GFP)–based selection studies, two p53 shRNA clones (p53.1 and p53.2) gave significant protein knockdown (>90%; Fig. 5A ). Control vectors with mutated shRNA sequences were identical to uninfected controls (data not shown). The functional knockdown of p53 protein in the WM793 cells was shown following treatment of the cells with Nutlin-3 (15 µmol/L, 48 h; Fig. 5A). In the WM793-p53 cells, there was little apoptosis following Nutlin-3 treatment and the cells continued to proliferate (sub-G1, 4%), whereas the control cells underwent apoptosis (sub-G1, 27%).
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To investigate the effects of DW1/2 on p53 regulation, cells were treated with drug for increasing periods before being probed for expression of p53, Mdm2, and Mdm4. It was noted that there was a rapid increase in p53 expression (<2 h) with a slower but marked reduction of Mdm2 and Mdm4 expression (>2 h; Fig. 5D). In contrast, cisplatin (40 µmol/L) induced a much slower up-regulation of p53 expression, which was seen only after 24 h of treatment (data not shown).
Pharmacologic activation of p53, but not p53 overexpression, induces apoptosis in p53-WT melanoma cells. Nutlin-3 activates p53 through the disruption of the p53/Mdm2 interaction (27). Here, we show that 24-h treatment of the p53-WT 1205Lu melanoma cells with Nutlin-3 leads to nuclear accumulation of p53 (Fig. 6A ). In contrast, treatment of p53-mut melanoma cells with Nutlin-3 had little effect on either p53 expression or nuclear localization (data not shown). Treatment of the p53-WT 1205Lu cell line with Nutlin-3 (10 µmol/L) was associated with up-regulation of the p53 transcriptional target p21 as well as p27 (Fig. 6B). In contrast, no up-regulation of either p21 or p27 was seen following Nutlin-3 treatment in the p53-mut cell line WM852 (Fig. 6B). Cell cycle analysis of p53-WT 1205Lu cells showed that Nutlin-3 induced a progressive accumulation of cells in the sub-G1 phase, indicating that this compound was inducing apoptosis (Fig. 6C). Similar experiments in the p53-mut cell line WM852 revealed little apoptosis following Nutlin-3 treatment (Fig. 6C). It was important to ascertain whether p53-dependent apoptosis in melanoma cells was the result of inhibiting the p53/Mdm2 complex or only increased p53 expression. To investigate this point, we infected p53-WT and p53-mut cells with an adenovirus for p53. It was noted that overexpression of p53 induced apoptosis in the p53-mut WM852 cells but not the p53-WT 1205Lu cells (Fig. 6D). Western blot analysis confirmed that the adenoviral infection led to increased p53 expression in both melanoma cell lines (data for 1205Lu is shown in Fig. 6D).
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Discussion |
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Here, we report for the first time the remarkable anticancer activity of DW1/2 on human melanoma cells. In most of the melanoma cell lines tested, 300 nmol/L of the DW1/2 were able to maximally inhibit cell growth. DW1/2 represents a member of a series of organometallic kinase inhibitors derived from the class of indolocarbazole alkaloids (e.g., staurosporine) with very high selectivity for GSK3ß (25, 26, 32).
All of the melanoma cells tested had high levels of the Ser9 phosphorylated form of GSK3ß, suggesting that this kinase was at least partly inactivated in melanoma cells, which is most likely a consequence of constitutive high Akt activity (33, 34). Although GSK3ß was Ser9 phosphorylated in the melanoma cells, DW1/2 treatment was still able to up-regulate ß-catenin expression in the 1205Lu melanoma cells, showing that the melanoma cells still possessed some GSK3ß activity that could be blocked by these compounds. The lower selectivity of DW2 over DW1 in the MTT assays shows that growth inhibition was directly correlated with the ability of these compounds to inhibit GSK3 in the in vitro kinase assay. Similar ß-catenin-stabilizing and growth-inhibitory results were also seen following treatment with two other GSK3ß inhibitors, SB216763 and LiCl.
Several studies have suggested that intracellular signaling is modified by the tumor microenvironment (31, 35, 36). Our previous studies have shown that melanoma cells derived from metastases become completely resistant to the growth-arresting activity of inhibitors of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein/ERK pathways when grown as three-dimensional collagen-implanted spheroid (31). Here, we show that the 1205Lu cells still respond well to the DW1/2 when grown under these conditions, suggesting that there is no acquisition of drug resistance to DW1/2 under three-dimensional culture conditions.
Treatment of melanoma cells that were WT for p53 (p53-WT) with DW1/2 led to the rapid induction apoptosis via the intrinsic caspase-9/caspase-3 mitochondrial pathway. Treatment of the melanoma cells with other GSK3ß inhibitors, such as LiCl and SB216763, were also found to induce apoptosis. The p53 pathway is a major activator of mitochondrial-dependent apoptosis. Consistent with this, treatment of p53-WT cells with DW1/2 led to the up-regulation of nuclear p53 expression. Interestingly, in p53-mut cells, the levels of p53 were high in the absence of the DW1/2 and were not altered following treatment, suggesting that mutations in p53 rendered the cells unresponsive to DW1/2. This was further shown by the fact that melanoma cells with mutated p53 (WM852, WM983A, and WM983B) did not undergo any (WM852) or very little (<10%; WM983A and WM983B) apoptosis following administration of DW1/2. The possible role of GSK3ß in the observed up-regulation of p53 expression in melanoma cells was also confirmed when cells were treated with LiCl and SB216763.
Tumor cells are genetically heterogeneous and harbor many mutations that work together to suppress apoptosis. To account for this, we further explored the role of p53 in DW1/2-induced apoptosis in a defined isogenic system where melanoma cells with p53-WT (WM793) had the p53 stably knocked down using a lentiviral shRNA (WM793-p53). Knockdown of p53 in WM793 cell line was found to significantly reduce the levels DW1/2-induced apoptosis by 48%, suggesting that p53 played an important role in DW1/2-mediated apoptosis. The fact that total suppression of apoptosis was not achieved can be either explained by the fact that the levels of p53 knockdown were not sufficient to completely abolish all DW1/2-mediated apoptosis or that there are other p53-independent apoptosis mechanisms involved in the activity of DW1/2. The fact that the WM793-p53 cells also display a blunted response to the p53/Mdm2 antagonist Nutlin-3 shows that levels of p53 knockdown achieved are sufficient to block p53-induced apoptosis, suggesting that DW1/2 may have additional mechanisms of apoptosis induction. The p53 dependency of GSK3ß inhibitor/chemotherapy–mediated apoptosis has been shown previously in two separate studies using the HCT116 colorectal carcinoma cell lines, which either express or were null for p53 (18, 19). In these studies, it was shown that a panel of GSK3ß inhibitors (SB 216763, purvalanol A, and LiCl) only induced p21 expression (indicative of p53 activity) and sensitized the cells to chemotherapy-induced apoptosis in the p53-expressing cells and not in the p53-null cells (18, 19).
To further investigate whether p53 activation was a bona fide approach for the induction of apoptosis in melanoma, we next tried Nutlin-3, another drug known to increase p53 activity, through disruption of the p53/Mdm2 interaction (16, 27). Like DW1/2, Nutlin-3 also increased nuclear accumulation of p53 in p53-WT melanoma cells. Like DW1/2, Nutlin-3 also increases the transcriptional activity of p53, as shown by the increased expression of p21Waf-1 in p53-WT cells but not in p53-mut cells. In a similar manner, Nutlin-3 also induced apoptosis in p53-WT melanoma cells but not p53-mut cells. In this instance, onset of apoptosis was slower (>48 h) than that seen to DW1/2, which induced significant apoptosis after 24 h of treatment.
To gain further insight into the mechanisms behind the proapoptotic activity of DW1/2 and Nutlin-3, we overexpressed p53 in both p53-WT and p53-mut melanoma cells using an adenoviral vector. It was found that p53 overexpression only induced apoptosis in the p53-mut cells and not in the p53-WT cells. This suggested that mere overexpression of p53 was not sufficient to induce apoptosis and that the p53-WT cells possessed other mechanisms to suppress p53-induced apoptosis. Indeed, others have shown the poor transcriptional activation of p53 target genes in p53-WT melanoma cells following DNA damage (37). It has also been shown that melanoma cells with p53-WT can tolerate high levels of exogenous p53 (9). It therefore seems likely that DW1/2 may function in a similar manner to Nutlin-3 in freeing the p53 from its suppressors rather than only increasing p53 expression. To investigate this further, we treated the 1205Lu cells with DW1/2 and looked for up-regulation of p53 and down-regulation of Mdm2 and Mdm4. Treatment with DW1/2 induced a rapid increase in p53 expression, a rapid down-regulation of Mdm4 expression (<2 h), and a slower down-regulation of Mdm2 (6 h). Recent studies have shown that both Mdm4 and Mdm2 play important roles in regulating p53 function, with Mdm4 regulating p53 activity and Mdm2 being more responsible for p53 stability (15). To our knowledge, this is the first report showing the pharmacologic down-regulation of Mdm4 expression.
Several recent studies have begun to shed light on the GSK3ß-mediated regulation of p53 expression. It has been suggested that GSK3ß cooperates with Mdm2 to directly regulate the export of p53 from the nucleus via phosphorylation of p53 at Ser315 and Ser376 (20). Our own studies revealed a transient increase in Ser6 but not Ser315 phosphorylation of p53 following DW1/2 treatment (data not shown). Others have shown that overexpression of GSK3ß directly reduces levels of p53 expression through the direct phosphorylation of Mdm2 (21).
Here, we have shown for the first time that activation of p53, using either the novel organometallic kinase inhibitor DW1/2 or the Mdm2/p53 antagonist Nutlin-3, led to the efficient induction of apoptosis in human melanoma cells. At present, GSK3ß inhibitors are being explored in both preclinical and clinical settings for a wide range of disease states from diabetes to neurodegeneration (38). The fact that melanomas harbor p53-WT, which can be activated pharmacologically, makes GSK3ß inhibitors and Nutlin-3 attractive novel therapeutic approaches.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 5/ 3/06. Revised 9/21/06. Accepted 10/ 9/06.
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-irradiation in many wild-type p53 human melanoma lines. Cancer Res 1996;56:840–7.This article has been cited by other articles:
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  X. Meng, M. L. Leyva, M. Jenny, I. Gross, S. Benosman, B. Fricker, S. Harlepp, P. Hebraud, A. Boos, P. Wlosik, et al. A Ruthenium-Containing Organometallic Compound Reduces Tumor Growth through Induction of the Endoplasmic Reticulum Stress Gene CHOP Cancer Res., July 1, 2009; 69(13): 5458 - 5466. [Abstract] [Full Text] [PDF]
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 H. Yu, R. McDaid, J. Lee, P. Possik, L. Li, S. M. Kumar, D. E. Elder, P. Van Belle, P. Gimotty, M. Guerra, et al. The Role of BRAF Mutation and p53 Inactivation during Transformation of a Subpopulation of Primary Human Melanocytes Am. J. Pathol., June 1, 2009; 174(6): 2367 - 2377. [Abstract] [Full Text] [PDF]
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 M. Lioni, K. Noma, A. Snyder, A. Klein-Szanto, J. A. Diehl, A. K. Rustgi, M. Herlyn, and K. S.M. Smalley Bortezomib induces apoptosis in esophageal squamous cell carcinoma cells through activation of the p38 mitogen-activated protein kinase pathway Mol. Cancer Ther., September 1, 2008; 7(9): 2866 - 2875. [Abstract] [Full Text] [PDF]
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 D. Yao, C. L. Alexander, J. A. Quinn, W.-C. Chan, H. Wu, and D. A. Greenhalgh Fos cooperation with PTEN loss elicits keratoacanthoma not carcinoma, owing to p53/p21WAF-induced differentiation triggered by GSK3{beta} inactivation and reduced AKT activity J. Cell Sci., May 15, 2008; 121(10): 1758 - 1769. [Abstract] [Full Text] [PDF]
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 D. J. Panka, D. C. Cho, M. B. Atkins, and J. W. Mier GSK-3 Inhibition Enhances Sorafenib-induced Apoptosis in Melanoma Cell Lines J. Biol. Chem., January 11, 2008; 283(2): 726 - 732. [Abstract] [Full Text] [PDF]
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