Denver T. Hendricks

The Karyopherin proteins, Crm1 and Karyopherin β1, 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 β1 (Kpnβ1) and Karyopherin α2 (Kpnα2), 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, Kpnβ1 and Kpnα2 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 Kpnβ1 in cancer cells significantly reduced cell proliferation, while Kpnα2 inhibition had no effect. Noncancer cells were unaffected by the inhibition of Crm1 and Kpnβ1. 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 Kpnβ1 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, Kpnβ1 and Kpnα2 are overexpressed in cervical cancer and that inhibiting the expression of Crm1 and Kpnβ1, not Kpnα2, induces cancer cell death, making Crm1 and Kpnβ1 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.

Oesophageal Cancer in Africa

Oesophageal cancer, the eighth most frequent cancer in the world occurs as two main subtypes, squamous cell carcinoma (more prevalent in developing countries) and adenocarcinoma (more common in developed countries). Certain populations of central, eastern, and southern Africa display very high frequencies of oesophageal squamous cell carcinoma, presenting a serious health burden to the continent. Most patients are diagnosed at a late stage because of the asymptomatic development of the disease, with associated poor prognosis. A better understanding of the aetiological agents and molecular mechanisms involved in the development of oesophageal squamous cell carcinoma may offer opportunities to reduce exposure to environmental risk factors and also allow early diagnosis or predict response to therapy. Epidemiologic studies have identified smoking, alcohol consumption, diets poor in fresh fruit and vegetables, consumption of foods contaminated with Fusarium verticillioides, and HPV infection as risk factors associated with the development of this disease in Africa. Although we have an incomplete understanding of the molecular events involved in the development of oesophageal squamous cell carcinoma, advances have been made that suggest lines of future exploration. South African patients with oesophageal squamous cell carcinoma display a lower incidence of point mutations in the p53 gene than described elsewhere, suggesting that the profile of aetiological agents may be different than described for other high‐risk areas for oesophageal cancer. Recent studies suggest that RAR βand COX II is frequently downregulated and upregulated, respectively, in oesophageal squamous cell carcinomas. These results suggest potential therapeutic opportunities that can be exploited to combat the high incidence of this disease in Africa.

Associations between p53 overexpression and multiple measures of clinical outcome in high-risk, early stage or suboptimally-resected, advanced stage epithelial ovarian cancers: A Gynecologic Oncology Group study

OBJECTIVE The Gynecologic Oncology Group (GOG) performed a detailed analysis of p53 overexpression in previously-untreated women with invasive early or advanced stage epithelial ovarian cancer (EOC). METHODS Women were eligible for the study if they provided a tumor block for translational research and participated in either GOG-157, a randomized phase III trial of three versus (vs.) six cycles of paclitaxel+carboplatin in high-risk, early stage EOC, or GOG-111, a randomized phase III trial of cyclophosphamide+cisplatin vs. paclitaxel+cisplatin in suboptimally-resected, advanced stage EOC. The N-terminal DO-7 p53 antibody was used to examine the expression of the major normal and mutant p53-isoforms. p53 overexpression was defined as >or=10% tumor cells exhibiting nuclear staining. RESULTS p53 was overexpressed in 51% (73/143) and 66% (90/136) of cases in the GOG-157 and GOG-111 cohorts, respectively. In the GOG-157 cohort, p53 overexpression was not associated with any clinical characteristics or overall survival (OS) but was associated with worse progression-free survival (PFS) (logrank test: p=0.013; unadjusted Cox modeling: p=0.015). In the GOG-111 cohort, p53 overexpression was associated with GOG performance status (p=0.018) and grade (p=0.003), but not with age, stage, cell type or with tumor response and disease status after primary chemotherapy, PFS or OS. Adjusted Cox regression modeling demonstrated that p53 overexpression was not an independent prognostic factor for PFS or OS in either cohort. CONCLUSIONS p53 overexpression assessed by DO-7 immunostaining is common in early and advanced stage EOC, but has limited prognostic value in women treated with surgical staging and platinum-based combination chemotherapy.

A Key Role for Early Growth Response-1 and Nuclear Factor-κB 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 p27Kip1 levels associated with a G2-M cell cycle arrest. EGR-1 knockdown also caused a marked induction in IκBα expression, an effect also observed in GROβ 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-κB inhibition with either sodium salicylate or p65 RNA interference led to a significant reduction in GROα and GROβ expression. These results indicate that EGR-1 and nuclear factor-κB mediate GRO/CXCR2 proliferative signaling in esophageal cancer and may represent potential target molecules for therapeutic intervention.(Mol Cancer Res 2009;7(5):755–64)

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. [Mol Cancer Ther 2007;6(9):2535–43]

Cytotoxicity of lapachol, β-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 β-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.

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.

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 β-hematin formation perhaps due to an increased number of quinoline moieties. The trimeric (12) and tetrameric (13) ferrocenyl compounds also inhibit β-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.

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.