Heinz-Herbert Fiebig

Comparison of 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) and 17-allylamino-17-demethoxygeldanamycin (17AAG) in vitro: effects on Hsp90 and client proteins in melanoma models

The heat shock protein Hsp90 is a potential target for drug discovery of novel anticancer agents. By affecting this protein, several cell signaling pathways may be simultaneously modulated. The geldanamycin analog 17AAG has been shown to inhibit Hsp90 and associated proteins. Its clinical use, however, is hampered by poor solubility and thus, difficulties in formulation. Therefore, a water-soluble derivative was desirable and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17DMAG) is such a derivative. Studies were carried out in order to evaluate the activity and molecular mechanism(s) of 17DMAG in comparison with those of 17-allylamino-demethoxygeldanamycin (17AAG). 17DMAG was found to be more potent than 17AAG in a panel of 64 different patient-derived tumor explants studied in vitro in the clonogenic assay. The tumor types that responded best included mammary cancers (six of eight), head and neck cancers (two of two), sarcomas (four of four), pancreas carcinoma (two of three), colon tumors (four of eight for 17AAG and six of eight for 17DMAG), and melanoma (two of seven). Bioinformatic comparisons suggested that, while 17AAG and 17DMAG are likely to share the same mode(s) of action, there was very little similarity with standard anticancer agents. Using three permanent human melanoma cell lines with differing sensitivities to 17AAG and 17DMAG (MEXF 276L, MEXF 462NL and MEXF 514L), we found that Hsp90 protein was reduced following treatment at a concentration associated with total growth inhibition. The latter occurred in MEXF 276L cells only, which are most sensitive to both compounds. The depletion of Hsp90 was more pronounced in cells exposed to 17DMAG than in those treated with 17AAG. The reduction in Hsp90 was associated with the expression of erbB2 and erbB3 in MEXF 276L, while erbB2 and erbB3 were absent in the more resistant MEXF 462NL and MEXF 514L cells. Levels of known Hsp90 client proteins such as phosphorylated AKT followed by AKT, cyclin D1 preceding cdk4, and craf-1 declined as a result of drug treatment in all three melanoma cell lines. However, the duration of drug exposure needed to achieve these effects was variable. All cell lines showed increased expression of Hsp70 and activated cleavage of PARP. No change in PI3K expression was observed and all melanoma cells were found to harbor the activating V599E BRAF kinase mutation. The results of our in vitro studies are consistent with both 17AAG and 17DMAG acting via the same molecular mechanism, i.e. by modulating Hsp90 function. Since 17DMAG can be formulated in physiological aqueous solutions, the data reported here strongly support the development of 17DMAG as a more pharmaceutically practicable congener of 17AAG.

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: activation of MET as one mechanism for drug resistance.

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.

In-vitro anti-tumor activity studies of bridged and unbridged benzyl-substituted titanocenes

The benzyl-substituted ansa-titanocenes [1,2-di(cyclopentadienyl)-1,2-di-(4-N,N-dimethylaminophenyl)ethanediyl] titanium dichloride (Titanocene X) and [1,2-di(cyclopentadienyl)-1,2-bis(m-dimethoxyphenyl)ethanediyl] titanium dichloride (Titanocene Z), and the benzyl-substituted unbridged titanocene bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) were tested on the growth of a wide variety of tumor cells in vitro on a panel of 36 human tumor cell lines containing 14 different tumor types investigated in a cellular proliferation assay. Titanocene Y with a mean IC50 value of 65.8×10−6 mol/l over the full panel of 36 cancer cell lines reaches the activity of cisplatin with 14.7×10−6 mol/l within a factor of 4, whereas Titanocene X and Z show significantly less cytotoxic activity. Titanocene Y is most effective on pleura mesothelioma, and uterine and renal cell cancer, where the IC50 values are comparable or significantly better than for cisplatin. In particular, in the case of renal cell cancer and pleura mesothelioma there is an obvious lack of chemotherapeutic reagents, which might be filled by Titanocene Y, where a very promising cytotoxic effect in comparison with cisplatin could be shown.

Xiamycin, a pentacyclic indolosesquiterpene with selective anti-HIV activity from a bacterial mangrove endophyte.

A novel pentacyclic indolosesquiterpene, named xiamycin (1), and its methyl ester (2) have been obtained from Streptomyces sp. GT2002/1503, an endophyte from the mangrove plant Bruguiera gymnorrhiza. The structures were established by 1D and 2D NMR, MS, and X-ray crystallography, and the absolute configuration of 1 was elucidated by the modified Mosher method. Compound 1 exhibits selective anti-HIV activity; it specifically blocks R5 but has no effects on X4 tropic HIV-1 infection. In a panel of cytotoxicity assays, compound 2 showed to be more potent (geometric mean IC(50)=10.13 μM) compared to compound 1 (geometric mean IC(50) >30 μM), with antitumor potency being generally less pronounced. Xiamycin represents one of the first examples of indolosesquiterpenes isolated from prokaryotes.

17-(allylamino)-17-demethoxygeldanamycin activity in human melanoma models

17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is a semisynthetic antitumor agent, which has entered phase I/II clinical trials. Melanoma cell lines in the NCI in vitro screen (mean GI50=84 nM) were relatively sensitive to the agent, which was therefore tested in vivo in four s.c. growing human melanoma xenografts (MEXF 276, 989, 462 and 514) in athymic mice. 17-AAG markedly inhibited tumor growth at doses of 80 (maximum tolerated dose) and 60 mg/kg/day in a qd×5 (h: 0, 6; i.p.) schedule in two of four xenograft models. Cell lines derived from the 17-AAG-sensitive MEXF 276 and -resistant MEXF 514 melanomas, MEXF 276L and 514L, were chosen to study the effects of 17-AAG on its target Hsp90 as well as the Hsp90 ‘client’ protein c-Raf-1 in vitro. Cells were exposed to drug concentrations which just cause total growth inhibition (total growth inhibition=375 nM in MEXF 276L and 10 μM in MEXF 514L). Pharmacokinetic determinations confirmed that 17-AAG concentrations producing growth inhibition invitro are readily achievable in vivo at the MTD (AUC0–∞ 1068 μM·min). Whilst 17-AAG treatment did not affect Hsp90 expression in the relatively resistant MEXF 514L cells, it caused a rapid transient decline in the markedly sensitive MEXF 276L cell line. In contrast, Hsp72 expression increased. Following Hsp90 depletion at 2–8 h in MEXF 276L cells, down-regulation of c-Raf-1 was seen starting at 16 h after drug addition. In MEXF 276 xenograft tissues treated with effective dose levels, loss of Hsp90 was seen and was associated with occurrence of apoptotic figures. The apoptotic index rose from 9% after 48 h, greater than 12% at 72 h to 45% at 10 days. These data support the hypothesis that in some melanoma models, a very good response (e.g. with tumor regressions) to 17-AAG may be associated with modulation of Hsp90 expression. The expression of this target should be followed in clinical studies with 17-AAG.

Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents.

A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate.

The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over-expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotests 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (Ferrosanol®). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect.

A family of multicyclic indolosesquiterpenes from a bacterial endophyte

Three novel indolosesquiterpenes, xiamycin B (1b), indosespene (2), and sespenine (3), along with the known xiamycin A (1a) were isolated from the culture broth of Streptomyces sp. HKI0595, a bacterial endophyte of the widespread mangrove tree Kandelia candel. Agar diffusion assays revealed moderate to strong antimicrobial activities against several bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, while no cytotoxicity against human tumor cell lines was observed. Together with the previously reported oridamycin, the endophyte metabolites represent the first indolosesquiterpenes isolated from prokaryotes.

2-benzoxazolyl and 2-benzimidazolyl hydrazones derived from 2-acetylpyridine: A novel class of antitumor agents

Here we describe the effects of novel benzoxazol‐2‐yl and benzimidazol‐2‐yl hydrazones derived from 2‐pyridinecarbaldehyde and 2‐acetylpyridine. The IC50 values for inhibition of cell proliferation in KB‐3‐1, CCRF‐CEM, Burkitts lymphoma, HT‐29, HeLa, ZR‐75 and MEXF276L by most of the novel compounds are in the nanomolar range. In colony‐forming assays with human tumor xenografts the compounds 2‐actylpyridine benzoxazol‐2‐ylhydrazone (EPH52), 2‐acetylpyridine benzoimidazol‐2‐ylhydrazone (EPH61) and 2‐acetylpyridine 1‐methylbenzoimidazol‐2‐ylhydrazone (EPH116) exhibited above‐average inhibition of colon carcinoma (IC50 = 1.3–4.56 nM); EPH52 and EPH116 also exhibited above‐average inhibition of melanoma cells. As shown with human liver microsomes, EPH116 is only moderately metabolized. The compound inhibited the growth of human colon cancer xenografts in nude mice in a dose‐dependent manner. Thiosemicarbazones derived from 2‐formylpyridines have been shown to be inhibitors of ribonucleotide reductase (RR). The following results show that RR is not the target of the novel compounds: cells overexpressing the M2 subunit of RR and resistant to the RR inhibitor hydroxyurea are not cross‐resistant to the novel compounds; inhibition of RR occurs at 6‐ to 73‐fold higher drug concentrations than that of inhibition of cell proliferation; the pattern of cell cycle arrest in S phase induced by the RR inhibitor hydroxyurea is not observed after treatment with the novel compounds; and a COMPARE analysis with the related compounds 2‐acetylpyrazine benzothiazol‐2‐ylhydrazone (EPH95) and 3‐acetylisoquinoline benzoxazol‐2‐ylhydrazone (EPH136) showed that the pattern of these compounds is not related to any of the standard antitumor drugs. Therefore, these novel compounds show inhibition of colon cancers and exhibit a novel mechanism of action.

P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models

P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be ∼30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was ∼26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G2-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non–small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G1. H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G1 with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non–small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule. [Mol Cancer Ther 2007;6(3):926–34]