Use of NQO1 status as a selective biomarker for oesophageal squamous cell carcinomas with greater sensitivity to 17-AAG.

BACKGROUND: Oesophageal squamous cell carcinoma (OSCC) is a major health burden in Sub-Saharan Africa, and novel chemotherapies are urgently required to combat this disease. The heat shock protein 90 (HSP90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) has previously been proposed as a possible candidate drug. NADPH quinone oxidoreductase 1 (NQO1) is known to increase the potency of 17-AAG, therefore we investigated the effects of 17-AAG in OSCC cell lines in the context of their NQO1 status. METHODS: We used MTT assays to compare the sensitivity of a panel of OSCC cell lines to 17-AAG. Western blotting, and RT-PCR were used to investigate NQO1 protein and mRNA levels, while an RFLP approach was used to investigate the NQO1 C609T SNP. RESULTS: Expression of NQO1 markedly increased sensitivity to 17-AAG in the OSCC cell lines, while normal fibroblasts, which expressed HSP90 at much lower levels, were more resistant to 17-AAG. In isolation, neither the C609T SNP, nor NQO1 mRNA levels was an accurate predictor of NQO1 protein levels. CONCLUSIONS: Since NQO1 greatly enhances the anti-cancer effects of 17-AAG, this could be used as a selective marker for patients that would benefit most from 17-AAG chemotherapy at low doses. Testing for the presence of the C609T SNP in both alleles could be used as a screen to exclude potentially poor responders to 17-AAG treatment at low dosages.

A key role for early growth response-1 and nuclear factor-kappaB in mediating and maintaining GRO/CXCR2 proliferative signaling in esophageal cancer.

Although early growth response-1 (EGR-1) has been shown as a key transcription factor in controlling cell growth, proliferation, differentiation, and angiogenesis, its role in the development of esophageal cancer is poorly understood despite the high frequency of this disease in many parts of the world. Here, immunohistochemistry showed that EGR-1 is overexpressed in 80% of esophageal tumor tissues examined. Furthermore, EGR-1 is constitutively expressed in all esophageal cancer cell lines analyzed. Esophageal squamous carcinoma WHCO1 cells stably transfected with EGR-1 short hairpin RNA displayed a 55% reduction in EGR-1 protein levels, 50% reduction in cell proliferation, a 50% reduction in cyclin-dependent kinase 4 levels, and a 2-fold induction in p27(Kip1) levels associated with a G(2)-M cell cycle arrest. EGR-1 knockdown also caused a marked induction in IkappaBalpha expression, an effect also observed in GRObeta RNA interference-expressing WHCO1 cells, because EGR-1 lies downstream of GRO/CXCR2 signaling. Furthermore, p65 mRNA levels were also reduced in cells treated with either short hairpin RNA EGR-1 or small interfering RNA EGR-1. Immunohistochemical analysis indicated that p65 is elevated in 78% (n = 61) of esophageal tumor sections analyzed. Moreover, nuclear factor-kappaB inhibition with either sodium salicylate or p65 RNA interference led to a significant reduction in GROalpha and GRObeta expression. These results indicate that EGR-1 and nuclear factor-kappaB mediate GRO/CXCR2 proliferative signaling in esophageal cancer and may represent potential target molecules for therapeutic intervention.

The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation.

The Karyopherin proteins are involved in nucleo-cytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest they are important in nuclear envelope component assembly, mitosis and replication. Since these are all critical cellular functions, alterations in the expression of the Karyopherins may have an impact on the biology of cancer cells. In this study, we examined the expression of the Karyopherins, Crm1, Karyopherin beta1 (Kpnbeta1) and Karyopherin alpha2 (Kpnalpha2), in cervical tissue and cell lines. The functional significance of these proteins to cancer cells was investigated using individual siRNAs to inhibit their expression. Microarrays, quantitative RT-PCR and immunofluorescence revealed significantly higher expression of Crm1, Kpnbeta1 and Kpnalpha2 in cervical cancer compared to normal tissue. Expression levels were similarly elevated in cervical cancer cell lines compared to normal cells, and in transformed epithelial and fibroblast cells. Inhibition of Crm1 and Kpnbeta1 in cancer cells significantly reduced cell proliferation, while Kpnalpha2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnbeta1. The reduction in proliferation of cancer cells was associated with an increase in a subG1 population by cell cycle analysis and Caspase-3/7 assays revealed increased apoptosis. Crm1 and Kpnbeta1 siRNA-induced apoptosis was accompanied by an increase in the levels of growth inhibitory proteins, p53, p27, p21 and p18. Our results demonstrate that Crm1, Kpnbeta1 and Kpnalpha2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnbeta1, not Kpnalpha2, induces cancer cell death, making Crm1 and Kpnbeta1 promising candidates as both biomarkers and potential anticancer therapeutic targets.

A growth-related oncogene/CXC chemokine receptor 2 autocrine loop contributes to cellular proliferation in esophageal cancer.

Growth-related oncogene (GRO), a member of the CXC chemokine subfamily, plays a major role in inflammation and wound healing. CXC chemokines have been found to be associated with tumorigenesis, angiogenesis, and metastasis. Although elevated expression of GRO has been reported in several human cancers, the expression and role of GRO and its receptor, CXCR2, in esophageal cancer are poorly understood. This study used real-time reverse transcription-PCR (RT-PCR) and immunohistochemical approaches to show that GROalpha, GRObeta, and CXCR2 are up-regulated in esophageal tumor tissue. Furthermore, GROalpha, GRObeta, and CXCR2 are constitutively expressed in WHCO1, an esophageal cancer cell line that was used as a model system here. GRObeta enhances transcription of EGR-1, via the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be blocked by a specific antagonist of CXCR2 (SB 225002) or specific antibody to GRObeta. WHCO1 cells treated with SB 225002 exhibited a 40% reduction in cell proliferation. A stable WHCO1 GROalpha RNA interference (RNAi) clone displayed a 43% reduction in GROalpha mRNA levels as determined by real-time RT-PCR, reduced levels of GROalpha by fluorescence microscopy, and a 60% reduction in the levels of phosphorylated ERK1/2. A stable clone expressing GRObeta RNAi displayed >95% reduction in GRObeta mRNA levels, reduced levels of GRObeta by fluorescence microscopy, and an 80% reduction in the levels of phosphorylated ERK1/2. Moreover, these GROalpha RNAi- and GRObeta RNAi-expressing clones displayed a 20% and 50% decrease in cell proliferation, respectively. Our results suggest that GROalpha-CXCR2 and GRObeta-CXCR2 signaling contributes significantly to esophageal cancer cell proliferation and that this autocrine signaling pathway may be involved in esophageal tumorigenesis.

Reactive oxygen species mediated apoptosis of esophageal cancer cells induced by marine triprenyl toluquinones and toluhydroquinones.

Marine invertebrates, algae, and microorganisms are prolific producers of novel secondary metabolites. Some of these secondary metabolites have the potential to be developed as chemotherapeutic agents for the treatment of a wide variety of diseases, including cancer. We describe here the mechanism leading to apoptosis of esophageal cancer cell lines in the presence of triprenylated toluquinones and toluhydroquinones originally isolated from the Arminacean nudibranch Leminda millecra. Triprenylated toluquinone-induced and toluhydroquinone-induced cell death is mediated via apoptosis after a cell cycle block. Molecular events include production of reactive oxygen species (ROS), followed by induction and activation of c-Jun (AP1) via c-Jun-NH2-kinase-mediated and extracellular signal-regulated kinase-mediated pathways. Partial resistance to these compounds could be conferred by the ROS scavengers Trolox and butylated hydroxyanisol, a c-Jun-NH2-kinase inhibitor, and inhibition of c-Jun with a dominant negative mutant (TAM67). Interestingly, the levels of ROS produced varied between compounds, but was proportional to the ability of each compound to kill cells. Because cancer cells are often more susceptible to ROS, these compounds present a plausible lead for new antiesophageal cancer treatments and show the potential of the South African marine environment to provide new chemical entities with potential clinical significance.

NDRG1 overexpression promotes the progression of esophageal squamous cell carcinoma through modulating Wnt signaling pathway.

N-myc down-regulated gene 1 (NDRG1) has been shown to regulate tumor growth and metastasis in various malignant tumors and also to be dysregulated in esophageal squamous cell carcinoma (ESCC). Here, we show that NDRG1 overexpression (91.9%, 79/86) in ESCC tumor tissues is associated with poor overall survival of esophageal cancer patients. When placed in stable transfectants of the KYSE 30 ESCC cell line generated by lentiviral transduction with the ectopic overexpression of NDRG1, the expression of transducin-like enhancer of Split 2 (TLE2) was decreased sharply, however ß-catenin was increased. Mechanistically, NDRG1 physically associates with TLE2 and ß-catenin to affect the Wnt pathway. RNA interference and TLE2 overexpression studies demonstrate that NDRG1 fails to active Wnt pathway compared with isogenic wild-type controls. Strikingly, NDRG1 overexpression induces the epithelial mesenchymal transition (EMT) through activating the Wnt signaling pathway in ESCC cells, decreased the expression of E-cadherin and enhanced the expression of Snail. Our study elucidates a mechanism of NDRG1-regulated Wnt pathway activation and EMT via affecting TLE2 and  ß-catenin expression in esophageal cancer cells. This indicates a pro-oncogenic role for NDRG1 in esophageal cancer cells whereby it modulates tumor progression.

Mono- and multimeric ferrocene congeners of quinoline-based polyamines as potential antiparasitics.

A series of mono- and multimeric polyamine-containing ferrocenyl complexes containing a quinoline motif were prepared. The complexes were characterised by standard techniques. The molecular structure of the monomeric salicylaldimine derivative was elucidated using single crystal X-ray diffraction and was consistent with the proposed structure. The antiplasmodial activity of the compounds were evaluated in vitro against both the NF54 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains of Plasmodium falciparum. The polyamine derivatives exhibit good resistance index values suggesting that these systems are beneficial in overcoming the resistance experienced by chloroquine. Mechanistic studies suggest that haemozoin formation may be the target of these quinoline complexes in the parasite. Some of the complexes exhibit moderate to high cytotoxicity against WHCO1 oesophageal cancer cells in vitro. The monomeric ferrocenyl-amine complexes exhibit potent activity against this particular cell line. The complexes were also screened against the G3 strain of Trichomonas vaginalis and the salicylaldimine complexes demonstrated promising activity at the tested concentration. All of these compounds show no inhibitory effect on several common normal flora bacteria, indicative of their selectivity for eukaryotic pathogens and cancer.

Antiesophageal cancer activity from Southern African marine organisms.

Squamous cell esophageal cancer presents a significant health burden in many developing countries around the world. In South Africa, this disease is one of the most common causes of cancer-related deaths in black males. Because this cancer is only modestly responsive to available chemotherapeutic agents, there is a need to develop more effective therapeutic agents for this cancer. Marine organisms are currently regarded as a promising source of unique bioactive molecules because they display a rich diversity of secondary metabolites. Some of these compounds have significant anticancer activity, with a few of these currently in phase I and II clinical trials. We report here an ongoing program to screen marine organisms collected from subtidal benthic communities off the coast of southern Africa for activity against cultured esophageal cancer cells. Of the 137 extracts tested, 2.2% displayed high activity (score = 3) and 11.7% displayed moderate activity (score = 2) against cultured esophageal cancer cells. Our results suggest that sponges had a higher hit rate (21.9%) than ascidians (7.1%). Using activity-directed purification, seven previously described compounds and four novel compounds, with varying activity against esophageal cancer cell lines, were isolated from the sponges Axinella weltneri, Aplysilla sulphurea, and Strongylodesma aliwaliensis. The results of this study suggest that subtidal benthic marine organisms collected off the coast of southern Africa hold potential for identifying possible drug leads for the development of agents with activity against esophageal cancer.

Plocoralides A-C, polyhalogenated monoterpenes from the marine alga Plocamium corallorhiza.

Three new polyhalogenated monoterpenes, plocoralides A-C (1-3) along with three known compounds (4-6) have been isolated from the organic extract of the red alga P. corallorhiza. Structures of the new compounds were characterized as 4,8-dibromo-1,1-dichloro-3,7-dimethyl-2E,6E-octadiene (1), 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene (2) and 4,8-dibromo-1,1,7-trichloro-3,7-dimethyl-2E,5Z-octadiene (3) on the basis of one- and two-dimensional NMR spectroscopic data and MS analyses. Compounds 2-6 show moderate cytotoxicity toward esophageal cancer cells.

Polyamine quinoline rhodium complexes: synthesis and pharmacological evaluation as antiparasitic agents against Plasmodium falciparum and Trichomonas vaginalis.

A series of mono- and bis-salicylaldimine ligands and their corresponding Rh(i) complexes were prepared. The compounds were characterised using standard spectroscopic techniques including NMR, IR spectroscopy and mass spectrometry. The salicylaldimine ligands and complexes were screened for antiparasitic activity against two strains of Plasmodium falciparum i.e. the NF54 CQ-sensitive and K1 CQ-resistant strain as well as against the G3 isolate of Trichomonas vaginalis. The monomeric salicylaldimine quinolines exhibited good activity against the NF54 strain and the dimeric salicylaldimine quinolines exhibited no cross resistance across the two strains. The binuclear 5-chloro Rh(i) complex displayed the best activity against the Trichomonas vaginalis parasite, possibly a consequence of its enhanced lipophilicity. The compounds were also screened for cytotoxicity in vitro against WHCO1 oesophageal cancer cells. The monomeric salicylaldimine quinolines exhibited high selectivity towards malaria parasites compared to cancer cells, while the dimeric compounds were less selective.

Elevated expression of the nuclear export protein, Crm1 (exportin 1), associates with human oesophageal squamous cell carcinoma.

The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Real­time RT­PCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.

Oncogenic but non-essential role of N-myc downstream regulated gene 1 in the progression of esophageal squamous cell carcinoma.

N-myc downstream regulated gene 1 (NDRG1/Cap43/Drg-1) has previously been shown to be dysregulated in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the role of NDRG1 in the neoplastic progression of ESCC using ectopic gain-of-function and loss-of-function approaches. Stable transfectants of the KYSE30 ESCC cell line with altered NDRG1 levels were generated by lentiviral transduction. Although no measurable effects on in vitro cell proliferation were observed with altered NDRG1 expression, the ectopic overexpression of NDRG1 was positively linked to recognized markers of metastasis, angiogenesis and apoptotic evasion. Accordingly, in the nude mouse xenograft model system, NDRG1 overexpression promoted the in vivo growth of KYSE30 derived xenografts, which could be attributed to the reduced apoptotic and enhanced angiogenic activities associated with this gene. These processes were mediated in part by increased NFκB activity in NDRG1 overexpressing cells. Nevertheless, no significant phenotypic changes were observed in response to NDRG1 knock-down, suggesting that this gene might not be essential for the neoplastic progression of ESCC. Taken together, our results suggest that NDRG1 may play positive but dispensable roles in the progression of esophageal squamous cell carcinoma.

Cytotoxicity of lapachol, ß-lapachone and related synthetic 1,4-naphthoquinones against oesophageal cancer cells.

Naphthoquinones have been found to have a wide range of biological activities, including cytotoxicity to cancer cells. The secondary metabolites lapachol, α- and ß-lapachone and a series of 25 related synthetic 1,4-naphthoquinones were screened against the oesophageal cancer cell line (WHCO1). Most of the compounds exhibited enhanced cytotoxicity (IC50 1.6-11.7 µM) compared to the current drug of choice cisplatin (IC50 = 16.5 µM). This study also established that the two new synthetic halogenated compounds 12a and 16a (IC50 = 3.0 and 7.3 µM) and the previously reported compound 11a (IC50 = 3.9 µM), were non-toxic to NIH3T3 normal fibroblast cells. Cell death of oesophageal cancer cells by processes involving PARP cleavage caused by 11a was shown to be associated with elevated c-Jun levels, suggesting a role for this pathway in the mechanism of action of this cohort of naphthoquinone compounds.

Synthesis, characterization, antiparasitic and cytotoxic evaluation of thioureas conjugated to polyamine scaffolds.

A series of mono- and multimeric 4-amino-7-chloroquinoline and ferrocenyl thioureas have been prepared by the reaction of a 7-chloroquinoline methyl ester and a ferrocenylimine methyl ester with various amines. These compounds were characterized using standard spectroscopic and analytical techniques. The compounds were evaluated against the NF54 (CQ-sensitive) and Dd2 (CQ-resistant) strains of Plasmodium falciparum. The quinoline compounds show enhanced activity compared to the ferrocene compounds against this parasite. Compound 5 displays the most promising activity against the NF54 strain. Compounds 5 and 6 are effective at inhibiting ß-hematin formation perhaps due to an increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also inhibit ß-hematin formation, albeit to a lesser degree compared to the quinoline thioureas. The compounds were also screened against the G3 strain of Trichomonas vaginalis and here the ferrocene-containing compounds show a slightly higher parasite growth inhibition compared to the quinoline thioureas. The quinoline compounds were also found to be more cytotoxic compared to the ferrocenyl compounds. Compound 6 displays good cytotoxicity against WHCO1 oesophageal cancer cells.

ß-Lactam synthon-interceded diastereoselective synthesis of functionalized octahydroindole-based molecular scaffolds and their in vitro cytotoxic evaluation.

A convenient and unprecedented synthesis of functionally enriched octahydroindole-based scaffolds has been developed via inter- and intra-molecular amidolysis of C-3 functionalized ß-lactams. The cytotoxic evaluation on oesophageal cancer cell line WHCO1 has revealed 7d as the most potent of the test compounds exhibiting an IC(50) value of 12.97 µM. The developed strategy further assumes significance as it entails the preparation of highly functionalized indoles without the aid of transition metal catalysis or pre-functionalization of substrates.

GROß and its downstream effector EGR1 regulate cisplatin-induced apoptosis in WHCO1 cells.

Cisplatin is one of the most widely used chemotherapeutic agents employed for treatment of a wide variety of solid tumors, including human esophageal squamous cell carcinoma (ESCC). However, a major limitation of cisplatin-based chemotherapy of ESCC is the rather low-effective rate. Understanding the molecular events of limited efficacy of cisplatin-based chemotherapy of ESCC could lead to strategies resulting in improved therapeutic benefits. The CXC chemokine family has been reported to be related to inflammatory reaction, injure recovery, cell proliferation, apoptosis and even to be involved in the regulation of chemotherapeutic agent-induced apoptosis. CXCL2 chemokine, also known as GROß (growth-related gene product ß), belongs to the CXC chemokine group. The known functions of GROß are related to attracting neutrophils to sites of inflammation, modulation of the neurotransmitter release, cell proliferation and apoptosis. However, little is known about the relationship between GROß and chemotherapeutic agent-induced apoptosis. This study was designed to provide insights into the possible role of GROß in the regulation of cisplatin-induced apoptosis in ESCCs. We report here that inhibition of expression of GROß can decrease cisplatin-induced apoptosis in WHCO1 cells. EGR1 is a downstream factor regulated by GROß. Silencing expression of EGR1 can also decrease cisplatin-induced apoptosis in WHCO1 cells. The activation of caspase 9 was delayed in cells in which GROß and EGR1 were knocked down after cisplatin treatment. All these results indicate that GROß and its downstream factor EGR1 are involved in regulating cisplatin-induced apoptosis in WHCO1 cells, and during this process the intrinsic apoptotic pathway is activated. It may be useful to examine the expression levels of GROß and EGR1 in ESCC patients to select those likely to respond well to cisplatin.

Clinical significance of serum expression of GROß in esophageal squamous cell carcinoma.

AIM: To determine the association between serum levels of growth-related gene product ß (GROß) and clinical parameters in esophageal squamous cell carcinoma (ESCC). METHODS: Using enzyme-linked immunosorbent assay, serum GROß levels were measured in ESCC patients (n = 72) and healthy volunteers (n = 83). The association between serum levels of GROß and clinical parameters of ESCC was analyzed statistically. RESULTS: The serum GROß levels were much higher in ESCC patients than in healthy controls (median: 645 ng/L vs 269 ng/L, P < 0.05). Serum GROß levels were correlated positively with tumor size, lymph node metastasis, and tumor-node-metastasis (TNM) staging, but not with gender or the histological grade of tumors in ESCC patients. The sensitivity and specificity of the assay for serum GROß were 73.61% and 56.63%, respectively. CONCLUSION: GROß may function as an oncogene product and contribute to tumorigenesis and metastasis of ESCC.

Identification and in vitro anti-esophageal cancer activity of a series of halogenated monoterpenes isolated from the South African seaweeds Plocamium suhrii and Plocamium cornutum.

Five known (1, 2, 4, 6 and 7) halogenated monoterpenes together with 1Z,3R∗,4S∗,5E,7Z)-1-bromo-3,4,8-trichloro-7-(dichloromethyl)-3-methylocta-1,5,7-triene (3) and (3R∗,4S∗)-3,4,6,7-tetrachloro-3,7-dimethyl-octen-1-ene (5) were isolated from the red macroalga Plocamium suhrii and their structures deduced from their spectroscopic data. The seven compounds from P. suhrii together with five related compounds from Plocamium cornutum have been evaluated for their cytotoxic effects on an esophageal cancer cell line (WHCO1). Compounds 1-6 showed greater cytotoxicity in this assay as compared to the known anticancer drug cisplatin.

Bioactive metabolites from the South African marine mollusk Trimusculus costatus.

A reinvestigation of extracts of the endemic South African intertidal limpet Trimusculus costatus yielded the known labdane diterpenes 6beta,7alpha-diacetoxylabda-8,13 E-dien-15-ol ( 1) and 2alpha,6beta,7alpha-triacetoxylabda-8,13 E-dien-15-ol ( 2) and three new metabolites, 6beta,7alpha,15-triacetoxylabda-8,13 E-diene ( 3), 3alpha,11-dihydroxy-9,11-seco-cholest-4,7-dien-6,9-dione ( 4), and cholest-7-en-3,5,7-triol ( 5). Chiral derivatization and X-ray analysis were used to confirm the labdane absolute configuration of 2. Compounds 1, 2 and 4 exhibited moderate activity (3-25 microM) against the WHCO1 human esophageal cancer cell line.

Halogenated monoterpene aldehydes from the South African marine alga Plocamium corallorhiza.

Four new halogenated monoterpene aldehydes (1-4) have been isolated from the South African marine red alga Plocamium corallorhiza, along with the known compounds 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene (5) and 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene (10). The structures of the new compounds were determined by interpretation of their spectroscopic data and synthesis and mass spectrometric analysis of their pentafluorobenzyloxime (PFBO) derivatives.