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The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Apoptosis has been shown to be induced in HCC by several agents, including sorafenib as well as by the naturally occurring K vitamins (VKs). As few nontoxic agents have activity against HCC growth, we evaluated the activity of sorafenib and VKs, both independently and together on the growth of HCC cells in vitro and in vivo. We found that when VK was combined with sorafenib, the concentration of sorafenib required for growth inhibition was substantially reduced. Conversely, VK enhanced sorafenib effects in several HCC cell lines on growth inhibition. Growth could be inhibited at doses of VK plus sorafenib that were ineffective with either agent alone, using vitamins K1, K2 and K5. Combination of VK1 plus sorafenib induced apoptosis on FACS, TUNEL staining and caspase activation. Phospho-extracellular signal-regulated kinase (ERK) levels were decreased as was myeloid cell leukemia sequence 1 (Mcl-1), an ERK target. Sorafenib alone inhibited growth of transplantable HCC in vivo. At subeffective sorafenib doses in vivo, addition of VK1 caused major tumor regression, with decreased phospho-ERK and Mcl-1 staining. Thus, combination of VK1 plus sorafenib strongly induced growth inhibition and apoptosis in rodent and human HCC and inhibited the RAF/mitogen-activated protein kinase kinase/ERK pathway. VK1 alone activated PKA, a mediator of inhibitory Raf phosphorylation. Thus, each agent can antagonize Raf: sorafenib as a direct inhibitor and VK1 through inhibitory Raf phosphorylation. As both agents are available for human use, the combination has potential for improving sorafenib effects in HCC., (Copyright © 2010 UICC.)

Unlabelled: Compound 5 (Cpd 5), a K vitamin analog, has been shown to inhibit Hep3B human hepatoma cell growth in cultures and rat hepatoma growth in vivo through prolonged epidermal growth factor receptor (EGFR)-extracellular response kinase (ERK) phosphorylation, and hepatocyte growth factor (HGF) synergizes with Cpd 5 to enhance the inhibition of Hep3B cell and rat hepatoma growth. To explore the mechanisms mediating the HGF/Cpd 5 synergy, we examined the possible involvement of the Grb2-associated binder-1 (Gab1) docking protein because it interacts with both EGFR and HGF receptor c-Met pathways. We found that HGF enhanced Cpd 5-induced c-Met phosphorylation at Tyr-1349, a binding site for Gab1, resulting in increased c-Met binding to Gab1, and induced strong and prolonged Gab1 tyrosine phosphorylation. Prolonged Gab1 phosphorylation by HGF/Cpd 5 in turn enhanced the ability of Gab1 to bind to protein tyrosine phosphatase SHP2 and enhanced the activation of its downstream mitogen-activated protein kinase pathway. In contrast, this same HGF/Cpd 5 treatment inhibited Gab1 binding to phosphatidylinositol 3-kinase (PI3K), leading to the inactivation of the PI3K-Akt pathway. The inhibition of Akt phosphorylation by HGF/Cpd 5 further activated the Raf-MEK-ERK signaling cascade via an Akt-Raf1 interaction, leading to strong and prolonged ERK phosphorylation. The transfection of Hep3B cells with mutated Gab1 (Gab1 Y627F), which had lost its ability to bind SHP2, antagonized HGF/Cpd 5-induced ERK phosphorylation, whereas the transfection of Hep3B cells with mutated Gab1 3YF, which lost its ability to bind PI3K, further enhanced HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition., Conclusion: Gab1 plays a central role in regulating HGF/Cpd 5 synergy in their actions on Hep3B cell growth inhibition.

We previously synthesized several K-vitamin derivatives, which are potent growth inhibitors of human tumor cells, including Hep3B human hepatoma cells. Among these, Cpd 5 was the most potent. However, being a quinone derivative, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a fluorinated derivative of Cpd 5, F-Cpd 5. The calculated reduction potential of F-Cpd 5 was much higher than that for Cpd 5 and it was not predicted to generate ROS. This was supported by our observation that F-Cpd 5 generated significantly lower ROS than Cpd 5. F-Cpd 5 was three times more potent than Cpd 5 in inhibiting Hep3B cell growth. Interestingly, under identical culture conditions, F-Cpd 5 inhibited mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold less potently than Hep3B cells. F-Cpd 5 was found to induce caspase-3 cleavage and nuclear DNA laddering, evidences for apoptosis. It preferentially inhibited the activities of the cell cycle controlling phosphatases Cdc25A and Cdc25B, by binding to their catalytic cysteines. Consequently, inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 were induced. F-Cpd 5 also induced phosphorylation of the MAPK proteins ERK1/2, JNK1/2 and p38 in Hep3B cells and the MAPK inhibitors (U0126, JNKI-II, and SB 203580) antagonized its growth inhibition. F-Cpd 5 inhibited the action of cytosolic ERK phosphatase activity, which likely caused the ERK phosphorylation. F-Cpd 5 thus differentially inhibited growth of normal and tumor cells by preferentially inhibiting the actions of Cdc25A and Cdc25B phosphatases and inducing MAPK phosphorylation.

A systemic vitamin K analog, compound 5 (Cpd 5), possesses the ability to inhibit cell growth of tumor cells. Therefore, we investigated the effect of Cpd 5 in human hepatocellular carcinoma (HCC) cell lines and evaluated its role in apoptosis. Human HCC cell lines were cultured and treated with Cpd 5. Apoptosis was assessed using DAPI staining and Annexin-V membrane staining. The expression of caspases, XIAP and Bcl-xL was also investigated. Cpd 5 decreased cell viability in a dose-dependent manner in two HCC cells (HLE and SK-Hep1) containing mutant p53, but not in the HepG2 cell line, which contained wild-type p53. Cpd 5-treated HLE and SK-Hep1 cells showed typical apoptotic features, nuclear condensation and nuclear fragmentation upon DAPI staining. Positive membranous staining for Annexin-V was also seen in these cells. Both caspase-8 and caspase-3 activities were up-regulated slightly. Pro-caspase-8 protein levels decreased slightly in both cells. Although the expression of Bcl-xL was not influenced by Cpd 5, that of XIAP decreased in HLE cells. However, the pan-caspase inhibitor, zVAD, could not significantly prevent Cpd 5-induced apoptosis and Cpd 5 could not augment TRAIL-induced apoptosis. These results demonstrate that Cpd 5 induced apoptosis in human HCC cell lines, mainly independently of caspase activities. This may contribute to its highly potent cytotoxicity toward HCC cells.

Based on the activity of menadione (M) in the human tumor stem cell assay, we conducted a phase I trial of M in patients with advanced cancer. Forty patients (19 men, 21 women) were treated with 90 courses of M; 82 treatment courses are evaluable for toxicity. The median patient age, Karnofsky performance status, and number of prior chemotherapy regimens were 61 years (range 32-74 years), 80% (range 50-100%), and two, respectively. M was given by a short (1-5 h) intravenous infusion every 3 weeks, starting at 40 mg/m2 and escalating by modified Fibonacci scheme to 1360 mg/m2. Toxicity was graded according to the Southwest Oncology Group toxicity scale with defined hypersensitivity reaction (HSR) scales. No grade > or =2 hematologic toxicity was observed. Non-hematologic toxicity consisted of a HSR syndrome of paresthesiae of the extremities, facial flushing, burning of the eyes and mucous membranes, chest pain and dyspnea. HSR was defined as Grade I toxicity by the presence of facial numbness, flushing, and/or a tingling sensation or burning of the eyes and mucous membranes. Grade II toxicity was defined as the presence of the same above symptoms plus chest tightness, paresthesiae of extremities and/or dyspnea and chest pain. These toxicities were grade 1 in 3 of 4 patients at a dose of 840 mg/m2. At 1360 mg/m2, 2 of 13 patients suffered grade 1 HSR and 7 of 13 grade 2 HSR. No objective partial or complete responses were observed. Plasma menadione concentrations peaked at 1.9-7.4 microM during the infusion in 3 patients receiving 1360 mg/m2. Further phase 1 and 2 combination trials using longer infusion durations have resulted from this trial.

We have previously shown that Compound 5 (Cpd 5), an inhibitor of protein phosphatase Cdc25A, inhibits Hep3B human hepatoma cell growth. We now show that hepatocyte growth factor (HGF), a hepatocyte growth stimulant, can strongly enhance Cpd 5-induced growth inhibition in Hep3B cells, and this enhancement in cell growth inhibition is correlated with a much stronger ERK phosphorylation when compared to cells treated with Cpd 5 or HGF separately. We found that HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition were mediated by Akt (protein kinase B) pathway, since combination HGF/Cpd 5 treatment of Hep3B cells inhibited Akt phosphorylation at Ser-473 and its kinase activity, which led to the suppression of Raf-1 phosphorylation at Ser-259. The suppression of Raf-1 Ser-259 phosphorylation caused the induction of Raf-1 kinase activity, as well as hyper-ERK phosphorylation. Transient transfection of Hep3B cells with dominant negative Akt c-DNA further enhanced both Cpd 5- and HGF/Cpd 5-induced ERK phosphorylation, while over-expression of wild-type Akt c-DNA diminished their effects. In contrast, HGF antagonized the growth inhibitory actions of Cpd 5 on normal rat hepatocytes, thus showing a selective effect on tumor cells compared to normal cells. Our data suggest that Akt kinase negatively regulates MAPK activity at the Akt-Raf level. Suppression of Akt activity by either combination HGF/Cpd 5 treatment or by dominant negative Akt c-DNA transfection antagonizes the Akt inhibitory effect on Raf-1, resulting in an enhancement of Cpd 5-induced MAPK activation and cell growth inhibition., (Copyright 2004 Wiley-Liss, Inc.)

Thioalkyl K vitamin derivatives, like 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone (Cpd 5), have been shown to inhibit both hepatoma cell growth and DNA synthesis in rat hepatocytes in vitro. We have here examined the tissue distribution, in vivo tolerance and growth inhibitory effects of a single injected dose of Cpd 5 in rats. Cpd 5 administered i.p. was sufficient to cause a 90% inhibition of the peak in DNA synthesis in rat liver 24 h after two-thirds partial hepatectomy (PH). However, DNA synthesis in post-PH, Cpd 5-treated rat livers did occur, but with a delay of 36 h. Dual phosphorylation of ERK2 was induced in rat liver dose-dependently as early as 0.5 h, but gradually returned to almost basal levels by 6 h after Cpd 5 treatment. The MEK1/2 inhibitor PD098059, administered in vivo 1 h prior to Cpd 5 treatment, antagonized both induction of ERK2 phosphorylation and inhibition of DNA synthesis in rat liver. Liver protein lysates post-PH exhibited protein phosphatase activity for phospho-ERK2, which was inhibited by Cpd 5. These results show that induction of ERK2 phosphorylation is likely involved in the mechanism by which Cpd 5 inhibits PH-induced DNA synthesis, probably as a result of its ability to inhibit the activity of ERK phosphatase(s).

Background/aims: A synthetic vitamin K analog, compound 5 (Cpd 5), is a potent inhibitor of cell growth. The aim was to investigate whether c-Myc was involved in Cpd 5-induced cell growth inhibition., Methods: Human hepotoma cells (Hep 3B) were cultured and treated with Cpd 5, and c-Myc protein expression and phosphorylation were investigated using Western blot analysis., Results: Cpd 5 was found to inhibit c-Myc protein expression and induce c-Myc phosphorylation in Hep 3B cells. The phosphorylation of c-Myc was induced by both Cpd 5-mediated persistent extracellular signal-regulated kinase (ERK) phosphorylation and Cpd 5 increased glycogen synthase kinase-3 (GSK-3) activity. When using GSK-3 inhibitor, SB216763, c-Myc phosphorylation was significantly decreased and c-Myc levels were restored in Cpd 5 treated cells, suggesting that Cpd 5-mediated increase of GSK-3 activity enhanced c-Myc degradation and resulted in reduction of c-Myc levels. The lower c-Myc levels were found to cause altered expression of two c-Myc target genes, growth arrest gene gadd45 and ornithine decarboxylase (ODC)., Conclusions: The results suggest that Cpd 5-mediated c-Myc phosphorylation resulted in enhanced c-Myc protein degradation and reduced c-Myc protein levels, which may contribute to cell growth inhibition by Cpd 5.

A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304-28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317-1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated cells. PD 49 also bound to protein phosphatases other than Cdc25. We found that the new analogue also inhibited Hep3B human hepatoma cell growth. This growth inhibition involved ERK1/2 phosphorylation and was inhibited by a MEK antagonist. The results demonstrate a direct interaction and binding between this growth-inhibiting K vitamin derivative with both purified as well as with cellular Cdc25A, Cdc25B, and Cdc25C.

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.

The main function of K vitamins is to act as co-factors for gamma-glutamyl carboxylase. However, they have also recently been shown to inhibit cell growth. We have chemically synthesized a series of K vitamin analogs with various side chains at the 2 or 3 position of the core naphthoquinone structure. The analogs with short thio-ethanol side chains are found to be more potent growth inhibitors in vitro of various tumor cell lines. Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone] is one of the most potent. The anti-proliferation activity of these compounds is antagonized by exogenous thiols but not by non-thiol antioxidants. This suggests that the growth inhibition is mediated by sulfhydryl arylation of cellular glutathione and cysteine-containing proteins and not by oxidative stress. The protein tyrosine phosphatases (PTP) are an important group of proteins that contain cysteine at their catalytic site. PTPs regulate mitogenic signal transduction and cell cycle progression. PTP inhibition by Cpd 5 results in prolonged tyrosine phosphorylation and activation of several kinases and transcription factors including EGFR, ERK1/2, and Elk1. Cpd 5 could activate ERK1/2 either by signaling from an activated EGFR, which is upstream in the signaling cascade, or by direct inhibition of ERK1/2 phosphatase(s). Prolonged ERK1/2 phosphorylation strongly correlates with Cpd 5-mediated growth inhibition. Cpd 5 can also bind to and inhibit the Cdc25 family of dual specific phosphatases. As a result, several Cdc25 substrates (Cdk1, Cdk2, Cdk4) involved in cell cycle progression are tyrosine phosphorylated and thereby inhibited by its action. Cpd 5 could also inhibit both normal liver regeneration and hepatoma growth in vivo. DNA synthesis during rat liver regeneration following partial hepatectomy, transplantable rat hepatoma cell growth, and glutathione-S-transferase-pi expressing hepatocytes after administration of the chemical carcinogen diethylnitrosamine, are all inhibited by Cpd 5 administration. The growth inhibitory effect during liver regeneration and transplantable tumor growth is also correlated with ERK1/2 phosphorylation induced by Cpd 5. Thus, Cpd 5-mediated inhibition of PTPs, such as Cdc25 leads to cell growth arrest due to altered activity of key cellular kinases involved in signal transduction and cell cycle progression. This prototype K vitamin analog represents a novel class of growth inhibitor based upon its action as a selective PTP antagonist. It is clearly associated with prolonged ERK1/2 phosphorylation, which is in contrast with the transient ERK1/2 phosphorylation induced by growth stimulatory mitogens., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2002 Wiley-Liss, Inc.)

The K-vitamin analog Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone] is a potent cell growth inhibitor in vitro and in vivo, likely due to arylation of enzymes containing a catalytic cysteine. This results in inhibition of protein tyrosine phosphatase (PTPase) activity with resultant hyperphosphorylation of EGF receptors (EGFR) and ERK1/2 protein kinases, which are downstream to EGFR in the MAPK pathway. We used NR6 fibroblast cells, which lack endogenous EGFR and its variant cells transfected with different EGFR mutants to assess the contribution of the EGFR-mediated signaling pathway to Cpd 5-mediated ERK activation and cell growth inhibition. Cpd 5 treatment resulted in enhanced phosphorylation of EGFR at carboxyl-terminal tyrosines. This phosphorylation and activation of EGFR were found to be necessary neither for growth inhibition nor for the activation of the downstream kinases ERK1/2, since both occurred in EGFR-devoid mutant cells. U0126 and PD 098059, specific inhibitors of MEK1/2, the ERK1/2 kinases, antagonized both cell growth inhibition and ERK1/2 phosphorylation mediated by Cpd5. Cpd 5 was also found to inhibit ERK1/2 phosphatase(s) activity in lysates from all the cells tested, irrespective of their EGFR status. These results show that EGFR-independent ERK1/2 phosphorylation was involved in the mechanism of Cpd5 mediated growth inhibition. This is likely due to the observed antagonism of ERK phosphatase activity. A candidate PTPase was found to be Cdc25A, a recently identified ERK phosphatase., (Copyright 2002 Wiley-Liss, Inc.)

Background/aims: Compound 5 (Cpd 5), a vitamin K analog, inhibits rat hepatocyte DNA synthesis and hepatoma cell growth. The aim of this study was to determine if the inhibitory effect of Cpd 5 on cell growth was related to apoptosis., Methods: Isolated rat hepatocytes were cultured with Cpd 5, and mitogen-activated signaling pathway and apoptosis pathway were investigated using Western blot analysis., Results: When rat hepatocytes were cultured with Cpd 5 for 48 h, apoptosis was evident, which included characteristic morphological changes, DNA fragmentation, and the activation of caspase 3 (CPP 32)-like protease. Examination of upstream events of apoptosis pathway showed that the expression of Bax was induced and bcl-2 was inhibited by Cpd 5 treatment. Concomitant with the induction of apoptosis, Cpd 5 activated the extracellular signal-regulated kinase (ERK) signaling pathway. PD 98059, a mitogen-activated protein kinase kinase inhibitor, and glutathione, an anti-thiol-oxidant, not only blocked Cpd 5-induced ERK phosphorylation, but also antagonized the activation of CPP-32, the altered Bcl-2/Bax expression, and DNA fragmentation., Conclusions: The data suggest that the ERK signaling pathway may be involved in the regulation of rat hepatocyte apoptosis induced by Cpd 5.

2-(2-hydroxy-ethylsulfanyl)-3-methyl-1,4-naphthoquinone or CPD-5, a K vitamin analog, was previously indicated to be a potent growth inhibitor for Hep 3B hepatoma cells in vitro. Here, we show that CPD-5 and two newly synthesized analogs, 2-(2-hydroxy-ethylsulfanyl)-3-methyl-5- nitro-1,4-naphthoquinone (PD-37) and 2-(2-hydroxy-ethylsulfanyl)-3- methyl-5-acetylamino-1,4-naphthoquinone (PD-42), are potent growth inhibitors of 13 different human cancer cell lines, with IC50 values in the range of 3-54 microM. Phospho-ERK was induced by each of three K vitamin analogs in every cell line in a dose-dependent manner, at growth inhibitory doses. ERK phosphorylation and growth inhibitory effects were strongly correlated, with p=0.0080 for CPD-5, p=0.0076 for PD-37 and p=0.0251 for PD-42. The induction of phospho-ERK and growth inhibition were antagonized by thiol-containing anti-oxidants, but not by catalase, consistent with a possible arylating mechanism. The data show a novel class of growth inhibitors with a wide spectrum of action that induces ERK hyper-phosphorylation, as a possible new growth inhibitory feature., (Copyright 2001 Academic Press.)

We previously found that K vitamin analogues caused cell growth inhibition in Hep3B hepatoma cells in vitro, which was associated with their inhibitory effects on protein tyrosine-phosphatases. In this study, we show that Cdc25A, a protein phosphatase, was inactivated by novel arylating K vitamin analogues. The inactivation of Cdc25A correlated with their effects on cell growth inhibition. Cyclin-dependent kinase (Cdk) 4, an important regulator for G(1) progression, was found to be tyrosine-phosphorylated by the arylating analogues, and this phosphorylation was correlated with the inhibitory effects of the analogues on Cdc25A activity. Furthermore, Cdk4 dephosphorylation experiments showed that Compound (Cpd) 5, a prototype arylating analogue, inhibited Cdc25A-mediated Cdk4 dephosphorylation, whereas Cpd 26, a nonarylating vitamin K analogue, had no effect on this event. We also examined Cdk4 kinase activity using retinoblastoma protein as a substrate and found that Cpd 5 inhibited retinoblastoma protein phosphorylation in a concentration-dependent manner, indicating that Cdk4 activity was inhibited by Cpd 5 treatment. Moreover, the thiol-antioxidants glutathione and N-acetyl-L-cysteine antagonized the Cpd 5-induced Cdk4 tyrosine phosphorylation, whereas the nonthiol-antioxidants catalase and superoxide dismutase did not. These results suggest that Hep3B cell growth inhibition by these K vitamin analogues may be related in part to inactivation of Cdc25A activity and support the hypothesis that Cdc25A is an attractive target for drugs designed to inhibit cancer cell growth.

Background/aims: To understand the mechanisms of liver regeneration or hepatoma apoptosis, it is important to estimate the turning point of the signal transduction by growth factor receptor. Since 2-(2-hydroxyethylsulfaryl) 3-methyl-1,4-naphthoquinone or CPD 5 has been shown to mediate the phosphorylation of epidermal growth factor (EGF) receptor in Hep3B hepatoma cells, the differences between EGF and CPD 5-mediated signal transduction were studied., Methods: DNA content was measured by Hoechst fluorescent assay. Phosphorylated proteins were described with Western blots or two-dimensional electrophoresis., Results: CPD 5-induced EGFR phosphorylation was functional to stimulate Ras pathway. However, CPD 5-mediated extracellular signal-regulated kinase (ERK) phosphorylation was not antagonized by inhibition of upstream activation with PD153035. CPD 5 inhibited ERK dephosphorylation in cell lysate, suggesting that ERK phosphorylation by CPD 5 was depending on kinase activity and phosphatase inhibition. Two-dimensional electrophoresis showed extra phospho ERK spot, which was indicated to have close association with CPD 5-induced growth inhibition, since U0126 antagonized growth inhibition and appearance of this spot., Conclusions: The turning point of EGFR pathway was proved to have close association with the expressed level of phosphorylated ERK. ERK phosphorylation was suggested to play a critical role in growth factor-induced signal transduction.

We have now found that the most potent, Cpd 5 [2-(2-mercaptoethanol)-3-methyl-1, 4-napthoquinone], inhibits growth of doxorubicin-resistant and doxorubicin-sensitive breast cancer cells (MCF 7r and MCF 7w) in culture. Growth inhibition by Cpd 5 was antagonized by the thiol antioxidants glutathione and cysteine, but not by catalase or superoxide dismutase, suggesting that growth inhibition is probably via conjugation of cellular thiols. In support of this, we found that Cpd 5 inhibited the activity of thiol containing cellular protein tyrosine phosphatase (PTP) enzyme, with consequent induction of various tyrosine phosphoproteins, but not serine or tyrosine phosphoproteins. The tyrosine phosphorylation was also inhibited by exogenous glutathione or cysteine and could be enhanced by depletion of cellular glutathione by BSO. This effect of Cpd 5 on protein tyrosine phosphorylation was highly selective, however. Tyrosine phosphorylation of EGF-R, Erb-B2, and ERK1/2 was increased, but not that of Insulin-R or JNK. ERK1/2 tyrosine phosphorylation and growth inhibition increased with increasing concentrations of Cpd 5. Furthermore, suppression of Cpd 5-mediated ERK1/2 phosphorylation by an ERK-kinase inhibitor antagonized growth inhibition. These results suggest a strong correlation between ERK1/2 phosphorylation by Cpd 5 and growth inhibition. This novel K-vitamin analog thus inhibits MCF 7 cell growth and induces selective protein tyrosine phosphorylation., (Copyright 2000 Wiley-Liss, Inc.)

In the present study, we show that 2-(2-hydroxyethylsulfaryl)-3-methyl-1,4-naphthoquinone, or CPD 5, is a potent growth inhibitor for pancreas cancer cell lines (ID(50): 21.4 +/- 3.8, 31.8 +/- 2.7 and 55.2 +/- 4.5 microM for MiaPaCa, Panc-1 and BxPc3, respectively). It induced protein tyrosine phosphor-ylation of hepatocyte growth factor (HGF) receptor (c-Met) or epidermal growth factor receptor (EGFR), which increased progressively to a maximum level at 30 min in Panc-1 cells. The receptor phosphorylation by CPD 5 was indicated to be functional, since these receptors were found to bind with Grb2 or SOS1 protein. CPD 5 was also suggested to induce phosphorylation of external signal-regulated kinase (ERK). EGF induced cell proliferation through ERK phosphorylation, since U0126, which is an inhibitor of ERK phosphorylation, abrogated the increase of cyclin D1 by EGF. HGF increased the amount of p27 protein, suggesting that it is associated with cell differentiation. By contrast, U0126 reduced CPD 5-induced cell death. On two-dimensional electrophoresis, we found an extra type of phospho-ERK, and this was completely and selectively abolished by U0126. These results suggest that ERK phosphorylation, especially the extra spot on two-dimensional gel, is critically associated with CPD 5-mediated cell death.

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., (Copyright 2000 Wiley-Liss, Inc.)

We recently found that a thioether analog of K vitamin (Cpd 5) inhibited the activity of protein-tyrosine phosphatases (PTPases) and induced protein-tyrosine phosphorylation in a human hepatoma cell line (Hep3B). We have now examined the structural requirements for induction of protein-tyrosine phosphorylation and PTPase inhibition by several K vitamin analogs. Thioether analogs with sulfhydryl arylation capacity, especially those with a hydroxy (Cpd 5) or a methoxy group at the end of the side chain, induced protein-tyrosine phosphorylation, but non-arylating analogs, such as those with an all-carbon or O-ether side chain, did not. Among the receptor-tyrosine kinases, epidermal growth factor receptors were tyrosine-phosphorylated by treatment with thioether analogs, whereas insulin and hepatocyte growth factor receptors were not. An increase in tyrosine-phosphorylated ERK2 mitogen-activated protein kinase was also observed. The activity of purified T cell PTPase was inhibited only by the thioether analogs, but not by non-arylating analogs. Furthermore, the epidermal growth factor receptor dephosphorylation activity of Hep3B cell lysates was inhibited by Cpd 5 treatment. A similar induction of protein-tyrosine phosphorylation by Cpd 5 was seen in other human hepatoma cell lines together with growth inhibition. However, one cell line (HepG2), which was relatively resistant to growth inhibition by Cpd 5, did not increase its phosphorylation levels upon Cpd 5 treatment. These results suggest that cell growth inhibition by thioether analogs is closely associated with inhibition of PTPases by sulfhydryl arylation and with tyrosine phosphorylation of selected proteins.

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Congeners of vitamin K are known to inhibit cell growth, although the precise mechanisms of growth inhibition are not well understood. To investigate the mechanisms involved, we synthesized several vitamin K analogs and examined their growth inhibitory activities for a human hepatoma cell line (Hep3B). The analogs included 2-methyl-1,4-naphthoquinone and trimethyl-benzoquinone, with and without aliphatic side chains at position 3. The side chains were all-carbon, thioethers, or O-ethers. Growth inhibition was potent in the compounds with short chains. The presence of a sulfur (thioether) or oxygen atom (O-ether) at the site of attachment of the side chain to the ring potentiated the activity. Apoptotic cell death was induced by the potent growth inhibitory compounds at low concentrations (20-60 microM), whereas necrotic cell death followed treatment with the same compounds at high concentrations. Expression of c-myc, which is thought to be associated with apoptosis, was increased by most of the compounds tested. Both reduced glutathione and cysteine almost completely abrogated the growth inhibitory effects of the thioether analogs as well as of vitamin K3. The effect of glutathione was less prominent for the all-carbon and O-ether analogs, and cysteine had no effect on these analogs. Catalase and deferoxamine mesylate had no significant effect on the thioether analogs, although they showed partial antagonistic effects on the growth inhibition of vitamin K3 and the all-carbon and O-ether analogs. Other non-thiol antioxidants tested had no effect on any of the analogs. Our results indicated that vitamin K-related quinoid compounds cause growth inhibition and both apoptotic and necrotic cell death and that the effects may be mediated by interaction at position 3 of their quinoid nuclei with cellular thiols.

A characteristic defect occurs in rat and human hepatocellular carcinoma (HCC) resulting in a loss of function of the vitamin K-dependent enzyme gamma-glutamyl-carboxylase in the tumor but not in the underlying liver. This causes the secretion of elevated levels of the immature or des-gamma-carboxylated form of prothrombin, which is used as a marker of HCC. We investigated whether, using the defined conditions of growing HCC cell lines in tissue culture, addition of the naturally occurring vitamins K1 or K2 or the synthetic vitamin K3 could influence the secretion of immature prothrombin. We found that vitamins K1, K2 and K3 all suppressed the secretion of immature prothrombin into the tissue culture medium. Vitamins K2 and K3 were also found to inhibit growth of the HCC cell line, in an apparently nontoxic and reversible manner. The influence of the vitamins K on the expression of some genes related to vitamin K action was examined and compared with that of another growth inhibitor, TGF beta 1 protein. The vitamins K were found to increase the expression of prothrombin and carboxylase messenger RNA and c-myc messenger RNA, but had no effects on the expression of TGF beta 1 messenger RNA. By contrast, TGF beta 1 increased TGF beta 1 messenger RNA levels, but had no effects on the other genes, suggesting a different pathway. The previously studied vitamin K3-mediated inhibition of growth was antagonized by the addition of catalase to the culture medium, but the inhibitory effects of vitamin K2 were not antagonized.(ABSTRACT TRUNCATED AT 250 WORDS)