Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Synthesis of N3-substituted thymidine analogues for measurement of cellular kinase activity.

N3-Substitued thymidine analogues that carry a carboranylalkyl moiety at the N3-position with various spacer lengths have been reported to be good substrates for thymidine kinase (TK1). As part of our continuing effort towards the development of new TK1 substrates for imaging tumor proliferative activity, we have synthesized a series of new N3-substituted analogues of thymidine that carry an aromatic ring with different spacer lengths. The overall yields for 6 and 7 were 13% and 39% in four steps and three steps, respectively, and those for 14, 16 and 18 were in the range of 13%-15% in six steps. The overall yield for 24 was 33% in three steps, and those for 25 and 26 were 64% and 58%, respectively, in one step. Most of these compounds have been tested for TK1 activity by enzymatic assay to identify a good substrate that can be radiolabeled for imaging. The phosphorylation rates of these compounds were 2%-6% compared with that of thymidine. The results from the in vitro enzymatic assays suggest that these N3-substituted thymidine analogues have some potential for imaging TK1 activity if radiolabeled with a suitable isotope.

Molecular imaging of EGFR kinase activity in tumors with 124I-labeled small molecular tracer and positron emission tomography.

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and (124)I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[(124)I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[(124)I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[(131)I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[(131)I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[(124)I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[(124)I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[(124)I]-IPQA, additional [(124)I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

Induction of apoptosis in MCF-7:WS8 breast cancer cells by beta-lapachone.

Beta-lapachone (beta-lap) affects a number of enzymes in vitro, including type I topoisomerase (Topo I); however, its exact intracellular target(s) and mechanism of cell killing remain unknown. We compared the cytotoxic responses of MCF-7:WS8 (MCF-7) human breast cancer cells after 4-h pulses of beta-lap or camptothecin (CPT), a known Topo I poison. A direct correlation between loss of survival and apoptosis was seen after beta-lap treatment (LD50 = 2.5 microM). A concentration-dependent, transient sub-2 N preapoptotic cell population was observed at 4-8 h. Estrogen deprivation-induced synchronization and bromodeoxyuridine-labeling studies revealed an apoptotic exit point near the G1-S border. Apoptosis activated by beta-lap was closely correlated with cleavage of lamin B but not with increases in p53/p21 or decreases in bcl-2. Loss of hyperphosphorylated forms of the retinoblastoma protein was observed within 5 h, but cyclins A, B1, and E levels were unaltered for up to 72 h after 5 microM beta-lap. Topo I and Topo IIalpha levels decreased at > 24 h. Logarithmic-phase MCF-7 cells were not affected by < or = 1 microM beta-lap. In contrast, dramatic and irreversible G2-M arrest with no apoptosis was observed in MCF-7 cells treated with 1 microM CPT, monitored for 6-10 days posttreatment. MCF-7 cells treated with supralethal doses of CPT (5 microM) resulted in only approximately 20% apoptosis. No correlation between apoptosis and loss of survival was observed. MCF-7 cells exposed to > 5 microM CPT arrested at key cell cycle checkpoints (i.e., G1, S, and G2-M), with little or no movement for 6 days. Ten-fold increases in p53/p21 and 2-5-fold decreases in bcl-2, Topo I, Topo IIalpha, and cyclins A and B1, with no change in cyclin E, were observed. Temporal decreases in bcl-2 and cleavage of lamin B corresponded to the minimal apoptotic response observed. Beta-lap activated apoptosis without inducing p53/p21 or cell cycle arrest responses and killed MCF-7 cells solely by apoptosis. In contrast, concentration-dependent increases in nuclear p53/p21 and various cell cycle checkpoint arrests were seen in MCF-7 cells after CPT. Despite dramatic p53/p21 protein induction responses, CPT-treated MCF-7 cells showed low levels of apoptosis, possibly due to protective cell cycle checkpoints or the lack of specific CPT-activated apoptotic pathways in MCF-7 cells.

Efficacy of dual-specific Bcr-Abl and Src-family kinase inhibitors in cells sensitive and resistant to imatinib mesylate.

Monotherapy of chronic myeloid leukemia (CML) with imatinib mesylate has been cast into shadow by the evolution of clinical resistance during therapy. Resistance to imatinib can arise by multiple mechanisms including amplification or mutation of Bcr-Abl, and continuity of imatinib therapy is probably a poor option for either of these patient groups. Recently, however, a structurally distinct new class of drugs, the pyrido[2,3-d]pyrimidines, has been described, and these compounds are predicted to make different molecular contacts in the Abl kinase domain. These drugs potently target both the Bcr-Abl and Src-family kinase activities, both of which are thought to be relevant to survival of the leukemic cell. We asked whether these drugs could selectively induce cell death in murine cell line models of CML cells sensitive and resistant to imatinib by different mechanisms. We show that whereas the pyrido[2,3-d] pyrimidines are indeed highly potent in suppressing proliferation of Bcr-Abl-overexpressing imatinib-resistant cells, they are almost completely ineffective against cells expressing the T315I mutant. This implies that despite structural differences from imatinib, these drugs are unlikely to be useful in patients expressing this mutant Bcr-Abl protein, but may be effective in cases where selection of cells overexpressing the oncoprotein leads to refractoriness to imatinib.

Schedule-dependent synergism between raltitrexed and irinotecan in human colon cancer cells in vitro.

The quinazoline folate analogue raltitrexed (ZD1694; Tomudex) and the camptothecin derivative irinotecan are two new agents showing clinical activity against colorectal cancer. To identify the optimal conditions to achieve synergistic cytotoxicity before the clinical development of their combination, we explored the interactions between ZD1694 and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), in vitro. Cytotoxicity was evaluated with a clonogenic assay using the human colon cancer cell line HCT-8. Different schedules of administration and different dose ratios of the two agents were compared and evaluated for synergism, additivity, or antagonism with a quantitative method based on the median-effect principle of Chou and Talalay (T. C. Chou and P. Talalay, Adv. Enzyme Regul., 22: 27-55, 1984). Sequential short-term (1 and 4-h) exposures to SN-38 followed by ZD1694 resulted in synergistic cytotoxicity at broad dose-effect ranges. At a high level of cell kill, the synergism was greater when either equiactive doses of the two agents or higher relative doses of ZD1694 were used. A 24-h interval between exposure to SN-38 and ZD1694 significantly enhanced the magnitude of the synergy (P = 0.001). The opposite sequence or simultaneous exposures produced significantly less potentiation or nearly additive interactions (P = 0.0006 and P < 0.0001). The synergism was completely lost under conditions of more prolonged drug exposure (24-h continuous exposure). In conclusion, in this in vitro model of human colon cancer, ZD1694 and SN-38 produced synergistic cytotoxicity. Our findings support the clinical use of this combination and provide a rationale for a clinical trial using sequential short-term exposures to equiactive doses of SN-38 and ZD1694 administered sequentially with a 24-h interval.

Co-administration of probenecid, an inhibitor of a cMOAT/MRP-like plasma membrane ATPase, greatly enhanced the efficacy of a new 10-deazaaminopterin against human solid tumors in vivo.

Earlier studies from this laboratory have shown that the uricosuric agent probenecid (PBCD) will inhibit the extrusion of folate analogues from tumor cells mediated by a plasma membrane ATPase resembling the canicular multispecific organic anion transporter/multidrug resistance-related protein (MRP) family of ATP binding cassette transporters. This inhibition of this outwardly directed membrane ATPase has been shown to have a favorable impact upon the cellular pharmacokinetics, cytotoxicity, and efficacy of methotrexate in vivo. In an extension of these earlier studies, which had focused only on murine ascites tumors, we now report that parental co-administration of PBCD will also enhance net intracellular accumulation in vitro and intracellular persistence in vivo of a new folate analogue, 10-propargyl-10-deazaaminopterin (PDX) in tumor cells. This resulted in marked enhancement of the efficacy of PDX against murine and human lung neoplasms and human prostate and mammary neoplasms growing as solid tumors in mice. As possible ATPases targeted by PBCD, all of these tumors expressed MRP-1, -4, and -7 genes, with expression of MRP-4 being greatest in each case. Four other MRP genes were expressed to a variable extent in some tumors but not others. The therapeutic enhancement of PDX by PBCD was manifested as tumor regression, where PDX alone was only growth inhibitory (A549 NSCL tumor), or as a substantial increase (3-4-fold) in overall regression and/or number of complete regressions (Lewis and LX-1 lung, PC-3 and TSU-PR1 prostate, and MX-1 mammary tumors) compared to PDX alone. Also, only in the case of PDX with PBCD, a significant number of mice transplanted with LX-1 or MX-1 tumors that experienced complete regression did not have regrowth of their tumor. In view of these results, clinical trials of this therapeutic modality appear to be warranted, especially in the case of new more efficacious folate analogues that are also permeants for this canicular multispecific organic anion transporter/MRP-like plasma membrane ATPase.

Cellular and molecular responses to topoisomerase I poisons. Exploiting synergy for improved radiotherapy.

The efficacy of topoisomerase (Topo) I-active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I-active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF-kappa B, a known anti-apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF-kappa B activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF-kappa B activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I-active drugs. NF-kappa B activation was intensified in S-phase and blocked by aphidicolin, suggesting that activation was a result of double-strand break formation due to Topo I poisoning and DNA replication. Dominant-negative I kappa B expression augmented Topo I poison-mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug-IR combinations may lead to improved radiotherapy by blocking anti-apoptotic NF-kappa B responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by beta-lapachone (beta-lap), a "reported" Topo I and II-alpha poison in vitro. In fact, beta-lap does not kill cells by poisoning either Topo I or II-alpha in vivo. Instead, the compound is "activated" by an IR (damage)-inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x-ray-inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, beta-lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by beta-lap appears to be noncaspase-mediated, involving calpain or a calpain-like protease. Thus, although Topo I poisons or beta-lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different.

Modulation of drug cytotoxicity in wild-type and multidrug-resistant tumor cells by stereoisomeric series of C-20'-vinblastine congeners that lack antimicrotubule activity.

Seven binary vinca alkaloid congeners were newly synthesized as the C14' or C16'(20') or C14'16'(20') stereoisomers of C20'-modified VBL. These congeners lacked detectable antimicrotubule activity in assays of polymerization of purified microtubule protein and of mitotic arrest induction. The compounds modulated the cytotoxicity of VBL, VCR, and DOX in sarcoma and colon-tumor cell lines. In wild-type cell lines, each congener elicited a concentration-dependent enhancement of cytotoxicity that was drug- and cell-type-selective. For example, C20'-deoxy C14'16'20'-epi VBL sensitized sarcoma S180 cells 19-fold to DOX and 11-fold to VCR but had no effect on VBL cytotoxicity. In the rat colon-cancer cell lines there was preferential enhancement of VCR cytotoxicity by most congeners. In two MDR cell strains of S180, the modulation potency of each congener was independent of specific drug or of resistance level. As a result, the amount of modulator (concentration) required for reversal was proportional to the drug-resistance level. Such properties were not displayed by the monomeric vinca alkaloid modulator vindoline. The potency of drug modulation in both wild-type and MDR cells strains was dependent on the stereoisomeric form of the congener and its C20'-substituents.

Mechanisms of action of ibogaine and harmaline congeners based on radioligand binding studies.

Assays using radioligands were used to assess the actions of ibogaine and harmaline on various receptor types. Ibogaine congeners showed affinity for opiate receptors whereas harmaline and harmine did not. The Ki for coronaridine was 2.0 microM at mu-opiate receptors. The Kis for coronaridine and tabernanthine at the delta-opiate receptors were 8.1 and 3.1 microM, respectively. Ibogaine, ibogamine, coronaridine and tabernanthine had Ki values of 2.08, 2.6, 4.3 and 0.15 microM, respectively, for kappa-opiate receptors. Long-lasting, dose-dependent behavioral effects of ibogaine have been reported. The possibility that these effects were due to irreversible binding properties of ibogaine at kappa-receptors was considered; however, radioligand wash experiments showed a rapid recovery of radioligand binding after one wash. A voltage-dependent sodium channel radioligand demonstrated Ki values in the microM range for all drugs tested. Using radioligand binding assays and/or 36Cl- uptake studies, no interaction of ibogaine or harmaline with the GABA receptor-ionophore was found. The kappa-activity of ibogaine (or an active metabolite) may be responsible for its putative anti-addictive properties whereas the tremorigenic properties of ibogaine and harmaline may be due to their effects on sodium channels.

Structural studies of vinblastine alkaloids by exciton coupled circular dichroism.

SCF-CI-dipole velocity MO calculations have shown that the bisignate circular dichroic curves of vinblastine/vincristine alkaloids at ca 210 and 220-230 nm are due to exciton coupling between the indoline and indole moieties. Furthermore, a combination of X-ray crystal structure data with MM2 local energy minimization provides a convenient means for estimation of the preferred solution conformation.

Bcl-2 protects against beta-lapachone-mediated caspase 3 activation and apoptosis in human myeloid leukemia (HL-60) cells.

We previously demonstrated that beta-lapachone (beta-lap) killed cancer cells solely by apoptosis. Beta-Lap induced apoptosis in HL-60 cells in a dose-dependent manner as measured by flow cytometry and DNA ladder formation. Cell cycle changes, such as accumulations in S and G2-phases, were not observed. Apoptosis was accompanied by activation of caspase 3 and concomitant cleavage of poly(ADP-ribose) polymerase (PARP) to an 89 kDa polypeptide. PARP cleavage was blocked by zDEVD-fmk or zVAD-fmk, caspase-specific cleavage site inhibitors. Retrovirally introduced bcl-2 prevented beta-lap-mediated caspase 3 activation and PARP cleavage and increased the viability of Bcl-2-expressing HL-60 cells compared to cells with vector alone. Various beta-lap-related analogs (e.g., dunnione and naphthoquinone derivatives) induced equivalent apoptosis in HL-60 cells, but no compound was more effective than beta-lap. These data provide further evidence that the primary mode of cell killing by beta-lap is by the initiation and execution of apoptosis in human cancer cells.

RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B.

Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Adenovirus 5 E1A enhances histone deacetylase inhibitors-induced apoptosis through Egr-1-mediated Bim upregulation.

Histone deacetylase inhibitors (HDACi) are potent anti-cancer agents for variety of cancer types. Suberoylanilide hydroxamic acid (SAHA) has been approved as a drug to treat cutaneous T cell lymphoma, and the combination of HDACi and other agents have been actively tested in many clinical trials. Adenovirus 5 early region 1A (E1A) has been shown to exhibit high tumor suppressor activity, and gene therapy using E1A has been tested in clinical trials. Here, we showed that proapoptotic activity of HDACi was robustly enhanced by E1A in multiple cancer cells, but not in normal cells. Moreover, we showed that combination of E1A gene therapy and SAHA showed high therapeutic efficacy with low toxicity in vivo ovarian and breast xenograft models. SAHA downregulated Bcl-XL and upregulated proapoptotic BH3-only protein Bim, whose expression was further enhanced by E1A in cancer cells. These alterations of Bcl-2 family proteins were critical for apoptosis induced by the combination in cancer cells. SAHA enhanced acetylation of histone H3 in Bim promoter region, while E1A upregulated Egr-1, which was directly involved in Bim transactivation. Together, our results provide not only a novel insight into the mechanisms underlying anti-tumor activity of E1A, but also a rationale for the combined HDACi and E1A gene therapy in future clinical trials.

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway.

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

Analysis of drug transport kinetics in multidrug-resistant cells using a novel coumarin-vinblastine compound.

We have synthesized an analogue of vinblastine wherein a coumarin molecule is attached to the C17 of the vindoline moiety via a succinimide bridge (cou-VBL). cou-VBL exhibits fluorescence similar to that exhibited by coumarin. Chinese hamster ovary fibroblasts (LR73 cells) that exhibit an IC50 for vinblastine (VBL) of about 100 nM in growth inhibition assays are similarly sensitive to the cou-VBL compound. LR73 cells transfected with the mu MDR 3 gene that were subsequently selected on vinblastine (MDR35 cells) exhibit resistance to cou-VBL that is similar to their VBL resistance. A large change in the quantum efficiency of cou-VBL fluorescence accompanies efflux from intact cells, and comparison between cou-VBL and [3H]VBL efflux from the MDR35 cells reveals that the transport kinetics of the fluorescent analogue is very similar (if not identical) to that exhibited by [3H]VBL. Thus, similar to continuous monitoring of fluorescence (CMF) studies performed with the naturally fluorescent chemotherapeutic doxorubicin (Roepe, 1992), cou-VBL may be used in CMF studies aimed at rigorously defining the kinetics of VBL efflux from multidrug-resistant (MDR) cells. Initial data suggest the following: (1) A single exponential term approximates efflux from sensitive cells, whereas two exponentials are required to fit efflux from MDR35 cells. (2) The faster MDR35 term is virtually identical to the single term for the sensitive cells, whereas the other defines a process that is 5-20 times slower than passive diffusion, depending on the drug concentration. (3) The slower component has a much steeper and nearly linear dependence on drug concentration, whereas the fast passive diffusion component becomes asymptotic near 0.3 pM exchangeable drug/microgram of cell protein.

Huanglian, A chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells.

Huanglian is an herb that is widely used in China for the treatment of gastroenteritis. We elected to determine whether huanglian could inhibit tumor cell growth by modulating molecular events directly associated with the cell cycle. Huanglian inhibited tumor growth and colony formation of gastric, colon, and breast cancer cell lines in a time- and dose-dependent manner. Cell growth was completely inhibited after 3 days of continuous drug exposure to 10 microg/ml of herb. This degree of growth inhibition was significantly greater than that observed with berberine, the major constituent of the herb. The inhibition of cell growth by huanglian was associated with up to 8-fold suppression of cyclin B1 protein. This resulted in complete inhibition of cdc2 kinase activity and accumulation of cells in G(2). The mRNA expression of cyclin B1 was not changed after huanglian treatment. There was no change in the protein expression of cyclins A or E. Therefore, the effect of huanglian on inhibiting tumor growth seems to be mediated by the selective suppression of cyclin B1, which results in the inhibition of cdc2 kinase activity. Inhibition of cyclin dependent kinase (cdk) activity is emerging as an attractive target for cancer chemotherapy. Huanglian represents a class of agents that can inhibit tumor cell growth by directly suppressing the expression of a cyclin subunit that is critical for cell cycle progression. These results indicate that traditional Chinese herbs may represent a new source of agents designed for selective inhibition of cyclin dependent kinases in cancer therapy.

Inhibition of human immunodeficiency virus type 1 replication by 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine, an in vivo metabolite of oltipraz.

Oltipraz, an inhibitor of human immunodeficiency virus type 1 replication in vitro (ED50 approximately 10 microM), undergoes extensive metabolism in vivo. Most of the orally administered drug undergoes opening of the dithiolethione ring, reduction, recyclization, and methylation to form 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-a]pyrazine ("metabolite III"). We report here that metabolite III inhibits viral replication in vitro (ED50 approximately 25 microM) in acutely infected H9 and CEM T cell lymphoma cell lines. Although both metabolite III and oltipraz were able to inhibit phorbol-12-myristate-13-acetate-stimulated viral replication in the chronically infected U1 promonocytic leukemia cell line, only metabolite III was able to inhibit phorbol-12-myristate-13-acetate-stimulated viral replication in chronically infected ACH-2 T cell lymphoma cells. The results with ACH-2 cells suggest that oltipraz inhibits an early stage of the viral life cycle, whereas metabolite III affects human immunodeficiency virus type 1 replication at a step distal to viral integration. This is consistent with the finding that oltipraz inhibits reverse transcriptase, whereas metabolite III does not. Although the mean ED50 for metabolite III in acutely infected peripheral blood mononuclear cells was 18 microM, the ED50 was below 5 microM in three of eight independent experiments. Studies of metabolite III in combination with oltipraz in acutely infected peripheral blood mononuclear cells demonstrated significant antiviral synergy. These results raise the possibility that the in vitro potency of oltipraz may underestimate its antiretroviral activity in vivo. Based on these results, the pharmacokinetics of oltipraz and metabolite III will be compared with the pharmacodynamic effects of orally administered oltipraz in a forthcoming phase I/II trial of oltipraz in patients with p24 antigenemia.