Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.

Reversal of P-glycoprotein-mediated multidrug resistance by XR9051, a novel diketopiperazine derivative.

XR9051 (N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phe nyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-pipera zinylidene) methylbenzamide) was identified as a potent modulator of P-glycoprotein-mediated multidrug resistance (MDR) following a synthetic chemistry programme based on a natural product lead compound. The activity of XR9051 was determined using a panel of human and murine drug-resistant cell lines (H69/LX4, 2780AD, EMT6/AR 1.0, MC26 and P388/DX Johnson). XR9051 was able to reverse resistance to a variety of cytotoxic drugs, including doxorubicin, etoposide and vincristine, which are associated with classical MDR. At a concentration of 0.3-0.5 microM, XR9051 was able to fully sensitize resistant cells to cytotoxics, whereas little or no effect was observed on the corresponding parental cell lines. No effect of XR9051 was observed on the response of cells to non-MDR cytotoxics such as methotrexate and 5-fluorouracil. XR9051 was consistently more potent than cyclosporin A (CsA) and verapamil (Vpm) in all assays used. XR9051 inhibited the efflux of [3H]daunorubicin from preloaded cells and, unlike CsA and Vpm, remained active for several hours after removal of resistance-modifying agent. In photoaffinity labelling experiments employing [3H]azidopine, XR9051 was able to displace binding to P-glycoprotein. In binding studies using [3H]vinblastine, XR9051 was shown to be a potent inhibitor of the binding of the cytotoxic to P-glycoprotein (EC50 = 1.4 +/- 0.5 nM). Taken together, the results indicate that XR9051 reverses the MDR phenotype through direct interaction with P-glycoprotein.

Synthesis and biological evaluation of a series of flavones designed as inhibitors of protein tyrosine kinases.

A series of flavones has been prepared, which are variously substituted in the 3,3',4',5 and 7 positions with halo-, alkoxy-, nitro-, amino-, hydroxy-, acyloxy- and azido-groups, for evaluation of their cytotoxicity to ANN-1 cells (3T3 murine fibroblasts transformed with the Abelson murine leukaemia virus) which contain a tyrosine kinase. This cytotoxicity was compared to their non-transformed 3T3 counterparts. 3'-Amino-4'-methoxyflavone was the most cytotoxic compound (IC50 = 1.6 microM) and was less inhibitory to the non-transformed parent 3T3 cell line (IC50 = 8 microM). The compound was inactive at 50 microM in assays of the inhibition of the cell-associated Abelson protein tyrosine kinase but inhibited an epidermal growth factor (EGF) protein tyrosine kinase by 42% at 50 microM. Quercetin (3,3',4',5,7-pentahydroxyflavone) was the most potent inhibitor of the Abelson protein tyrosine kinase but showed no selective inhibition of the growth of ANN-1 cells compared to the parent 3T3 cell line. Different structure-activity relationships were observed between the results of the cytotoxicity assays and inhibition of protein tyrosine kinases. Inhibitors of the Abelson protein tyrosine kinase which were competitive with respect to ATP showed different potencies for inhibition of the EGF receptor kinase.

Comparison of effects of bryostatins 1 and 2 and 12-O-tetradecanoylphorbol-13-acetate on protein kinase C activity in A549 human lung carcinoma cells.

Activators of protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatins 1 and 2, inhibit the growth of A549 cells. At high concentrations the bryostatins do not affect cell growth. Here the hypothesis has been tested that modulation of A549 cell growth is the consequence of agent-induced changes in location or extent of cellular PKC activity. PKC activity was measured after semi-purification with nondenaturing polyacrylamide gel electrophoresis in the cytosol and the particulate fraction of A549 cells. When cells were exposed to TPA or mezerein, PKC activity underwent rapid and concentration-dependent translocation from the cytosol to the membrane. TPA at 0.1 microM or mezerein at 1 microM caused almost complete translocation within 30 min. Incubation with bryostatins 1 or 2 also led to enzyme translocation, which was, however, much weaker than that observed with the tumor promoters. Neither 4 alpha-phorboldidecanoate nor the synthetic diacylglycerols 1,2-sn-dioctanoylglycerol or 1-oleoyl-2-acetyl-sn-glycerol mimicked TPA in this way. Exposure of cells to TPA or the bryostatins for longer than 30 min caused the gradual disappearance of total cellular PKC activity. PKC downregulation was concentration dependent and complete after 24 h. A549 cells which had acquired temporary resistance toward the growth-arresting potential of TPA were completely devoid of any measurable PKC activity. The bryostatins were potent inhibitors of the binding of [3H]phorbol-12,13-dibutyrate to its receptors in intact cells, and the inhibition was dependent on bryostatin concentration. The results support the contention that PKC is involved in the mediation of growth inhibition caused by TPA or the bryostatins. However, the relationship between growth arrest and PKC translocation or downregulation seems to be a complex one.

Studies on bioactive compounds. 13. Synthesis and lack of growth-inhibitory properties of cyclohexane-1,2,4-triol 1,2-diesters, which resemble ring C of the phorbol ester molecule.

It has been suggested that ring C of biologically active phorbol esters is an essential structural feature of the pharmocophore which confers activity on these compounds. In this study the hypothesis has been tested that compounds which resemble ring C of the phorbol ester molecule mimic the ability of phorbol esters to inhibit cell growth at nontoxic concentrations. All four diastereoisomers of (+/-)-1,2-di-O-octanoylcyclohexane-1,2,4-triol have been prepared from cyclohexen-4-ol and tested for growth-inhibitory and cytotoxic properties. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate inhibited the growth of A549 human lung carcinoma cells by 50% at a concentration of 0.2 nM and exerted cytotoxicity at concentrations of greater than 1 microM. Diacylglycerols are the physiological ligands and activators of protein kinase C, the receptor via which phorbol esters are thought to mediate their effects. The diacylglycerols 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol and the cyclohexanetriol diesters inhibited the growth of A549 cells only at concentrations of 10(-5) to 10(-4) M, at which they were also cytotoxic. A computer-assisted analysis of the goodness of fit between the cyclohexanetriol diesters and ring C of the phorbol moiety revealed possible energetic grounds for conformational dissimilarities. The results suggest that activation of protein kinase C alone is probably not sufficient to reproduce phorbol ester induced growth arrest in A549 cells and that the cyclohexanetriol diesters may lack pivotal elements of the phorbol ester pharmacophore.

Effects of activators of protein kinase C, including bryostatins 1 and 2, on the growth of A549 human lung carcinoma cells.

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit the growth of A549 human lung carcinoma cells at non-toxic concentrations, whereas 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol, synthetic analogues of the physiological ligands of protein kinase C (PKC), do not. Experiments were conducted to test the hypothesis that other activators of PKC are capable of interfering with A549 cell growth. The non-phorboid tumour promotor mezerein mimicked the growth-inhibitory effect of TPA in that it arrested growth for 5 days, after which cells proliferated again in the continued presence of the agent. TPA was 20 times more potent as a growth inhibitor than was mezerein. Bryostatin 1 at 10 nM and bryostatin 2 at 100 nM also arrested A549 cell growth and inhibited DNA replication as measured by incorporation of [methyl-3H]-thymidine into cells. Inhibition of DNA synthesis to between 90 and 75% of control values developed during the first hour of incubation of the cells with TPA, mezerein or the bryostatins. The extent of inhibition changed little during the subsequent 5 hr of incubation, after which it increased further to reach maximal values within 12 hr. At concentrations above those which caused maximal growth inhibition, the bryostatins abolished both their own inhibition of DNA synthesis and the anti-replicative effect of TPA and mezerein. The results show that activators of PKC other than phorbol esters are capable of inhibiting the growth of A549 cells. The bryostatins not only interfere with A549 cell growth but can also counter the growth-inhibitory effect of PKC activators, presumably via interaction with a target separate from the phorbol ester receptor site.