Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

BACKGROUND: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents. METHODS: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation. RESULTS: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNß-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model. CONCLUSIONS: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

A chemical biology approach identifies a beta-2 adrenergic receptor agonist that causes human tumor regression by blocking the Raf-1/Mek-1/Erk1/2 pathway.

A chemical biology approach identifies a beta 2 adrenergic receptor (beta2AR) agonist ARA-211 (Pirbuterol), which causes apoptosis and human tumor regression in animal models. beta2AR stimulation of cAMP formation and protein kinase A (PKA) activation leads to Raf-1 (but not B-Raf) kinase inactivation, inhibition of Mek-1 kinase and decreased phospho-extracellular signal-regulated kinase (Erk)1/2 levels. ARA-211 inhibition of the Raf/Mek/Erk1/2 pathway is mediated by PKA and not exchange protein activated by cAMP (EPAC). ARA-211 is selective and suppresses P-Erk1/2 but not P-JNK, P-p38, P-Akt or P-STAT3 levels. beta2AR stimulation results in inhibition of anchorage-dependent and -independent growth, induction of apoptosis in vitro and tumor regression in vivo. beta2AR antagonists and constitutively active Mek-1 rescue from the effects of ARA-211, demonstrating that beta2AR stimulation and Mek kinase inhibition are required for ARA-211 antitumor activity. Furthermore, suppression of growth occurs only in human tumors where ARA-211 induces cAMP formation and decreases P-Erk1/2 levels. Thus, beta2AR stimulation results in significant suppression of malignant transformation in cancers where it blocks the Raf-1/Mek-1/Erk1/2 pathway by a cAMP-dependent activation of PKA but not EPAC.

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter.

Recently, we have shown that RhoB suppresses EGFR-, ErbB2-, Ras- and Akt-mediated malignant transformation and metastasis. In this paper, we demonstrate that the novel antitumor agents farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase I inhibitors (GGTIs) upregulate RhoB expression in a wide spectrum of human cancer cells including those from pancreatic, breast, lung, colon, bladder and brain cancers. RhoB induction by FTI-277 and GGTI-298 occurs at the transcriptional level and is blocked by actinomycin D. Reverse transcription-PCR experiments documented that the increase in RhoB protein levels is due to an increase in RhoB transcription. Furthermore, treatment with FTIs and GGTIs of cancer cells results in HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter. Thus, promoter acetylation is a novel mechanism by which RhoB expression levels are regulated following treatment with the anticancer agents FTIs and GGTIs.

p21(WAF1/CIP1) is upregulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor beta- and Sp1-responsive element: involvement of the small GTPase rhoA.

We have recently reported that the geranylgeranyltransferase I inhibitor GGTI-298 arrests human tumor cells at the G1 phase of the cell cycle and increases the protein and RNA levels of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Here, we show that GGTI-298 acts at the transcriptional level to induce p21(WAF1/CIP1) in a human pancreatic carcinoma cell line, Panc-1. This upregulation of p21(WAF1/CIP1) promoter was selective, since GGTI-298 inhibited serum responsive element- and E2F-mediated transcription. A functional analysis of the p21(WAF1/CIP1) promoter showed that a GC-rich region located between positions -83 and -74, which contains a transforming growth factor beta-responsive element and one Sp1-binding site, is sufficient for the upregulation of p21(WAF1/CIP1) promoter by GGTI-298. Electrophoretic mobility shift assays showed a small increase in the amount of DNA-bound Sp1-Sp3 complexes. Furthermore, the analysis of Sp1 transcriptional activity in GGTI-298-treated cells by using GAL4-Sp1 chimera or Sp1-chloramphenicol acetyltransferase reporter revealed a significant increase in Sp1-mediated transcription. Moreover, GGTI-298 treatment also resulted in increased Sp1 and Sp3 phosphorylation. These results suggest that GGTI-298-mediated upregulation of p21(WAF1/CIP1) involves both an increase in the amount of DNA-bound Sp1-Sp3 and enhancement of Sp1 transcriptional activity. To identify the geranylgeranylated protein(s) involved in p21(WAF1/CIP1) transcriptional activation, we analyzed the effects of the small GTPases Rac1 and RhoA on p21(WAF1/CIP1) promoter activity. The dominant negative mutant of RhoA, but not Rac1, was able to activate p21(WAF1/CIP1). In contrast, constitutively active RhoA repressed p21(WAF1/CIP1). Accordingly, the ADP-ribosyl transferase C3, which specifically inhibits Rho proteins, enhanced the activity of p21(WAF1/CIP1). Taken together, these results suggest that one mechanism by which GGTI-298 upregulates p21(WAF1/CIP1) transcription is by preventing the small GTPase RhoA from repressing p21(WAF1/CIP1) induction.

The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis.

Farnesyltransferase inhibitors (FTIs) represent a novel class of anticancer drugs that exhibit a remarkable ability to inhibit malignant transformation without toxicity to normal cells. However, the mechanism by which FTIs inhibit tumor growth is not well understood. Here, we demonstrate that FTI-277 inhibits phosphatidylinositol 3-OH kinase (PI 3-kinase)/AKT2-mediated growth factor- and adhesion-dependent survival pathways and induces apoptosis in human cancer cells that overexpress AKT2. Furthermore, overexpression of AKT2, but not oncogenic H-Ras, sensitizes NIH 3T3 cells to FTI-277, and a high serum level prevents FTI-277-induced apoptosis in H-Ras- but not AKT2-transformed NIH 3T3 cells. A constitutively active form of AKT2 rescues human cancer cells from FTI-277-induced apoptosis. FTI-277 inhibits insulin-like growth factor 1-induced PI 3-kinase and AKT2 activation and subsequent phosphorylation of the proapoptotic protein BAD. Integrin-dependent activation of AKT2 is also blocked by FTI-277. Thus, a mechanism for FTI inhibition of human tumor growth is by inducing apoptosis through inhibition of PI 3-kinase/AKT2-mediated cell survival and adhesion pathway.

Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice.

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGbeta (IC50 values > 100 microM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 microM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor-binding molecules with potent anticancer and antiangiogenic activity.

Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression.

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src(-/-) bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.

Species- and length of exposure-dependent differences in the benzo(a)pyrene:DNA adducts formed in embryo cell cultures from mice, rats, and hamsters.

The activation of benzo(a)pyrene (BaP) to DNA-binding metabolites in early-passage embryo cell cultures prepared from various species of rodents was investigated by exposing cells from mice (BALB/c and Sencar), rats (Wistar and Fischer 344), and Syrian hamsters to [3H]BaP for various lengths of time. The BaP:DNA adducts containing cis-vicinal hydroxyl groups such as those formed from 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) were separated from the other types of BaP:DNA adducts by immobilized boronate chromatography, and the individual adducts were analyzed by high-performance liquid chromatography. A number of BaP:DNA adducts were present in the DNA from the cultures from all three species after 5 h of BaP treatment. After a 24-h exposure to BaP, the mouse and hamster embryo cell DNA contained a large amount of the adduct formed by reaction of (+)-anti-BaPDE with the 2-amino group of deoxyguanosine (dGuo) and a small amount of a 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:dGuo adduct. A large number of BaP:DNA adducts derived from 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and other unidentified BaP metabolites were present in rat embryo cell cultures at all times. Neither the Fischer 344 nor the Wistar rat embryo cell cultures had a significant amount of (+)-anti-BaPDE:dGuo adduct after 5 h of BaP treatment, and in the Wistar rat cells larger amounts of other adducts were present even after a 96-h exposure to BaP. In cell cultures from all three species the proportion of (+)-anti-BaPDE:dGuo adduct increased as the length of time of exposure to BaP increased. There are major differences in the metabolic activation of BaP to DNA binding metabolites in embryo cells from various species of rodents. However, the variations between cell cultures from different strains of rats or mice are not as great as the variations between cell cultures from different species. The time-dependent alterations in the BaP:DNA adducts indicate that analysis after various lengths of time of exposure to BaP is essential to characterize accurately the pathways of metabolic activation of BaP in cells from various species and tissues.

Ras CAAX peptidomimetic FTI 276 selectively blocks tumor growth in nude mice of a human lung carcinoma with K-Ras mutation and p53 deletion.

Farnesylation of the oncoprotein Ras is required for its cancer-causing activity. We have designed farnesyltransferase inhibitor (FTI)-276, a tetrapeptide mimetic of the carboxyl terminus of K-Ras4B, as a highly potent and selective inhibitor of Ras farnesylation in vitro and in vivo. FTI-276 blocked the growth in nude mice of a human lung carcinoma that expresses the two most prevalent genetic alterations in human cancers (K-Ras oncogenic mutation and deletion in the tumor suppressor gene p53). In contrast, FTI-276 did not inhibit tumor growth of a human lung carcinoma that harbors no Ras mutations. Furthermore, FTI-276 inhibited oncogenic signaling and tumor growth of NIH 3T3 cells transformed with the ras but not the raf oncogene. Inhibition of tumor growth in vivo was dose dependent and correlated with inhibition of Ras processing in tumors in vivo. The work described here identifies FTI-276 as a highly selective suppressor of Ras-dependent oncogenicity and suggests that a broad spectrum of human cancers with aberrant Ras function could benefit from farnesyltransferase inhibitor treatment.

Analysis of benzo(a)pyrene:DNA adducts formed in cells in culture by immobilized boronate chromatography.

A chromatographic procedure using boronic acid residues linked to a cellulose support [(N-(N'-[m-(dihydroxyboryl)-phenyl]succinamyl)amino]ethyl cellulose), used by Sawicki et al. (Cancer Res., 43: 3212-3218, 1983) for analysis of 7,12-dimethylbenz(a)anthracene:DNA adducts, was modified to allow the analysis of benzo(a)pyrene (BaP):DNA adducts formed in cells in culture. Adducts resulting from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) contain cis-vicinal hydroxyl groups that complex with the boronic acid residues; adducts resulting from 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BaPDE) do not. A mixture of [3H]-syn-BaPDE:deoxyguanosine (dGuo) adduct and [14C]-anti-BaPDE:dGuo adduct was completely resolved on a column of boronate:cellulose. Early-passage cultures of Sencar mouse, Syrian hamster, and Wistar rat embryo cells and a culture of a human hepatoma cell line (Hep G2) were exposed to [3H]BaP, and the BaP:DNA adducts were resolved by boronate chromatography and high-performance liquid chromatography. The Hep G2 cells and mouse embryo cells contained two major adducts, a (+)-anti-BaPDE:dGuo adduct and a syn-BaPDE:dGuo adduct. Boronate chromatography permitted the resolution of an additional minor syn-BaPDE:deoxyribonucleoside adduct in the mouse embryo cells. The hamster and rat embryo cells contained a number of major BaP-DNA adducts that were resolved by boronate chromatography followed by high-performance liquid chromatography. The rat embryo cells contained three syn-BaPDE:deoxyribonucleoside adducts and approximately equal amounts of two adducts tentatively identified as dGuo adducts of the (+) and (-) enantiomers of anti-BaPDE. The boronate chromatography-high-performance liquid chromatography procedure improves the separation of the BaP:DNA adducts formed in biological systems and facilitates the identification of the BaP metabolite(s) responsible for the formation of these adducts.

In vivo circumvention of human colon carcinoma resistance to bleomycin.

Metabolic inactivation of bleomycin (BLM) by cysteine proteinase-like enzymes is thought to be a major mechanism of BLM tumor resistance. We now report that the human colon carcinoma COLO-205 is highly resistant to BLM and that E-64, a cysteine proteinase inhibitor, sensitizes COLO-205 to BLM. Treatment of COLO-205-bearing nude mice with either E-64 (40 mg/kg) or BLM (10 mg/kg) alone did not inhibit COLO-205 growth. However, pretreatment with E-64 prior to BLM prevented these xenografts from growing. Analysis by high performance liquid chromatography of in vivo BLM metabolism following [3H]BLM A2 treatment of COLO-205-bearing nude mice showed a different metabolic profile among the various organs and the tumor. Whereas [3H]BLM A2 was the only major radioactive peak detected in sera and tumors, several metabolites, including deamido-BLM A2, were found in kidney, liver, and lung as early as 15 min. Pretreatment of mice with E-64 inhibited tumor, kidney, and lung BLM A2 metabolism. Furthermore, pretreatment with E-64 increased BLM A2 accumulation in tumors (6.1-fold), kidney (4.0-fold), lung (2.8-fold), liver (1.8-fold), and serum (1.7-fold). E-64 pretreatment did not enhance the major toxicity of BLM, pulmonary fibrosis, as determined by both lung hydroxyproline levels and histopathology. Thus, the cysteine proteinase inhibitor E-64 affects the metabolic fate and the levels of accumulation of BLM in vivo. These results demonstrate that resistance of human COLO-205 tumors to BLM can be circumvented by E-64 without enhancement of the major side effect of BLM, suggesting a possible clinical use of this combination therapy.

GGTI-298 induces G0-G1 block and apoptosis whereas FTI-277 causes G2-M enrichment in A549 cells.

The mechanism by which the geranylgeranyltransferase I inhibitor GGTI-298 and the farnesyltransferase inhibitor FTI-277 inhibit human tumor growth is not known. Herein, we demonstrate that in the human lung adenocarcinoma A549 cells, GGTI-298 induced a G1-G0 block whereas FTI-277 induced an enrichment in the G2-M phase of the cell cycle. Although FTI-277, GGTI-298, and compactin inhibited A549 cell growth, only GGTI-298 and compactin induced apoptosis as demonstrated by four criteria: 4',6-diamidine-2-phenylindoledihydrochloride staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, DNA fragmentation assay, and flow cytometry. Furthermore, the involvement of geranylgeranylated proteins in apoptotic pathways was confirmed by demonstrating that geranylgeraniol was able to block the ability of compactin to induce apoptosis. These results suggest that protein geranylgeranylation is critical for the control of programmed cell death and that, in A549 cells, farnesylated and geranylgeranylated proteins are involved in G2-M and G0-G1, respectively.

Metabolic inactivation: a mechanism of human tumor resistance to bleomycin.

The mechanism of human tumor resistance to the antineoplastic drug bleomycin (BLM) is not known. We now provide evidence implicating metabolic inactivation in the resistance of human Burkitt's (Daudi) lymphoma to BLM. Daudi lymphoma and human head and neck squamous cell carcinoma (A-253) cells grown (s.c.) in nude mice were found to be resistant and sensitive to BLM treatment, respectively. Within 1 h of s.c. injection of [3H]BLM A2, Daudi xenografts accumulated less BLM and metabolized this drug to a much greater extent than did A-253 xenografts. The BLM-resistant Daudi xenografts metabolized BLM A2 to at least 6 metabolites and only a small proportion of the drug remained as unmetabolized BLM A2. In the BLM-sensitive A-253 xenografts, however, BLM A2 remained the major component. Incubation of BLM A2 with Daudi cytosolic fractions resulted in a complex mixture of metabolites similar to that formed by Daudi xenografts in nude mice. This BLM metabolite mixture was biologically inactive in plasmid DNA degradation assays. Treatment of mice bearing Daudi xenografts with an inhibitor of BLM hydrolase, L-trans-epoxysuccinyl-leucylamido-(4-guanidino)butane (E-64), prior to [3H]BLM A2 treatment did not affect the amount of BLM accumulated but inhibited BLM A2 metabolism in the xenografts. Furthermore, although E-64 alone did not inhibit the growth of Daudi xenografts, it potentiated the antitumor activity of BLM. These results indicate that Daudi tumors resist BLM by metabolically inactivating it and that inhibition of BLM metabolism in vivo enhances the antitumor activity of BLM and hence overcomes resistance.

The farnesyltransferase inhibitor FTI-277 radiosensitizes H-ras-transformed rat embryo fibroblasts.

Many tumor cells have a greater resistance to ionizing radiation than their normal counterparts, suggesting that the development of drugs that can reduce that radioresistance would potentiate the efficacy of radiation therapy. Because activated H-ras expression has been shown to markedly increase radiation resistance in some transformed cells, the inactivation of H-ras would then be predicted to radiosensitize these tumor cells, while leaving normal cells unaffected. H-ras depends for activity upon farnesylation, which can be blocked by farnesylation inhibitors, including the compound FTI-277. In keeping with this prediction, inhibition of H-ras processing using FTI-277 resulted in higher levels of apoptosis after irradiation and increased radiosensitivity in H-ras-transformed rat embryo cells but did not affect control cells. These experiments suggest that farnesylation inhibitors may prove clinically useful as radiosensitizers of tumors that depend on ras function.

Synthesis and evaluation of fluoromycin: a novel fluorescence-labeled derivative of talisomycin S10b.

We have synthesized fluoromycin (FLM), a novel fluorescein-labeled derivative of talisomycin S10b (TLM S10b), and used it to evaluate cellular drug accumulation and distribution in bleomycin (BLM)-sensitive and -resistant cell lines. The fluorescence intensity of FLM was 300- to 400-fold greater than that of BLM A2, TLM S10b, or the lipophilic BLM analogue, liblomycin. FLM possessed an antiproliferative potency similar to liblomycin in BLM-sensitive human A-253 squamous carcinoma cells but was less potent than BLM A2 or TLM S10b. C-10E cells, a clone of A-253 cells with 40- to 50-fold resistance to BLM A2 and TLM S10b, were 50-fold resistant to FLM. A partially revertant cell population (C-10E ND) regained sensitivity to BLM A2, TLM S10b, and FLM. FLM like BLM cleaved pGEM-3Z plasmid DNA in vitro in a concentration-dependent manner. Flow cytometric analysis of FLM content in C-10E and C-10E ND cell lines showed 4-fold and 2-fold lower fluorescence intensity, respectively, compared with A-253 cells. Similar results were seen by fluorescence spectrophotometry with cell extracts. Fluorescence microscopy indicated heterogeneous distribution among A-253 cells with at least 50% of the cells exhibiting marked nuclear fluorescence localization. In contrast, C-10E cells displayed lower cellular fluorescence and predominantly cytoplasmic localization. C-10E ND cells exhibited a mixed population of nuclear and cytoplasmic vesicular localization with fluorescence levels that were intermediate between A-253 and C-10E cells. Thus, BLM-resistant cells have reduced levels of FLM and appear to have a lower nuclear:cytoplasmic ratio of FLM. FLM may be useful in studying the intracellular fate of BLM-like drugs as well as providing a tool to detect and isolate BLM-resistant cells.

Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine.

Ras malignant transformation requires posttranslational modification by farnesyltransferase (FTase). Here we report on the design and antitumor activity, in monotherapy as well as in combination therapy with cytotoxic agents, of a novel class of non-thiol-containing peptidomimetic inhibitors of FTase and the closely related family member geranylgeranyltransferase I (GGTase I). The non-thiol-containing FTI-2148 is highly selective for FTase (IC50, 1.4 nM) over GGTase I (IC50, 1700 nM), whereas GGTI-2154 is highly selective for GGTase I (21 nM) over FTase (IC50, 5600 nM). In whole cells, the corresponding methylester prodrug FTI-2153 is >3000-fold more potent at inhibiting H-Ras (IC50, 10 nM) than Rap1A processing, whereas GGTI-2166 is over 100-fold more selective at inhibiting Rap1A (IC50, 300 nM) over H-Ras processing. Furthermore, FTI-2153 was highly effective at suppressing oncogenic H-Ras constitutive activation of mitogen-activated protein kinase and human tumor growth in soft agar. FTI-2148 suppressed the growth of the human lung adenocarcinoma A-549 cells in nude mice by 33, 67, and 91% in a dose-dependent manner. Combination therapy of FTI-2148 with either cisplatin, gemcitabine, or Taxol resulted in a greater antitumor efficacy than monotherapy. GGTI-2154 in similar antitumor efficacy experiments is less potent than FTI-2148 and inhibits tumor growth by 9, 27, and 46%. Combination therapy of GGTI-2154 with cisplatin, gemcitabine, or Taxol is also more effective. Finally, FTI-2148 and GGTI-2154 are 30- and 33-fold more selective and 30- and 16-fold more potent in whole cells than our previously reported thiol-containing FTI-276 and GGTI-297, respectively. Thus, our results demonstrate that this highly potent and selective novel class of non-thiol-containing peptidomimetics inhibits human tumor growth in whole animals and that combination therapy with cytotoxic agents is more beneficial than monotherapy.

Inhibiting Ras prenylation increases the radiosensitivity of human tumor cell lines with activating mutations of ras oncogenes.

The influence of activated ras oncogenes on the sensitivity of human tumor cells to killing by radiation has been an unresolved question in radiobiology. We have examined this question by measuring the radiation sensitivity of human tumor cell lines with oncogenic mutations in their H- or K-ras genes after treatment with prenyltransferase inhibitors that prevent the posttranslational modification of ras required for its activity. Using two measures of clonogenic survival, we have demonstrated radiosensitization in cell lines with oncogenic H-ras mutations or with oncogenic K-ras mutations when ras processing was inhibited by prenyltransferase inhibitor treatment. In contrast, the inhibition of ras processing in cell lines expressing wild-type ras had no effect on radiation-induced cell death. The prenyltransferase inhibitors themselves inhibited clonogenic survival in some cases, but this inhibition did not correlate with ras mutational status. Although treatment with prenyltransferase inhibitors and radiation resulted in a greater reduction of clonogenicity than either treatment alone in cells with wild-type ras, treatment with both agents had a synergistic effect on cell killing in tumor cells with ras mutations. Our results demonstrate that the inhibition of oncogenic ras activity in human tumor cells can reduce the radiation survival of these cells, suggesting that oncogenic ras can contribute to radiation resistance in human tumors. These results further demonstrate the potential of using prenyltransferase inhibitors in combination with radiotherapy in the treatment of human malignancies.

CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and p27KIP1 expression and apoptosis and inhibits the growth of the human lung adenocarcinoma A-549 in nude mice.

The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH-(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.

Design of growth factor antagonists with antiangiogenic and antitumor properties.

This review describes our recent efforts in the development of novel therapies for cancer. Our primary approach is to design synthetic agents that antagonize the function of growth factors that are critically involved in oncogenesis and angiogenesis. We achieve this by designing synthetic molecules that can recognize the exterior surface of the growth factor and so block the interaction with its receptor tyrosine kinase. A key step is the construction of synthetic agents that contain a large (> 400A2) and functionalized surface area to recognize a complementary surface on the target growth factor. In the course of this work we have discovered a molecule, GFB-111, that binds to PDGF, prevents it from binding to its receptor tyrosine kinase, blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases and DNA synthesis. The binding affinity for PDGF is high (IC50=250 nM) and selective over EGF, IGF-1, aFGF, bFGF and HRGbeta. In nude mouse models GFB-111 also shows significant inhibition of tumor growth and angiogenesis.

Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies.

In 1990, more than 10 years after the discovery that the low molecular weight GTPase Ras is a major contributor to human cancer, farnesylation, a lipid posttranslational modification required for the cancer-causing activity of Ras, emerged as a major target for the development of novel anticancer agents. However, it took only 5 years from 1993, when the first farnesyltransferase inhibitors (FTIs) were reported, to 1998 when results from the first phase I clinical trials were described. This rapid progress was due to the demonstration of outstanding antitumor activity and lack of toxicity of FTIs in preclinical models. Although, many FTIs are currently in phase H and at least one is in phase III clinical trial, the mechanism of FTI antitumor activity is not known. In this review a brief summary of the development of FTIs as antitumor agents will be given. The focus of the review will be on important mechanistic and bench-to-bedside translational issues. Among the issues that will be addressed are: evidence for and against inhibition of the prenylation of Ras and RhoB proteins in the mechanism of action of FTIs; implications of the alternative prenylation of K-Ras by geranylgeranyl-transferase I (when FTase is inhibited) in cancer therapy; GGTase I inhibitors (GGTIs) as antitumor agents; effects of FTIs and GGTIs on cell cycle machinery and progression and potential mechanisms by which FTIs and GGTIs induce apoptosis in human cancer cells. A thorough discussion about bench-to-bedside issues relating to hypothesis-driven clinical trials with proof-of-principle in man will also be included. This section will cover issues relating to whether the biochemical target (FTase) is inhibited and the level of inhibition of FTase required for clinical response; are signaling pathways such as H-Ras/PI3K/Akt and/or K-Ras/Raf/MEK/Erk relevant biological readouts?; is Ras (particularly N-Ras and H-Ras) mutation status a good predictor of clinical response?; in phase I trials should effective biological dose, not maximally tolerated dose, be used to determine phase II dose?; and finally, in phase II/III trials what are the most appropriate clinical end points for anti-signaling molecules such as FTIs? Parts of this topic have been recently reviewed (Sebti and Hamilton, 2000c).