A2780 Ovarian Carcinoma Research Articles

Abstract 4494: MPT0G066, a novel microtubule-destabilizing agent, induces cell apoptosis and potentiates antineoplastic effects of cisplatin in human ovarian cancer cells in vitro and in vivo.

Abstract Ovarian cancer is one of the leading causes of death in women with gynecological malignancy in the United States. About 70% of ovarian cancer cases are diagnosed at an advanced stage resulting in poor survival rate. In the present study, we provide the data showing that MPT0G066, a novel synthetic arylsulfonamide compound, inhibited cell growth and viability in human ovarian cancer cell lines as evidenced by sulphorodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle distribution was also determined by flow cytometry, the data revealed that MPT0G066 induced cell cycle arrest at multipolyploidy (MP) phase (SKOV3 cells) or at G2/M phase (A2780 cells) in 24 hours and ultimately increased the cell population in sub-G1 phase in 48 hours. Moreover, in vitro tubulin polymerization assay, in situ labeling of β-tubulin and western blot analysis showed that MPT0G066, similar to colchicine and vincristine, promoted tubulin depolymerization and altered the expression of G2/M cell cycle regulatory proteins. Mechanistically, MPT0G066 induced JNK activation and altered Bcl-2 family proteins by increasing the expression of p-Bcl-2, Bax, Bim, Puma and decreasing the level of Bcl-2 and Mcl-1. Disruption of mitochondrial proteins triggered intrinsic apoptotic pathways through the cleavage of caspase-3, -7, -9, and poly (ADP-ribose) polymerase (PARP). Moreover, treatment with SP600125, a JNK inhibitor, abolished the effects of MPT0G066 on cell viability, Bcl-2 family proteins and apoptotic cell death. Finally, we used xenograft mouse model to demonstrate that MPT0G066 is able to suppress the growth of ovarian carcinoma A2780 cells and potentiated the antineoplastic effects of cisplatin in vivo. Immunohistochemistry data also confirmed MPT0G066-induced JNK activation in tumor sections. In summary, these results suggested that MPT0G066 could disrupt microtubule dynamics, and lead to cell cycle arrest at MP phase or G2/M phase. MPT0G066 induced JNK activation and altered Bcl-2 family proteins, which ultimately caused apoptosis through the intrinsic apoptotic pathways. These finding indicated that MPT0G066 is a potential anticancer agent worthy for further development and may foster novel treatment for patients with ovarian cancer. Citation Format: Chun-Han Chen, Ya-Chi Li, Li-Hsun Chang, Jing-Ping Liou, Shiow-Lin Pan, Che-Ming Teng. MPT0G066, a novel microtubule-destabilizing agent, induces cell apoptosis and potentiates antineoplastic effects of cisplatin in human ovarian cancer cells in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2013-4494

Micellar formulations of pro-apoptotic DM-PIT-1 analogs and TRAIL in vitro and in vivo

We have developed and characterized micellar formulations of analogs to the recently developed inhibitor of the phosphatidylinositol-3-kinase (PI3K) pathway (N-[(2-hydroxy-5-nitrophenyl)amino]carbonothioyl-3,5-dimethylbenzamide (DM-PIT-1)) for their physicochemical, loading and cytotoxic properties. The first generation inhibitor DM-PIT-1 is a non-lipid, small molecule inhibitor of phosphatidylinositol-3,4,5-triphosphate/Pleckstrin homology (PIP3/PH) binding capable of inhibiting the growth of tumor cells both in vitro and in vivo. A second generation of improved and druggable analogs has been developed. All compounds were successfully loaded (>70%) in PEG2000-PE micelles of 16–20 nm in size with several analogs demonstrating favorable cytotoxic activity against A2780 ovarian carcinoma. These compounds were also successfully incorporated into polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles combined with surface-bound tumor necrosis factor related apoptosis inducing ligand (TRAIL). The resulting multifunctional combination micelles were able to significantly enhance cytotoxic activity in the TRAIL-resistant A2780 cell line. Additionally, analogs NCL-176 and NCL-240 were effective in inhibiting tumor growth in an in vivo subcutaneous tumor model of A2780. These results indicate the utility of delivering TRAIL and PI3K pathway inhibitors in a combined micellar preparation.

Pre-Treatment of platinum resistant ovarian cancer cells with an MMP-9/MMP-2 inhibitor prior to cisplatin enhances cytotoxicity as determined by high content screening.

Platinum resistance is a major cause of treatment failure in ovarian cancer. We previously identified matrix metalloproteinase 9 (MMP-9) as a potential therapeutic target of chemoresistant disease. A2780cis (cisplatin-resistant) and A2780 (cisplatin-sensitive) ovarian carcinoma cell lines were used. The cytotoxic effect of MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) alone or in combination with cisplatin was determined using high content screening. Protein expression was examined using immunohistochemistry and ELISA. Co-incubation of cisplatin and an MMP-9/MMP-2 inhibitor, (2R)-2-[(4-Biphenylsulfonyl) amino]-3 phenylpropionic acid (C21H19NO4S) resulted in significantly greater cytotoxicity as compared to either treatment alone in a cisplatin resistant MMP-9 overexpressing cell line; A2780cis. In addition, pre-incubating with MMP-9i prior to cisplatin further enhances the cytotoxic effect. No significant difference was observed in MMP-9 protein in tissue but a trend towards increased MMP-9 was observed in recurrent serum. We propose that MMP-9/MMP-2i may be utilized in the treatment of recurrent/chemoresistant ovarian cancers that overexpress MMP-9 mRNA but its role in vivo remains to be evaluated.

Low-Cytotoxic Synthetic Bromorutaecarpine Exhibits Anti-Inflammation and Activation of Transient Receptor Potential Vanilloid Type 1 Activities

Rutaecarpine (RUT), the major bioactive ingredient isolated from the Chinese herb Evodia rutaecarpa, possesses a wide spectrum of biological activities, including anti-inflammation and preventing cardiovascular diseases. However, its high cytotoxicity hampers pharmaceutical development. We designed and synthesized a derivative of RUT, bromo-dimethoxyrutaecarpine (Br-RUT), which showed no cytotoxicity at 20 μM. Br-RUT suppressed nitric oxide (NO) production and tumor necrosis factor-α release in concentration-dependent (0~20 μM) manners in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages; protein levels of inducible NO synthase (iNOS) and cyclooxygenase-2 induced by LPS were downregulated. Br-RUT inhibited cell migration and invasion of ovarian carcinoma A2780 cells with 0~48 h of treatment. Furthermore, Br-RUT enhanced the expression of transient receptor potential vanilloid type 1 and activated endothelial NOS in human aortic endothelial cells. These results suggest that the synthetic Br-RUT possesses very low cytotoxicity but retains its activities against inflammation and vasodilation that could be beneficial for cardiovascular disease therapeutics.

Synthesis and evaluation of a backbone biodegradable multiblock HPMA copolymer nanocarrier for the systemic delivery of paclitaxel

The performance and safety of current antineoplastic agents, particularly water-insoluble drugs, are still far from satisfactory. For example, the currently widely used Cremophor EL®-based paclitaxel (PTX) formulation exhibits pharmacokinetic concerns and severe side effects. Thus, the concept of a biodegradable polymeric drug-delivery system, which can significantly improve therapeutic efficacy and reduce side effects is advocated. The present work aims to develop a new-generation of long-circulating, biodegradable carriers for effective delivery of PTX. First, a multiblock backbone biodegradable N-(2-hydroxypropyl)methacrylamide(HPMA) copolymer-PTX conjugate (mP-PTX) with molecular weight (Mw) of 335kDa was synthesized by RAFT (reversible addition-fragmentation chain transfer) copolymerization, followed by chain extension. In vitro studies on human ovarian carcinoma A2780 cells were carried out to investigate the cytotoxicity of free PTX, HPMA copolymer-PTX conjugate with Mw of 48kDa (P-PTX), and mP-PTX. The experiments demonstrated that mP-PTX has a similar cytotoxic effect against A2780 cells as free PTX and P-PTX. To further compare the behavior of this new biodegradable conjugate (mP-PTX) with free PTX and P-PTX in vivo evaluation was performed using female nu/nu mice bearing orthotopic A2780 ovarian tumors. Pharmacokinetics study showed that high Mw mP-PTX was cleared more slowly from the blood than commercial PTX formulation and low Mw P-PTX. SPECT/CT imaging and biodistribution studies demonstrated biodegradability as well as elimination of mP-PTX from the body. The tumors in the mP-PTX treated group grew more slowly than those treated with saline, free PTX, and P-PTX (single dose at 20mgPTX/kg equivalent). Moreover, mice treated with mP-PTX had no obvious ascites and body-weight loss. Histological analysis indicated that mP-PTX had no toxicity in liver and spleen, but induced massive cell death in the tumor. In summary, this biodegradable drug delivery system has a great potential to improve performance and safety of current antineoplastic agents.

Study of the betulin enriched birch bark extracts effects on human carcinoma cells and ear inflammation

BackgroundPentacyclic triterpenes, mainly betulin and betulinic acid, are valuable anticancer agents found in the bark of birch tree. This study evaluates birch bark extracts for the active principles composition.ResultsNew improved extraction methods were applied on the bark of Betula pendula in order to reach the maximum content in active principles. Extracts were analyzed by HPLC-MS, Raman, SERS and 13C NMR spectroscopy which revealed a very high yield of betulin (over 90%). Growth inhibiting effects were measured in vitro on four malignant human cell lines: A431 (skin epidermoid carcinoma), A2780 (ovarian carcinoma), HeLa (cervix adenocarcinoma) and MCF7 (breast adenocarcinoma), by means of MTT assay. All of the prepared bark extracts exerted a pronounced antiproliferative effect against human cancer cell lines. In vivo studies involved the anti-inflammatory effect of birch extracts on TPA-induced model of inflammation in mice.ConclusionsThe research revealed the efficacy of the extraction procedures as well as the antiproliferative and anti-inflammatory effects of birch extracts.

Abstract 3738: A rationale for combining the targeted investigational agents TAK-733, a MEK1/2 inhibitor, with alisertib (MLN8237), an Aurora A kinase inhibitor, for cancer therapy

Abstract MEK1/2 kinases have diverse functions in cell proliferation and survival, including the regulation of cell cycle transitions between the G1, S, and G2 phases and mediation of DNA damage response pathways. Therefore, inhibition of MEK1/2 may facilitate the antiproliferative effects of other therapies that disrupt cell cycle progression leading to DNA damage, including antimitotic therapies. AAK is a serine/threonine protein kinase essential for normal mitotic progression. Inhibition of AAK leads to inappropriate progression through mitosis in the presence of misaligned chromosomes resulting in aneuploidy and DNA damage, which leads to apoptosis or senescence. Here, we evaluated the combination of the allosteric MEK1/2 inhibitor TAK-733 with alisertib, the selective AAK inhibitor. Proliferation assays in multiple tumor cell lines showed a concentration-dependent decrease in the IC50 of TAK-733 and alisertib with increasing concentrations of the second agent, suggesting increased anti-proliferative activity of these agents when combined. This result was corroborated by measuring changes in cell confluence over time, a surrogate for proliferation rate, using an IncuCyte live-cell imaging system. The decreased proliferation rate of the combination was due in part to induction of apoptosis, as demonstrated by Annexin V and sub-diploid DNA content analysis. Cell cycle progression differences of the TAK-733/alisertib combination relative to the single agents were assessed using time-lapse video microscopy by measuring the time to the first mitotic event after drug addition, time spent in mitosis, and time between mitotic events. Of these, the only observed difference in cell-cycle progression time in the A2780 ovarian carcinoma cell line was the time between mitotic events, which was 10 hours longer for the combination relative to single agent alisertib. Moreover, the combination of TAK-733 and alisertib significantly reduced the ability of the cells to re-enter the cell cycle after one mitosis compared to either single agent; 30% for the combination, compared to 90% for TAK-733 and 76% for alisertib. Flow cytometry DNA content analysis showed that TAK-733 suppressed the formation of >4N/tetraploid cells induced by alisertib alone in multiple cell lines. This observed decrease in the proportion of >4N cells is greater than the decrease that could be extrapolated solely from the cell cycle-slowing effect of the combination, suggesting that TAK-733 reduces the proliferative potential of the alisertib-generated aneuploid cells via cell-cycle arrest and apoptosis. Collectively, these findings demonstrate an added benefit of TAK-733 over alisertib alone and provide a pre-clinical rationale for further investigation of the combination of TAK-733 and alisertib for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3738. doi:1538-7445.AM2012-3738

Increased apoptosis in cancer cells in vitro and in vivo by ceramides in transferrin-modified liposomes

Lysosomes are a promising therapeutic target for induction apoptosis in cancer cells due to lysosomal membrane permeabilization (LMP) leading to leakage of hydrolytic enzymes, especially the cathepsins, into the cytoplasm. We hypothesized that with the modification of the ceramide-loaded liposomes with transferrin (Tf), we would achieve both tumor targeting and increased delivery of lysosome-destabilizing agents, such as ceramides to lysosomes, to initiate LMP-induced apoptosis. We prepared Tf-modified (TL) and plain (PL) liposomes loaded with short (C6) or long (C16) N-acyl chain ceramides. Uptake, intracellular localization of liposomes, stability of the lysosomal membrane and release of cathepsin D were investigated on Hela cells by fluorescence microscopy and flow cytometry. Apoptosis was evaluated by binding of fluorescently-labeled Annexin V. Antitumor and pro-apoptotic effects of C6Cer-loaded Tf-liposomes were demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. TL were internalized specifically via the TfR-dependent endocytic pathway and localized within the endosome-lysosomal compartment. Ceramide-loaded Tf-liposomes significantly increased apoptosis compared with ceramide-free and ceramide-loaded non-modified liposomes. The treatment of cancer cells with TL led to increased LMP and cytoplasmic relocation of the intralysosomal cathepsin D. A strong antitumor and pro-apoptotic effect of C6Cer-loaded TL was also demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. The lysosomal accumulation of ceramides delivered by Tf-liposomes initiates the permeabilization of the lysosomal membranes required for the release of lysosomal cathepsins into the cytoplasm and initiation of the cancer cell apoptosis both in vitro and in vivo.

Abstract C171: TriplatinNC, a nucleolar-targeting platinum agent. Heparan sulfate-mediated cell entry, localization, and apoptosis.

Abstract TriplatinNC, a “non-covalent” derivative of the phase II clinical drug BBR3464, exhibits a distinct mode of DNA binding mediated through “phosphate clamps”, which display structural analogy to the “polyarginine” (arginine fork) DNA/RNA recognition motif. TriplatinNC shows an improved pharmacokinetic profile and increased cellular accumulation compared to BBR3464. Furthering the “polyarginine” analogy, the cellular accumulation of TriplatinNC, unlike cisplatin or oxaliplatin, is dependent upon the presence of cell surface glycosaminoglycans (GAGs), shown through comparison of platinum level accumulation in wild-type Chinese Hamster Ovary (CHO) cell lines to those lacking heparan sulfate (CHO-pgsD-677) or both heparan sulfate and chondroitin sulfate (CHO-pgsA-745). The cellular localization of TriplatinNC and cisplatin was visualized in confocal microscopy experiments utilizing the fluorescently-tagged derivatives, TriplatinNC-NBD or cisplatin-NBD. TriplatinNC, but not cisplatin, shows distinct localization to the nucleolar region of human colon and ovarian carcinoma cell lines, HCT116 and A2780, respectively. It was hypothesized that this pattern of localization, combined with nucleic acid affinity, would allow TriplatinNC to disrupt rRNA synthesis, the primary function within the nucleolus. Using 32P-metabolic labeling to monitor the rate of pre-rRNA transcript formation after cellular treatment, it was observed that both 47S pre-RNA and processed rRNAs, 32S, 28S, and 18S levels decreased dramatically compared to the untreated control in a dose-dependent manner. While the localization and abundance of nucleolar proteins, RNA pol I and UBTF, were unaffected by treatments with TriplatinNC at early timepoints, levels of the clinical relevant proliferative marker Ki-67, decreased markedly within one hour post-treatment. The morphological characteristics of apoptosis, including reduction in cell size, membrane blebbing, and cytosolic vacuolization, become visible 10hrs after drug treatment. This apoptotic process is not dependent on p53, as western blot analysis showed cleavage of procaspases 8, 9, and 3, followed by Parp-1 in HCT116 p53+/+ and p53−/− cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C171.

Tumor Accumulation of NIR Fluorescent PEG–PLA Nanoparticles: Impact of Particle Size and Human Xenograft Tumor Model

Cancer therapies are often terminated due to serious side effects of the drugs. The cause is the nonspecific distribution of chemotherapeutic agents to both cancerous and normal cells. Therefore, drug carriers which deliver their toxic cargo specific to cancer cells are needed. Size is one key parameter for the nanoparticle accumulation in tumor tissues. In the present study the influence of the size of biodegradable nanoparticles was investigated in detail, combining in vivo and ex vivo analysis with comprehensive particle size characterizations. Polyethylene glycol-polyesters poly(lactide) block polymers were synthesized and used for the production of three defined, stable, and nontoxic near-infrared (NIR) dye-loaded nanoparticle batches. Size analysis based on asymmetrical field flow field fractionation coupled with multiangle laser light scattering and photon correlation spectroscopy (PCS) revealed narrow size distribution and permitted accurate size evaluations. Furthermore, this study demonstrates the constraints of particle size data only obtained by PCS. By the multispectral analysis of the Maestro in vivo imaging system the in vivo fate of the nanoparticles next to their accumulation in special red fluorescent DsRed2 expressing HT29 xenografts could be followed. This simultaneous imaging in addition to confocal microscopy studies revealed information about the accumulation characteristics of nanoparticles inside the tumor tissues. This knowledge was further combined with extended size-dependent fluorescence imaging studies at two different xenograft tumor types, the HT29 (colorectal carcinoma) and the A2780 (ovarian carcinoma) cell lines. The combination of two different size measurement methods allowed the characterization of the dependence of nanoparticle accumulation in the tumor on even rather small differences in the nanoparticle size. While two nanoparticle batches (111 and 141 nm in diameter) accumulated efficiently in the human xenograft tumor tissue, the slightly bigger nanoparticles (diameter 166 nm) were rapidly eliminated by the liver.

Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg(483)-Val(484) with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON-Fc fusion protein, whereas hepsin-cleaved MSP bound with a K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.

Combination therapy with the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin achieves complete remissions and improves tolerability in an ovarian A2780 xenograft model

We recently reported on the in superior in vivo antitumor efficacy of the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206, formerly DOXO-EMCH), an albumin-binding prodrug of doxorubicin with acid-sensitive properties (see Fig. 1), in three tumor models in this journal, i.e. in three xenograft models (breast carcinoma M3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreatic carcinoma model (AsPC-1) [1] Previously, INNO-206 has shown significantly improved activity over doxorubicin in two xenograft breast cancer models and an orthotopic murine renal cell carcinoma model [2] and recently against resistant and non resistant myeloma (see http//:www.cytrx.com). INNO-206 binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site due to incorporation of an acid-sensitive hydrazone bond. The accumulation of INNO-206 in tumor tissue combined with a favorable biodistribution [3] is due to the pathophysiology of tumor tissue, characterized by a high metabolic turnover, angiogenesis, hypervasculature, a defective vascular architecture and an impaired lymphatic drainage, the so-called EPR (enhanced and retention) effect [4]. As a carrier for INNO-206, we selected albumin, which is emerging as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptideor protein-based drugs [5]. Albumin is a potential carrier for tumor targeting because it accumulates in solid tumors. In addition, we discovered that the HS-group of cysteine-34 of albumin is a unique and accessible functional group of a plasma protein for in situ prodrug conjugation considering that free thiol groups are absent from most circulating serum proteins except for albumin, which makes this group by far the most abundant thiol group in human plasma After injection directly into the blood stream, INNO-206 binds in situ to the cysteine-34 position of circulating albumin and is then transported in its albumin-bound form to the tumor site. INNO-206 is a prodrug of the anticancer agent doxorubicin in which doxorubicin is derivatized at its C-13 keto-position with a thiol-binding spacer molecule, i.e. 6-maleimidocaproic acid hydrazide. It contains an acidsensitive hydrazone linker that allows doxorubicin to be released either extracellularly in the slightly acidic environment often present in tumor tissue (pH range 6.0-6.8) [6] or intracellularly in acidic endosomal (pH 5.0-6.5) or lysosomal (pH 4.0-5.0) compartments after cellular uptake of the albumin conjugate by the tumor cell. INNO-206 was selected as an investigational product for clinical evaluation after toxicology studies in mice, rats, and dogs had shown a 2.5to 5-fold increase in the maximum tolerated dose (MTD) when compared to conventional doxoElectronic supplementary material The online version of this article (doi:10.1007/s10637-011-9686-5) contains supplementary material, which is available to authorized users.

Effects of Noncovalent Platinum Drug–Protein Interactions on Drug Efficacy: Use of Fluorescent Conjugates as Probes for Drug Metabolism

The overall efficacy of platinum based drugs is limited by metabolic deactivation through covalent drug-protein binding. In this study the factors affecting cytotoxicity in the presence of glutathione, human serum albumin (HSA) and whole serum binding with cisplatin, BBR3464, and TriplatinNC, a "noncovalent" derivative of BBR3464, were investigated. Upon treatment with buthionine sulfoximine (BSO), to reduce cellular glutathione levels, cisplatin and BBR3464-induced apoptosis was augmented whereas TriplatinNC-induced cytotoxicity was unaltered. Treatment of A2780 ovarian carcinoma cells with HSA-bound cisplatin (cisplatin/HSA) and cisplatin preincubated with whole serum showed dramatic decreases in cytotoxicity, cellular accumulation, and DNA adduct formation compared to treatment with cisplatin alone. Similar effects are seen with BBR3464. In contrast, TriplatinNC, the HSA-bound derivative (TriplatinNC/HSA), and TriplatinNC pretreated with whole serum retained identical cytotoxic profiles and equal levels of cellular accumulation at all time points. Confocal microscopy of both TriplatinNC-NBD, a fluorescent derivative of TriplatinNC, and TriplatinNC-NBD/HSA showed nuclear/nucleolar localization patterns, distinctly different from the lysosomal localization pattern seen with HSA. Cisplatin-NBD, a fluorescent derivative of cisplatin, was shown to accumulate in the nucleus and throughout the cytoplasm while the localization of cisplatin-NBD/HSA was limited to lysosomal regions of the cytoplasm. The results suggest that TriplatinNC can avoid high levels of metabolic deactivation currently seen with clinical platinum chemotherapeutics, and therefore retain a unique cytotoxic profile after cellular administration.

Abstract 2623: ST3595, a new histone deacetylase (HDAC) inhibitor endowed with a peculiar mechanism of action

Abstract The novel hydroxamic acid-based compound ST3595, was identified within an in vitro screening for its inhibitory activity on histone deacetylase's (HDAC's) (Pisano C. et al., Clin Cancer Res 2010, 16 (15): 3944-53; Dalla Valle S et al., Eur J Med Chem 2009, 44 (5): 1900-12). With aim to define its antitumor potential, we investigated the in vivo activity of ST3595 in a variety of tumor models. In addition, we performed a comparative study of ST3595 and vorinostat (SAHA) to better understand the cellular/molecular basis of the antiproliferative activity. In in vivo experiments, ST3595, delivered orally or i.p. at well-tolerated doses (100 mg/kg) induced a significant tumor growth inhibition (ranging from 57 to 87%) in a variety of human tumor xenografts (H460 non-small cell lung, HCT116 colon and A2780 ovarian carcinoma). In in vitro studies, ST3595 exhibited arrest of H460 cells (wt p53) in S phase associated with a large extent of apoptosis. Under conditions producing comparable antiproliferative effects, treatment with vorinostat resulted in a G2/M arrest with a marginal induction of apoptosis. In HT29 cells (mutant p53) ST3595 caused arrest in S and G2/M phases with apoptosis induction, whereas vorinostat induced G1 accumulation without evidence of apoptosis, thus suggesting only antiproliferative effects. To explore the basis of the different cellular effects, we investigated the modulation of various factors implicated in DNA damage response and repair, cell cycle control and regulation of apoptosis. In H460 cells, we found that, in contrast to vorinostat, ST3595 was an effective inducer of p53 acetylation, supporting a role of this p53 modification in the proapoptotic activity of ST3595. In addition, in contrast to vorinostat, ST3595 strongly affected the expression of critical proteins involved in cell cycle control and DNA damage response. In particular, ST3595 induced activation and phosphorylation of H2AX, and of the checkpoint kinases chk1 and chk2, thus supporting an interference of the HDAC inhibitor in the processes controlling DNA integrity. In the light of the use of broad-specificity HDAC inhibitors in cancer therapy and in combination with other antitumor agents, the ability of ST3595 to modulate DNA damage response and it proapoptotic efficacy may have relevant implications in enhancing antitumor activity of well-established cytotoxic agents. Preclinical studies of ST3595 with genotoxic agents support this interpretation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2623. doi:10.1158/1538-7445.AM2011-2623

Folate-decorated maleilated pullulan–doxorubicin conjugate for active tumor-targeted drug delivery

A novel folate-decorated maleilated pullulan–doxorubicin conjugate (abbreviated as FA–MP–DOX) for active tumor targeting was set up. The structure of this conjugate was confirmed by 1H NMR analysis. Furthermore, the conjugation efficiency, drug release property and stability of the conjugate were determined. The cellular uptake and cytotoxicity were assessed by using ovarian carcinoma A2780 cells as in vitro cell model. In vitro DOX release from FA–MP–DOX conjugate occurred at a faster rate at acidic pH compared to neutral pH (7.4). After 30 h of incubation at pH 2.5, 5.0 and 7.4 the released free DOX was about 68.71%, 50.08% and 26%, respectively. Based on the IC 50 values, the conjugate was found more effective with ovarian carcinoma A2780 cells than the parent drug after 48 h culture. These results suggested that FA–MP–DOX conjugate could be a promising doxorubicin carrier for its targeted and intracellular delivery.

Transcription factor Nrf2 maintains the basal expression of Mdm2: An implication of the regulation of p53 signaling by Nrf2

Co-operated regulation of oxidative stress–response transcription factors would be an important issue for animals to determine the cell fate under environmental stress. This notion raises a possibility that NF-E2-related factor 2 (Nrf2), which confers cytoprotection against oxidative stress, and p53 can have a direct co-regulation network. In the current study, we have indentified that the expression of murine double minute 2 (Mdm2) is repressed in nrf2-deleted murine embryonic fibroblasts (MEFs). This was confirmed by microarray, RT-PCR, and immunoblot analyses, and further promoter analysis showed that Nrf2 is directly involved in the basal expression of Mdm2 through the antioxidant response element, which is located in the first intron of this gene. This linkage between Nrf2 and Mdm2 appears to cause the accumulation of p53 protein in nrf2-deficent MEFs. In addition, we show that ovarian carcinoma A2780 cells with Nrf2 shRNA expression displayed higher levels of p53 activation in response to hydrogen peroxide treatment, leading to increased cell death. Collectively, our results suggest novel evidence that the inhibition of Nrf2 can suppress Mdm2 expression, which may result in p53 signaling modulation. In addition, this observation supports the concept that Nrf2 inhibition in cancer cells can facilitate apoptotic response upon environmental stress.

Pt(ii) complexes with bidentate and tridentate pyrazolyl-containing chelators: synthesis, structural characterization and biological studies

A series of four Pt(II) complexes anchored by bidentate or tridentate pyrazolyl-alkylamine chelators bearing different substituents at the azolyl rings has been prepared with the aim to assess their interest in the design of novel anticancer drugs. All complexes have been fully characterized by classical analytical methods and three of them were characterized also by X-ray diffraction analysis. Their solution behavior, together with lipophilicity measurements, cell uptake, antiproliferative properties, DNA interaction have been evaluated. Albeit all the complexes were less active than cisplatin on ovarian carcinoma A2780 cell line, greatly retained their activity in the cisplatin-resistant A2780cisR cell line and presented a lower resistance factor compared to cisplatin. Moreover, the Pt(II) complexes under investigation were less prone to undergo deactivation by glutathione, believed to be the major cellular target of cisplatin that inactivates the drug by binding to it irreversibly.

Expression of Notch3 and Notch intracellular domain in ovarian carcinoma and effect of N-[N-(3,5-difluorophenyl) acetyl-L-alanyl]-S-phenyl glycine t-butyl ester on ovarian carcinoma cell

To study the expression and clinical significance of Notch3 and Notch intracellular domain (NICD) in ovarian carcinoma and the effects of N-[N-(3,5-difluorophenyl) acetyl-L-alanyl]-S-phenyl glycine t-butyl ester (DAPT), a γ-secretase inhibitor on the proliferation and apoptosis in OVCAR3, A2780 ovarian carcinoma cell lines. Western blot was used to detect the expression of NICD in the tissues from 58 ovarian carcinomas patients and 21 normal ovarie, who were admitted in Peking University First Hospital from July 2006 to June 2009.Immunohistochemistry was also used to detect the expression of Notch3 in these tissues. The relationship with clinical features of ovarian carcinoma was also analyzed. Proliferation of OVCAR3 and A2780 ovarian cancer cells was determined by methyl thiazolyl tetrazolium (MTT) assay, cell cycles and apoptosis and index of proliferation were detected by flow cytometry method. The expression of NICD in OVCAR3 and A2780 cells incubated with DAPT was detected by western blot. (1) The expression level of NICD in ovarian carcinomas was significantly higher than that in normal ovarian tissues (1.64 ± 0.19 vs. 0.98 ± 0.20; P < 0.05). The NICD expression was higher in ovarian cancers with low grade or advanced stage than those in high-middle grade or early stage, respectively (1.90 ± 0.22 vs. 1.25 ± 0.21, 1.80 ± 0.21 vs. 1.21 ± 0.15; all P < 0.05). The Notch3 protein was stained positively in cytoplasm, nuclear and cell membrane. The expression of Notch3 was higher in ovarian carcinomas than that in normal ovaries [78% (45/58) vs. 24% (5/21); P < 0.01]. While, there were no stasistical difference in different pathological types, stages, differentiation of ovarian carcinoma. There was no difference between the patients with adjuvant chemotherapy or not. (2) After OVCAR3 and A2780 cells incubated with DAPT 24, 48, 72 hours, NICD expression was significantly lower than that in control group (P < 0.05). The effects of DAPT inhibited the proliferation and prompted the apoptosis of OVCAR3 and A2780 cells were depended on the concentrations and times. Notch3 and NICD may play a key role in the occurrence and progress of ovarian carcinoma. The mechanism of DAPT inhibited the proliferation and prompted the apoptosis of OVCAR3 and A2780 cells may be due to decreased the formation of NICD.

Antiproliferative Pt(IV) complexes: synthesis, biological activity, and quantitative structure–activity relationship modeling

Several Pt(IV) complexes of the general formula [Pt(L)2(L')2(L'')2] [axial ligands L are Cl-, RCOO-, or OH-; equatorial ligands L' are two am(m)ine or one diamine; and equatorial ligands L'' are Cl- or glycolato] were rationally designed and synthesized in the attempt to develop a predictive quantitative structure-activity relationship (QSAR) model. Numerous theoretical molecular descriptors were used alongside physicochemical data (i.e., reduction peak potential, Ep, and partition coefficient, log Po/w) to obtain a validated QSAR between in vitro cytotoxicity (half maximal inhibitory concentrations, IC50, on A2780 ovarian and HCT116 colon carcinoma cell lines) and some features of Pt(IV) complexes. In the resulting best models, a lipophilic descriptor (log Po/w or the number of secondary sp3 carbon atoms) plus an electronic descriptor (Ep, the number of oxygen atoms, or the topological polar surface area expressed as the N,O polar contribution) is necessary for modeling, supporting the general finding that the biological behavior of Pt(IV) complexes can be rationalized on the basis of their cellular uptake, the Pt(IV)-->Pt(II) reduction, and the structure of the corresponding Pt(II) metabolites. Novel compounds were synthesized on the basis of their predicted cytotoxicity in the preliminary QSAR model, and were experimentally tested. A final QSAR model, based solely on theoretical molecular descriptors to ensure its general applicability, is proposed.

Insulin-like Growth Factor 2 Expression Modulates Taxol Resistance and Is a Candidate Biomarker for Reduced Disease-Free Survival in Ovarian Cancer

This study was undertaken to examine the role of the insulin-like growth factor (IGF) signaling pathway in the response of ovarian cancer cells to Taxol and to evaluate the significance of this pathway in human epithelial ovarian tumors. The effect of Taxol treatment on AKT activation in A2780 ovarian carcinoma cells was evaluated using antibodies specific for phospho-AKT. To study the drug-resistant phenotype, we developed a Taxol-resistant cell line, HEY-T30, derived from HEY ovarian carcinoma cells. IGF2 expression was measured by real-time PCR. A type 1 IGF receptor (IGF1R) inhibitor, NVP-AEW541, and IGF2 small interfering RNA were used to evaluate the effect of IGF pathway inhibition on proliferation and Taxol sensitivity. IGF2 protein expression was evaluated by immunohistochemistry in 115 epithelial ovarian tumors and analyzed in relation to clinical/pathologic factors using the chi(2) or Fisher's exact tests. The influence of IGF2 expression on survival was studied with Cox regression. Taxol-induced AKT phosphorylation required IGF1R tyrosine kinase activity and was associated with upregulation of IGF2. Resistant cells had higher IGF2 expression compared with sensitive cells, and IGF pathway inhibition restored sensitivity to Taxol. High IGF2 tumor expression correlated with advanced stage (P < 0.001) and tumor grade (P < 0.01) and reduced disease-free survival (P < 0.05). IGF2 modulates Taxol resistance, and tumor IGF2 expression is a candidate prognostic biomarker in epithelial ovarian tumors. IGF pathway inhibition sensitizes drug-resistant ovarian carcinoma cells to Taxol. Such novel findings suggest that IGF2 represents a therapeutic target in ovarian cancer, particularly in the setting of Taxol resistance.