Meifang Wang

Persistent ERK Phosphorylation Negatively Regulates cAMP Response Element-binding Protein (CREB) Activity via Recruitment of CREB-binding Protein to pp90RSK

Compound 5 (Cpd 5) or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is an inhibitor of protein phosphatase Cdc25A and causes persistent activation of extracellular signal-regulated kinase (ERK) and cell growth inhibition. To study the mechanism(s) by which persistent ERK phosphorylation might induce cell growth inhibition, we used Cpd 5 as a tool to examine its effects on the activity of CREB (cAMP response element-binding protein) transcription factor in Hep3B human hepatoma cells. We found that CREB activity, including its DNA binding ability and phosphorylation on residue Ser-133, was strongly inhibited by Cpd 5, followed by suppression of CRE-mediated transcription of cyclin D1 and Bcl-2 genes. Cpd 5-mediated suppression of CREB phosphorylation and transcriptional activity was antagonized by mitogen-activated protein kinase kinase inhibitors PD 98059 and U-0126, implying that this inhibition of CREB activity was regulated at least in part by the ERK pathway. The phosphorylation of ribosomal S6 kinase (pp90RSK), a CREB kinase in response to mitogen stimulation, was also found to be inhibited by Cpd 5 action. This inhibition of pp90RSKphosphorylation is likely the result of its increased association with CREB-binding protein (CBP), which subsequently caused inhibition of CREB phosphorylation and activity. To support the hypothesis that Cpd 5 effects on Cdc25A inhibition with subsequent ERK activation could cause CREB inhibition, we examined the effects of Cdc25A inhibition without the use of Cpd 5. Hep3B cells were transfected with C430S Cdc25A mutant, and ERK was found to be phosphorylated in a constitutively activated manner, which was accompanied by decreased CREB phosphorylation and increased recruitment of CBP to pp90RSK. These data provide evidence that CBP·RSK complex formation in response to persistent ERK phosphorylation by Cpd 5 down-regulates CREB activity, leading to inhibition of both cAMP response element-mediated gene expression and cell growth.

Transient and sustained ERK phosphorylation and nuclear translocation in growth control

Growth stimulation and inhibition are both associated with tyrosine phosphorylation. We examined the effects of epidermal growth factor (EGF), a growth stimulant, and compound 5 (Cpd 5), a protein‐tyrosine phosphatase (PTPase) inhibitor, which inhibits the growth of the same Hep3B hepatoma cells. We found that both EGF and Cpd 5 induced tyrosine phosphorylation of EGF receptor (EGFR) and ERK. However, the phosphorylation caused by EGF was transient and that caused by Cpd 5 was prolonged. Furthermore, Cpd 5 action caused a strong nuclear phospho‐ERK signal and induced phospho‐Elk‐1, a nuclear target of ERK activation, in contrast to the weak effects of EGF. An ERK kinase assay demonstrated that ERK activated by Cpd 5 could phosphorylate its physiological substrate, Elk‐1. The MEK inhibitors PD098056 and U0126 abrogated both the induction by Cpd 5 of phospho‐ERK, its nuclear translocation and phospho‐Elk‐1 and also antagonized its growth inhibitory effects. Furthermore, phospho‐ERK phosphatase and phospho‐Elk‐1 activities were lost from nuclear extracts from Cpd 5 treated, but not EGF treated cells. In conclusion, the data show that Cpd 5 causes growth inhibition as a consequence of prolonged ERK and Elk‐1 phosphorylation, likely a result of inhibition of multiple PTPases, including those acting on phospho‐EGFR, on phospho‐ERK, and on phospho‐Elk‐1, in contrast to the kinase driven transient activation resulting from EGF.

PM-20, a novel inhibitor of Cdc25A, induces extracellular signal–regulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo

We have synthesized several new phenyl maleimide compounds, which are potent growth inhibitors of several human tumor cell lines. Among these, PM-20 was the most potent with an IC50 of 700 nmol/L for Hep3B human hepatoma cell growth. Two other derivatives, PM-26 and PM-38, did not inhibit Hep3B cell growth even at 100 μmol/L. Interestingly, under identical experimental conditions, PM-20 inhibited DNA synthesis of primary cultures of normal hepatocytes at a 10-fold higher concentration than that needed to inhibit the DNA synthesis of the Hep3B hepatoma cells. PM-20 affected two cellular signaling pathways in Hep3B cells: Cdc25 phosphatase and extracellular signal–regulated kinase (ERK) 1/2. It competitively inhibited the activity of Cdc25 (preferentially Cdc25A) by binding to the active site, likely through the catalytic cysteine, but did not inhibit PTP1B, CD45, or MKP-1 phosphatases. As a result of its action, tyrosine phosphorylation of the cellular Cdc25A substrates Cdk2 and Cdk4 was induced. It also induced strong and persistent phosphorylation of the Cdc25A substrate ERK1/2. Hep3B cell lysates were found to contain ERK2 phosphatase(s) activity, which was inhibited by the actions of PM-20. However, activity of exogenous dual-specificity ERK2 phosphatase MKP1 was not inhibited. Induction of ERK1/2 phosphorylation correlated with the potency of growth inhibition in tumor cell lines and inhibition of ERK1/2 phosphorylation by the mitogen-activated protein kinase (MAPK)/ERK kinase 1/2 inhibitor U0126 or overexpression of the cdc25A gene in Hep3B cells antagonized the growth inhibitory actions of PM-20. Growth of transplantable rat hepatoma cells in vivo was also inhibited by PM-20 action with a concomitant induction of pERK in the tumors. The mechanism(s) of growth inhibition of Hep3B hepatoma cells by the phenyl maleimide PM-20 involves prolonged ERK1/2 phosphorylation, likely resulting from inhibition of the ERK phosphatase Cdc25A. PM-20 thus represents a novel class of tumor growth inhibitor that inhibits mainly Cdc25A, is dependent on ERK activation, and has a considerable margin of selectivity for tumor cells compared with normal cells. [Mol Cancer Ther 2006;5(6):1511–9]

JS‐K, a novel non‐ionic diazeniumdiolate derivative, inhibits Hep 3B hepatoma cell growth and induces c‐Jun phosphorylation via multiple MAP kinase pathways

JS‐K, a non‐ionic diazeniumdiolate derivative, is capable of arylating nucleophiles and spontaneously generating nitric oxide (NO) at physiological pH. This recently synthesized low molecular weight compound is shown here to be an inhibitor of cell growth with concomitant activation of mitogen‐activated protein kinase (MAPK) members ERK, JNK, and p38 and their downstream effectors c‐Jun and AP‐1. Inhibitors of these MAPK pathways abrogated the growth inhibitory actions of JS‐K. In addition to the well‐described actions of JNK as a kinase for c‐Jun, we show that c‐Jun is also an ERK target. Furthermore, JS‐K generated NO in culture and NO inhibitors antagonized both MAPK induction and the growth inhibitory effects of JS‐K. These results suggest two possible mechanisms for the mediation of JS‐K growth inhibitory actions, namely NO‐induction of MAPK pathway constituents as well as possible arylation reactions. The data support the idea that prolonged MAPK activation by JS‐K action is important in mediating its growth‐inhibitory actions. JS‐K thus represents a promising platform for novel growth inhibitory analog synthesis. J. Cell. Physiol. 197: 426–434, 2003© 2003 Wiley‐Liss, Inc.

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal‐regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2‐(2‐mercaptoethanol)‐3‐methyl‐1,4‐napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de‐phosphorylated phospho‐ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper‐phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over‐expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock‐down by Cdc25A siRNA resulted in a constitutive‐like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR‐devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho‐ERK is likely another Cdc25A substrate, and Cpd 5‐caused ERK phosphorylation is probably regulated by both EGFR‐dependent and EGFR‐independent pathways.

Changes in TGF‐β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF‐β receptors associated with increased sensitivity to TGF‐β‐mediated growth inhibition

To clarify the role of transforming growth factor‐β (TGF‐β) and its receptors in hepatocyte growth, we studied the expression of TGF‐β1 and its receptors and the sensitivity to growth inhibition by TGF‐β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF‐β type II receptor (TβR‐II), insulin‐like growth factor‐II/mannose 6‐phosphate receptor (IGF‐II/M‐6‐PR), and TGF‐β1 increased with time in primary culture. The cell surface TGF‐β receptor proteins (TβR‐I, II, and III), examined by the receptor affinity‐labeling assay using 125I‐TGF‐β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF‐β1 protein was added at later times in culture, corresponding to the presence of increased TGF‐β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR‐I, TβR‐II, TβR‐III, IGF‐II/M‐6‐PR, and TGF‐β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF‐β receptor affinity‐labeling assay, hepatocytes from PH livers were found to have an increase in TβR‐I, II, and III proteins, with a peak at 4 days post‐PH, compared with hepatocytes from resting livers. When TGF‐β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF‐β receptors and sensitivity to growth inhibition by TGF‐β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998.

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen‐activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2‐(2‐mercaptoethanol)‐3‐methyl‐1,4‐naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)‐induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen‐activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time‐ and dose‐dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP‐GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5‐altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho‐extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5‐induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth. J. Cell. Physiol. 183:338–346, 2000.

Novel hydroxyl naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties.

Cdc25 phosphatases are important in cell cycle control and activate cyclin-dependent kinases (Cdk). Efforts are currently under way to synthesize specific small-molecule Cdc25 inhibitors that might have anticancer properties. NSC 95397, a protein tyrosine phosphatase antagonist from the National Cancer Institute library, was reported to be a potent Cdc25 inhibitor. We have synthesized two hydroxyl derivatives of NSC 95397, monohydroxyl-NSC 95397 and dihydroxyl-NSC 95397, which both have enhanced activity for inhibiting Cdc25s. The new analogues, especially dihydroxyl-NSC 95397, potently inhibited the growth of human hepatoma and breast cancer cells in vitro. They influenced two signaling pathways. The dual phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was induced, likely due to inhibition of the ERK phosphatase activity in Hep 3B cell lysate but not the dual specificity ERK phosphatase MKP-1. They also inhibited Cdc25 enzymatic activities and induced tyrosine phosphorylation of the Cdc25 target Cdks. Addition of hydroxyl groups to the naphthoquinone ring thus enhanced the potency of NSC 95397. These two new compounds may be useful probes for the biological functions of Cdc25s and have the potential for disrupting the cell cycle of growing tumor cells.

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a non-quinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 μM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo.

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.