Kyu Kwang Kim

Cranberry proanthocyanidins are cytotoxic to human cancer cells and sensitize platinum-resistant ovarian cancer cells to paraplatin.

Polyphenolic extracts of the principal flavonoid classes present in cranberry were screened in vitro for cytotoxicity against solid tumor cells lines, identifying two fractions composed principally of proanthocyanidins (PACs) with potential anticancer activity. Matrix‐Assisted Laser Desorption/Ionization Time‐Of‐Flight Mass Spectrometry (MALDI‐TOF‐MS) analysis of the proanthocyanidins (PACs) fractions indicated the presence of A‐type PACs with 1–4 linkages containing between 2–8 epicatechin units with a maximum of 1 epigallocatechin unit. PACs exhibited in vitro cytotoxicity against platinum‐resistant human ovarian, neuroblastoma and prostate cancer cell lines (IC50 = 79–479 µg/mL) but were non‐cytotoxic to lung fibroblast cells (IC50 > 1000 µg/ml). SKOV‐3 ovarian cancer cells treated with PACs exhibited classic apoptotic changes. PACs acted synergistically with paraplatin in SKOV‐3 cells. Pretreatment of SKOV‐3 cells with PACs (106 µg/ml) resulted in a significant reduction of the paraplatin IC50 value. Similarly, in a BrdU incorporation assay, co‐treatment of SKOV‐3 cells with PACs and paraplatin revealed reduced cell proliferation at lower concentrations than with either individually. In SKOV‐3 cell cultures co‐treated with PAC‐1 and paraplatin, an HPLC analysis indicated differential quantitative presence of various PAC oligomers such as DP‐8, ‐9, ‐11 and ‐14 indicating either selective binding or uptake. Cranberry proanthocyanidins exhibit cell‐line specific cytotoxicity, induce apoptotic markers and augment cytotoxicity of paraplatin in platinum‐resistant SKOV‐3 ovarian cancer cells. Copyright

Iron(III)-Salophene: An Organometallic Compound with Selective Cytotoxic and Anti-Proliferative Properties in Platinum- Resistant Ovarian Cancer Cells

Background In this pioneer study to the biological activity of organometallic compound Iron(III)-salophene (Fe-SP) the specific effects of Fe-SP on viability, morphology, proliferation, and cell-cycle progression on platinum-resistant ovarian cancer cell lines were investigated. Methodology/Principal Findings Fe-SP displayed selective cytotoxicity against SKOV-3 and OVCAR-3 (ovarian epithelial adenocarcinoma) cell lines at concentrations between 100 nM and 1 µM, while the viability of HeLa cells (epithelial cervix adenocarcinoma) or primary lung or skin fibroblasts was not affected. SKOV-3 cells in contrast to fibroblasts after treatment with Fe-SP revealed apparent hallmarks of apoptosis including densely stained nuclear granular bodies within fragmented nuclei, highly condensed chromatin and chromatin fragmentation. Fe-SP treatment led to the activation of markers of the extrinsic (Caspase-8) and intrinsic (Caspase-9) pathway of apoptosis as well as of executioner Caspase-3 while PARP-1 was deactivated. Fe-SP exerted effects as an anti-proliferative agent with an IC50 value of 300 nM and caused delayed progression of cells through S-phase phase of the cell cycle resulting in a complete S-phase arrest. When intra-peritoneally applied to rats Fe-SP did not show any systemic toxicity at concentrations that in preliminary trials were determined to be chemotherapeutic relevant doses in a rat ovarian cancer cell model. Conclusion/Significance The present report suggests that Fe-SP is a potent growth-suppressing agent in vitro for cell lines derived from ovarian cancer and a potential therapeutic drug to treat such tumors in vivo.

HE4 (WFDC2) gene overexpression promotes ovarian tumor growth

Selective overexpression of Human epididymal secretory protein E4 (HE4) points to a role in ovarian cancer tumorigenesis but little is known about the role the HE4 gene or the gene product plays. Here we show that elevated HE4 serum levels correlate with chemoresistance and decreased survival rates in EOC patients. HE4 overexpression promoted xenograft tumor growth and chemoresistance against cisplatin in an animal model resulting in reduced survival rates. HE4 displayed responses to tumor microenvironment constituents and presented increased expression as well as nuclear translocation upon EGF, VEGF and Insulin treatment and nucleolar localization with Insulin treatment. HE4 interacts with EGFR, IGF1R, and transcription factor HIF1α. Constructs of antisense phosphorothio-oligonucleotides targeting HE4 arrested tumor growth in nude mice. Collectively these findings implicate increased HE4 expression as a molecular factor in ovarian cancer tumorigenesis. Selective targeting directed towards the HE4 protein demonstrates therapeutic benefits for the treatment of ovarian cancer.

Anti‐proliferative and Pro‐apoptotic Properties of 3‐Bromoacetoxy Calcidiol in High‐risk Neuroblastoma

The cytotoxic, anti‐proliferative and apoptotic effects of 3‐Bromoacetoxy Calcidiol (B3CD), a derivative of vitamin D3 precursor calcidiol, on human neuroblastoma (NB) cells were examined. NB, predominantly a tumor of early childhood, is the most common extracranial solid tumor. Despite aggressive treatments, survival for advanced stages remains low and novel treatment strategies are needed. B3CD‐induced apoptosis in various neuroblastic cells via caspases‐3 and ‐9 activation. B3CD upregulated mitochondrial pro‐apoptotic Bax and anti‐apoptotic Bcl‐2 expression, caused cytochrome c release, downregulated N‐Myc expression and activated pro‐survival marker Akt. Accordingly, B3CD treatment dose dependently reduced the viability of NB cells with IC50 values between 1 and 3 μm. The cytotoxicity of B3CD was significantly higher than for the calcemic parent‐compound vitamin D3 (IC50 between 10 and 30 μm). Further studies revealed that B3CD treatment inhibits the proliferation of NB cells at low concentrations (IC50 between 30 and 100 nm). Cell cycle analysis showed a dramatic increase in the apoptotic sub‐diploidal population along with a cell cycle block. In summary, the present study shows that B3CD is toxic to NB cells via suppression of cell proliferation and cell viability by caspase activation and regulation of survival signals. These results suggest that B3CD could be developed as a treatment for NB.

A novel indole ethyl isothiocyanate (7Me-IEITC) with anti-proliferative and pro-apoptotic effects on platinum-resistant human ovarian cancer cells

OBJECTIVE A novel indole ethyl isothiocyanate derivative (7Me-IEITC) was defined as a potent growth-suppressing agent to cell lines derived from ovarian cancers. Key mechanisms of the cellular response in vitro were studied and suggest a potential of 7Me-IEITC as a therapeutic drug. METHODS The viability of ovarian cancer cell lines (SKOV-3, OVCAR-3) in comparison to pancreatic and prostate cancer cell lines, primary fibroblast and immortalized trophoblasts after treatment with 7Me-IEITC was analyzed. Morphological and apoptotic responses of SKOV-3 were studied by fluorescence microscopy (DAPI staining, TUNEL assay). SKOV-3 proliferation was estimated by a standardized BrdU incorporation assay. The phosphorylation of MAP-Kinases, pro-survival factors and the activation of caspases and PARP-1 were analyzed by western blotting. Changes of the mitochondrial transmembrane-potential and in cell-cycle progression were studied by FACS analysis. MAP-Kinase and caspase inhibitors were employed in cytotoxicity studies. RESULTS 7Me-IEITC selectively reduced the viability of SKOV-3, OVCAR-3, BXPC-3 and PC-3 cells (IC(50) values < or = 5 microM), while the viability of fibroblasts or trophoblasts remained un-affected at concentrations below 20 microM. 7Me-IEITC treatment down-regulated pro-survival kinases and transcription factors (STAT-3, IKKalpha and NF-kappaB), caused rapid loss of the mitochondrial transmembrane-potential and inactivation of PARP-1 along with activation of caspases. The use of p38 MAP-Kinase-and caspase inhibitors suppressed the cytotoxicity of the drug. 7Me-IEITC acted as an anti-proliferative agent and arrested the cell-cycle progression of SKOV-3 in G2/M phase. CONCLUSION 7Me-IEITC is a potent and growth-suppressing agent to cell lines derived from ovarian cancers by causing deactivation of survival signals, apoptosis, and cell-cycle arrest.

Apoptotic and chemotherapeutic properties of iron(III)-salophene in an ovarian cancer animal model

The cytotoxicity of organometallic compounds iron(III)-, cobalt(III)-, manganese(II)-, and copper(II)-salophene (-SP) on platinum-resistant ovarian cancer cell lines was compared. Fe-SP displayed selective cytotoxicity (IC50 at ~1 μM) against SKOV-3 and OVCAR-3 cell lines while Co-SP caused cytotoxic effects only at higher concentrations (IC50 at 60 μM) and Cu-SP effects were negligible. High cytotoxicity of Mn-SP (30–60 μM) appeared to be nonspecific because the Mn-chloride salt reduced cell viability similarly. The effect of Fe-SP at 1 μM proved to be ovarian cancer cell selective when compared to a panel of cell lines derived from different tumors. The first irreversible step in the induction of cell death by Fe-SP occurred after 3 hrs as indicated by the mitochondrial transmembrane potential (ΔΨm) and was mainly linked to apoptotic, not necrotic events. To evaluate the toxicity of Fe-SP in vivo we conducted an acute toxicity study in rats. The LD50 of Fe-SP is >2000 mg/kg orally and >5.5 mg/kg body weight by intraperitoneal injection. An ovarian cancer animal model showed that the chemotherapeutic relevant dose of Fe-SP in rats is 0.5–1 mg/kg body weight. The present report suggests that Fe-SP is a potential therapeutic drug to treat ovarian cancer.

HE4 expression is associated with hormonal elements and mediated by importin-dependent nuclear translocation

Antiestrogens including tamoxifen and fulvestrant have been evaluated as chemotherapeutics for ovarian cancer, particularly in cases of platinum resistant disease. Human epididymis protein 4 (HE4) is highly overexpressed in women with ovarian cancer and overexpression of HE4 has been found to correlate with platinum resistance. However, the role of HE4 in modulating responses to hormones and hormonal therapy has not been characterized in ovarian cancer. Here we demonstrate that 17β-estradiol, tamoxifen, and fulvestrant induce nuclear and nucleolar translocation of HE4 and that HE4 overexpression induces resistance to antiestrogens. HE4 was found to interact with estrogen receptor-α (ER-α), and HE4 overexpression resulted in ER-α downregulation in vitro and in human ovarian cancers. We identified a novel role for importin-4 in governing the nuclear transport of HE4. Treatment with ivermectin, an importin inhibitor, blocked HE4/importin-4 nuclear accumulation and sensitized HE4-overexpressing ovarian cancer cells to fulvestrant and tamoxifen.

Organometallic Iron(III)-Salophene Exerts Cytotoxic Properties in Neuroblastoma Cells via MAPK Activation and ROS Generation

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as p38 and JNK MAPK activation, DNA degradation, and elevated generation of reactive oxygen species (ROS) in SMS-KCNR NB cells. In contrast to Fe-SP, non-complexed salophene or Cu(II)-SP did not raise ROS levels in NB or SKOV-3 ovarian cancer control cells. Cytotoxicity of Fe-SP and activation of caspase-3, -7, PARP, pro-apoptotic p38 and JNK MAPK could be prevented by co-treatment with antioxidants suggesting ROS generation is the primary mechanism of cytotoxic action. We report here that Fe-SP is a potent growth-suppressing and cytotoxic agent for in vitro NB cell lines and, due to its high tolerance in previous animal toxicity studies, a potential therapeutic drug to treat NB tumors in vivo.

Lipophilic aroylhydrazone chelator HNTMB and its multiple effects on ovarian cancer cells

BackgroundMetal chelators have gained much attention as potential anti-cancer agents. However, the effects of chelators are often linked solely to their capacity to bind iron while the potential complexation of other trace metals has not been fully investigated. In present study, we evaluated the effects of various lipophilic aroylhydrazone chelators (AHC), including novel compound HNTMB, on various ovarian cancer cell lines (SKOV-3, OVCAR-3, NUTU-19).MethodsCell viability was analyzed via MTS cytotoxicity assays and NCI60 cancer cell growth screens. Apoptotic events were monitored via Western Blot analysis, fluorescence microscopy and TUNEL assay. FACS analysis was carried out to study Cell Cycle regulation and detection of intracellular Reactive Oxygen Species (ROS)ResultsHNTMB displayed high cytotoxicity (IC50 200-400 nM) compared to previously developed AHC (oVtBBH, HNtBBH, StBBH/206, HNTh2H/315, HNI/311; IC50 0.8-6 μM) or cancer drug Deferoxamine, a hexadentate iron-chelator (IC50 12-25 μM). In a NCI60 cancer cell line screen HNTMB exhibited growth inhibitory effects with remarkable differences in specificity depending on the cell line studied (GI50 10 nM-2.4 μM). In SKOV-3 ovarian cancer cells HNTMB treatment led to chromatin fragmentation and activation of the extrinsic and intrinsic pathways of apoptosis with specific down-regulation of Bcl-2. HNTMB caused delayed cell cycle progression of SKOV-3 through G2/M phase arrest. HNTMB can chelate iron and copper of different oxidation states. Complexation with copper lead to high cytotoxicity via generation of reactive oxygen species (ROS) while treatment with iron complexes of the drug caused neither cytotoxicity nor increased ROS levels.ConclusionsThe present report suggests that both, non-complexed HNTMB as a chelator of intracellular trace-metals as well as a cytotoxic HNTMB/copper complex may be developed as potential therapeutic drugs in the treatment of ovarian and other solid tumors.

Isothiocyanate NB7M causes selective cytotoxicity, pro-apoptotic signalling and cell-cycle regression in ovarian cancer cells

The present report identifies indole-3-ethyl isothiocyanate NB7M as a potent cytotoxic agent with selective activity against cell lines derived from various tumour types. Ovarian cancer cell lines showed sensitivity to NB7M (60–70% cytotoxicity at 2.5 μM), in contrast to control cells (TCL-1 and HTR-8; IC50 ∼15 μM). In a screen performed by the National Cancer Institute (NCI) (NCI60 cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC50 between 0.1 and 10 μM depending on the cell line studied. Using SKOV-3 ovarian cancer cells as a model, mechanisms of cytotoxicity were analysed. NB7M caused hallmarks of apoptosis such as PARP-1 deactivation, chromatin condensation, DNA nicks, activation of caspases-9, -8, -3, loss of mitochondrial transmembrane depolarisation potential and upregulation of pro-apoptotic mitogen activated protein kinases (p38, SAP/JNK). NB7M downregulated phosphorylation of prosurvival kinases (PI-3K, AKT, IKKα), transcription factor NF-κB, and expression of DNA-Pk and AXL receptor tyrosine kinase. Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression. The present report suggests that NB7M is a selective cytotoxic agent in vitro for cell lines derived from ovarian and certain other tumours. In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.