Evofosfamide Is Effective against Pediatric Aggressive Glioma Cell Lines in Hypoxic Conditions and Potentiates the Effect of Cytotoxic Chemotherapy and Ionizing Radiations.

Hypoxia is a hallmark of many solid tumors and is associated with resistance to anticancer treatments. Hypoxia-activated prodrugs (HAPs) were developed to target the hypoxic regions of these tumors. Among 2nd generation HAPs, Evofosfamide (Evo, also known as TH-302) exhibits preclinical and clinical activities against adult glioblastoma. In this study, we evaluated its potential in the field of pediatric neuro-oncology. We assessed the efficacy of Evo in vitro as a single drug, or in combination with SN38, doxorubicin, and etoposide, against three pediatric high-grade glioma (pHGG) and three diffuse intrinsic pontine glioma (DIPG) cell lines under hypoxic conditions. We also investigated radio-sensitizing effects using clonogenic assays. Evo inhibited the growth of all cell lines, mainly under hypoxia. We also highlighted a significant synergism between Evo and doxorubicin, SN38, or etoposide. Finally, Evo radio-sensitized the pHGG cell line tested, both with fractionated and single-dose irradiation schedules. Altogether, we report here the first preclinical proof of evidence about Evofosfamide efficiency against hypoxic pHGG and DIPG cells. Since such tumors are highly hypoxic, and Evo potentiates the effect of ionizing radiation and chemotherapy, it appears as a promising therapeutic strategy for children with brain tumors.

Synthesis and biological activity of ferrocenyl indeno[1,2-c]isoquinolines as topoisomerase II inhibitors.

Three series of indeno[1,2-c]isoquinolines bearing a ferrocenyl entity were synthesized and evaluated for DNA interaction, topoisomerase I and II inhibition, and cytotoxicity against breast human cancer cell lines. In the first and second series, the ferrocenyl scaffold was inserted as a linker between the two nitrogen atoms. In the last series, it was introduced at the end of the carbon chain. The present study showed that the ferrocenyl entity enhanced the topoisomerase II inhibition. Most compounds showed a potent growth inhibitory effect on MDA-MB-231 cell line with the IC50 in µM range.

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Low level of baseline circulating VEGF-A is associated with better outcome in patients with vascular sarcomas receiving sorafenib: an ancillary study from a phase II trial.

We have carried out a stratified phase II study of sorafenib (So) in patients with advanced angiosarcoma (n = 32) and epithelioid hemangioendothelioma (n = 13). This report concerns the correlative analysis of the predictive values of circulating pro/anti-angiogenetic biomarkers. Using the ELISA method (R&D Systems), circulating biomarkers (VEGF-A, in picograms per milliliter), thrombospondin-1 (TSP1, in micrograms per milliliter), stem cell factor (SCF, in picograms per milliliter), placental growth factor (PlGF, in picograms per milliliter), VEGF-C (in picograms per milliliter), and E-selectin (in nanograms per milliliter) were measured before So treatment and after 7 days. VEGF-A (mean value 475 vs. 541, p = 0.002), TSP1 (16 vs. 24, p = 0.0002), and PlGF (20.9 vs. 40.7, p = 0.0001) significantly increased during the treatment. Treatment did not affect the levels of SCF, VEGF-C, and E-selectin. Only two biomarkers were associated with better outcome as follows: VEGF-A and PlGF. Best objective response and non-progression at 180 days were associated with low level of VEGF-A at baseline (p = 0.04 and 0.03, respectively). There was a correlation between the circulating level of VEGF-A and time to progression (TTP) (r = -0.47, p = 0.001). Best objective response and non-progression at 180 days were not associated with baseline level of PIGF, but there was a correlation between the circulating level of PIGF at baseline and TTP. Low level of VEGF-A at baseline (<500) was significantly associated with better outcome.

Cilengitide targets pediatric glioma and neuroblastoma cells through cell detachment and anoikis induction.

The prognosis of children with high-grade glioma or high-risk neuroblastoma remains poor. Cilengitide is a selective antagonist of αvß3 and αvß5 integrins, which are involved in tumor growth and development of metastasis. We have evaluated the effects of cilengitide on pediatric glioma and neuroblastoma cell lines for the first time. Expression levels of αvß3 and αvß5 were determined by flow cytometry in three neuroblastoma and five pediatric glioma cell lines compared with adult U87-MG before and after irradiation. Cell detachment, cytotoxicity, and cell growth under nonadhesive conditions were measured using the MTS assay. Cell death and apoptosis were assessed by annexin-V/propidium iodide staining. The varying αvß3 and αvß5 expression levels were unrelated to tumor grade. Irrespective of the αvß5 expression level, the pediatric cells expressing αvß3 were dose dependently sensitive to cilengitide. UW479 cells expressed only αvß5 integrin and were not sensitive to cilengitide, suggesting that cilengitide's action largely depends on αvß3 inhibition. Cell detachment resulted in a higher cytotoxicity in pediatric glioma compared with U87-MG cells, which seem able to grow despite the significant cilengitide-induced cell detachment. Growth kinetics on polyHEMA showed that only pediatric glioma cells were sensitive to anoikis and so died after cilengitide-induced detachment. Furthermore, irradiation of glioma cells increased αvß3 expression slightly but not cilengitide sensitivity. Cilengitide's action on glioma and neuroblastoma cells appears to be dependent on αvß3 expression and sensitivity to anoikis. Cilengitide is able to target pediatric glioma and neuroblastoma cells in vitro directly and efficiently. Tumor context could validate these promising observations.

New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1µM CA IX inhibitor.

Novel stable camptothecin derivatives replacing the E-ring lactone by a ketone function are potent inhibitors of topoisomerase I and promising antitumor drugs.

The E-ring lactone is the Achilles' heel of camptothecin derivatives: although it is considered necessary for the inhibition of the enzyme topoisomerase I (topo1), the opening of the lactone into a carboxylate abolishes the generation of topo1-mediated DNA breaks. S38809 is a novel camptothecin analog with a stable 5-membered E-ring ketone; therefore, it lacks the lactone function. DNA relaxation and cleavage assays revealed that S38809 functions as a typical topo1 poison by stimulating DNA cleavage at T downward arrow G sites. The activity was strongly dependent on the stereochemistry of the C-7 carbon atom that bears the hydroxy group. S38809 proved to be a potent cytotoxic agent, with a mean IC50 of 5.4 nM versus 11.6 nM for topotecan and 3.3 nM for SN38 (the active metabolite of irinotecan) on a panel of 31 human tumor cell lines. The cytotoxicity of S38809 and its ability to stabilize cleavable complexes was considerably reduced in camptothecin-resistant cells that express a mutated topo1, confirming that topo1 is its primary target. Cell death induced by topo1 poisoning requires the conversion of DNA single-strand breaks into double-strand breaks that can be detected by the formation of phosphorylated histone H2AX. In HCT116 cells, topotecan, SN38, and S38809 induced histone H2AX phosphorylation in S phase of the cell cycle, with S38809 being as potent as SN38 and 5-fold more potent than topotecan. In vivo, S38809 showed a marked antitumor activity against HCT116 xenografts. These findings open a new route for improving the pharmacological properties of camptothecin derivatives.

Lamellarin D: a novel potent inhibitor of topoisomerase I.

We report the identification and characterization of a novel potent inhibitor of DNA topoisomerase I: lamellarin D (LAM-D), initially isolated from a marine mollusk, Lamellaria sp., and subsequently identified from various ascidians. This alkaloid, which displays potent cytotoxic activities against multidrug-resistant tumor cell lines and is highly cytotoxic to prostate cancer cells, bears a 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-one pentacyclic planar chromophore, whereas its synthetic 5,6-dehydro analogue, LAM-501, has a significantly tilted structure. DNA binding measurements by absorbance, fluorescence, and electric linear dichroism spectroscopy show that LAM-D is a weak DNA binder that intercalates between bp of the double helix. In contrast, the nonplanar analogue LAM-501 did not bind to DNA and failed to inhibit topoisomerase I. DNA intercalation may be required for the stabilization of topoisomerase I-DNA complexes by LAM-D. In the DNA relaxation assay, LAM-D strongly promoted the conversion of supercoiled DNA into nicked DNA in the presence of topoisomerase I. The marine product was approximately 5 times less efficient than camptothecin (CPT) at stabilizing topoisomerase I-DNA complexes, but interestingly, the two drugs exhibited slightly distinct sequence specificity profiles. Topoisomerase I-mediated DNA cleavage in the presence of LAM-D occurred at some sites common to CPT, but a few specific sites identified with CPT but not with LAM-D or conversely unique sites cleaved by LAM-D but not by CPT were detected. The distinct specificity profiles suggest that LAM-D and CPT interact differently with the topoisomerase I-DNA interface. A molecular modeling analysis provided structural information on the orientation of LAM-D within the topoisomerase I-DNA covalent complex. The marine alkaloid did not induce DNA cleavage by topoisomerase II. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped on DNA by LAM-D in P388 and CEM leukemia cells. P388/CPT5 and CEM/C2 cell lines, both resistant to CPT and expressing a mutated top1 gene, were cross-resistant to LAM-D. Collectively, the results identify LAM-D as a novel lead candidate for the development of topoisomerase I-targeted antitumor agents.