Synergistic Effect of SN-38 in Combination with Cetuximab on Angiogenesis and Cancer Cell Invasion.

BACKGROUND: The combination of irinotecan, a topoisomerase I inhibitor with cetuximab, an antibody against epidermal growth factor receptor, produces synergistic and beneficial effects in patients with irinotecan-refractory colorectal cancer. Our hypothesis was that synergistic effects could be due to anti-angiogenesis and anti-invasion, but not to cytotoxicity. MATERIALS AND METHODS: Cytotoxicity was assessed by viability test and flow cytometry. Anti-angiogenesis, anti-invasion were studied by the endothelial cell capillary-like network formation and transmigration through an extracellular matrix. Protein kinase B (PKB, frequently cited as AKT), and extracellular signal-regulated kinases (ERK) activation was assayed by cell-based enzyme-linked immunosorbent assay (ELISA). RESULTS: Combinations of SN-38 (the active of irinotecan) and cetuximab did not induce any synergistic cytotoxicity confirmed by viability test and cell-cycle analyses. Interestingly, their combination produced synergistic anti-angiogenesis and anti-invasion activities revealed by endothelial cell capillary-like network formation and cell invasion tests. Subsequently, their combination attenuated either expression or phosphorylation of AKT and ERK1/2 using cell-based ELISA. CONCLUSION: SN-38/cetuximab combination has synergistic anti-angiogenesis and anti-invasion activities mediated by down-regulation of phosphatidylinositol-3-kinases/AKT and mitogen-activated protein kinase/ERK pathways.

Synthesis and biological evaluation of 4-arylcoumarin analogues of combretastatins.

A series of A-ring polymethoxylated neoflavonoids was prepared by ligand coupling reactions involving either Suzuki or Stille reactions. Cytotoxicity studies indicated a potent activity against a CEM leukemia cell line for the compounds presenting a substitution pattern related to that of combretastatin A-4. The two compounds having a 3'-OH and a 4'-OCH(3) substituents on the 4-phenyl B-ring have no effect on human topoisomerases I and II but potently inhibit, in vitro, microtubule assembly. At the cell level, the active compounds were characterized as proapoptotic agents, but they can also trigger cell death via a nonapoptotic pathway.

Plasma stability of two glycosyl indolocarbazole antitumor agents.

In recent years, several glycosyl indolocarbazole derivatives have been developed as antitumor agents targeting the topoisomerase I-DNA complex and a few of them were evaluated in clinical trials. The lead drug in the series is compound A which bears a formylamino substituent on the N-imide F-ring. This compound has shown promising antitumor activities in vivo and was tested clinically but it has been recently replaced with a more active analogue, J-107088, bearing a (hydroxymethyl-2-hydroxy) ethylamino substituent on the N-imide F-ring. We have compared the plasma stability of two molecules in this series, compounds A and D, which only differ by the nature of the group on the imide ring. The conversion of the compounds into the anhydride species B was studied by HPLC and the resulting metabolite, formed both in human plasma ultrafiltrate and in water, was characterized by NMR and mass spectrometry. Absorption measurements provided a facile method to follow the conversion of compounds A and D into their metabolite product B. Altogether, the experimental data demonstrate that the replacement of the NHCHO substituent of compound A with a hydrophilic NHCH(CH(2)OH)(2) chain preserves the intact imide function that is known to be essential for topoisomerase I inhibition and cytotoxicity. The transformation of compound A into the anhydride metabolite B (or its diacid open form) occurs much more slowly compared to compound D. Half-life parameter t(1/2) of 67 and 245 min(-1) were calculated for compounds A and D, respectively. A molecular modeling analysis, performed to compare the conformation and electronic properties of compounds A and D, offers a rational explanation for the gain of chemical stability of the indolocarbazole derivative D. The data provide important information for the rational design of antitumor indolocarbazole derivatives.

Induction of apoptosis in HL-60 leukemia and B16 melanoma cells by the acronycine derivative S23906-1.

The benzoacronycine derivative S23906-1 is a highly potent antitumor agent with a broad spectrum of activity against different human solid tumor xenografts. The marked cytotoxic potential of this drug may be the result of its interaction with DNA but the precise mechanism of action remains unclear at present. We have investigated the induction of apoptosis in human promyelocytic leukemia HL-60 and murine melanoma B16 cells treated with S23906-1. With both cell lines, the drug induces cell cycle perturbations (G2/M arrest) and triggers apoptosis as revealed by the externalization of Annexin V-targeted PS residues at the periphery of the cells. But the biochemical pathways leading to apoptosis are different for the two cancer cell lines. In HL-60 cells, the drug induces significant variations of the Delta Psi(mt), measured by flow cytometry using the fluorochromes JC-1 and cm-X-ros. Activation of caspase-3 and chromatin condensation in HL-60 cells exposed to submicromolar concentrations of S23906-1 for 24hr were also clearly seen by flow cytometry and confocal microscopy experiments. In contrast, the extent of apoptosis induced by S23906-1 was found to be much more limited in B16 cells. No significant variations of Delta Psi(mt) and no cleavage of the fluorescent caspase-3 substrate GDEVDGI (PhiPhiLux-G(1)D(2) probe) could be detected by cytometry in B16 cells exposed to S23906-1. In addition, we characterized the mitochondrial production of reactive oxygen species (ROS) using the probe dihydroethidine (HE) and the variations of the mitochondrial mass using the cardiolipin-interacting probe nonyl acridine orange (NAO). S23906-1 stimulates the production of ROS in both cell lines but the number of mitochondria seems to increase only in drug-treated B16 cells. Collectively these findings identify S23906-1 as a potent inducer of cell apoptosis in the leukemia cells and to a lower extent in the melanoma cells. The results help to understand the downstream cytotoxic actions of this new anticancer agent which is currently undergoing preclinical development.

Synthesis, antiproliferative activity and tubulin targeting effect of acridinone and dioxophenothiazine derivatives.

The synthesis of new acridinone and dioxophenothiazine derivatives along with their tubulin polymerization inhibitory and antiproliferative activities is reported. The analysis of correlation for cytotoxic and antitubulin potential of tested compounds showed that 4-methoxyphenylethyl derivatives 18a and 19a were highly cytotoxic but were regarded to have no significant antitubulin activity. However, the introduction of a 3-hydroxy substituent leading to compounds 18e and 19e, strongly increased the antitubulin potential but was associated with a loss of the antiproliferative activity. Modeling studies, topoisomerase inhibition assays and cell cycle analysis have been performed to better investigate the mechanism of action of such compounds.

Mechanisms underlying resistance to cetuximab in the HNSCC cell line: role of AKT inhibition in bypassing this resistance.

EGFR is frequently overexpressed in head and neck squamous cell cancer (HNSCC). Cetuximab is a monoclonal antibody designed to interact with EGFR, block its activation, reduce the downstream signaling pathways and induce EGFR internalization. This study aims to investigate the role of the EGFR signaling pathway and EGFR internalization in a cetuximab-resistant cell line and to propose a new therapeutic strategy to optimize treatment of HNSCC. The HNSCC cell line, CAL33 was sensitive to gefitinib but resistant to cetuximab. Cetuximab induces an unexpected EGFR phosphorylation in CAL33 cells similarly to EGF but this EGFR activation does not trigger EGFR internalization/degradation, the process currently implicated in the response to cetuximab. Cetuximab inhibits ERK and AKT phosphorylation in cetuximab-sensitive A431 cells, whereas the level of AKT phosphorylation is unmodified in cetuximab-resistant cells. Interestingly, CAL33 cells harbor a PIK3CA mutation. The treatment of CAL33 cells with PI3K inhibitor and cetuximab restores the inhibition of AKT phosphorylation and induces growth inhibition. Our results indicate that EGFR internalization is impaired by cetuximab treatment in CAL33 cells and that the AKT pathway is a central element in cetuximab resistance. The combination of cetuximab with a PI3K inhibitor could be a good therapeutic option in PIK3CA-mutated HNSCC.

Unusual cellular uptake of cytotoxic 4-hydroxymethyl-3-aminoacridine.

Aminoacridine derivatives display interesting chemical and biological properties in the field of antitumor agents. The synthesis of 4-hydroxymethyl-3-aminoacridine and its iodo labelled analogue allows the study of cell distribution using two innovative, complementary and powerful techniques, real time fluorescence microscopy and dynamic secondary ion mass spectrometry (SIMS). All the data point to lysosomal localization of the active molecule.

Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production.

BACKGROUND: Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types. METHODS: The inhibitory effect of ANA on the epidermal growth factor receptor (EGFR) levels expressed on the EGF-stimulated prostatic cancer cells LNCaP, DU145, and PC3 was estimated by ELISA tests. The anti-proliferative and cytotoxic effects of ANA were also evaluated on these human prostatic cancer cell lines by growth tests, flow cytometric analyses, trypan blue dye exclusion assays combined with the Papanicolaou cytological staining method. RESULTS: ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB(1) receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G(1) arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production. CONCLUSIONS: The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.

Chromophore-modified bisnaphthalimides: DNA recognition, topoisomerase inhibition, and cytotoxic properties of two mono- and bisfuronaphthalimides.

Bisnaphthalimides represent a promising group of DNA-targeted anticancer agents. In this series, the lead compounds elinafide and bisnafide have reached clinical trials, and the search for more potent analogues remains a priority. In the course of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the naphthalimide skeleton, different dimeric molecules containing two tetracyclic neutral DNA intercalating chromophores were synthesized. The naphthalimide unit has been fused to a benzene ring (azonafide derivatives), an imidazole, a pyrazine, or, as reported here, a furan ring which increases the planar surface of the chromophore and enhances its stacking properties. We report a detailed investigation of the DNA binding capacity of the dimeric molecule MCI3335 composed of two furonaphthalimide units connected by a 12 A long amino alkyl linker [(CH(2))(2)-NH-(CH(2))(3)-NH-(CH(2))(2)] identical to that of elinafide. Qualitative and quantitative binding studies, in particular using surface plasmon resonance, establish that the dimer binds considerably more tightly to DNA (up to 1000 times) than the corresponding monomer and exhibits a higher sequence selectivity for GC-rich sequences. DNase I footprinting experiments attest that the dimer, and to a lesser extent the monomer, preferentially intercalate at GC sites. The strong binding interaction between the drugs and DNA perturbs the relaxation of supercoiled DNA by topoisomerases, but the test compounds do not promote DNA cleavage by topoisomerase I or II. Despite the lack of poisoning effect toward topoisomerase II, MCI3335 displays a very high cytotoxicity toward CEM human leukemia cells, with an IC(50) in the low nanomolar range, approximately 4 times inferior to that of the reference drug elinafide. Confocal microscopy observations indicate that the monomer shows a stronger tendency to accumulate in the cell nuclei than the dimer. The extremely high cytotoxic potential of MCI3335 is attributed to its enhanced capacity to bind to DNA and to inhibit DNA synthesis, as evidenced by flow cytometry experiments using the BrdU assay. The results provide novel mechanistic information that furthers the understanding of the structure-activity relationships in the bisnaphthalimide series and identify MCI3335 as a novel lead compound for further preclinical investigations.