Prognostic Significance of CD26 in Patients with Colorectal Cancer

Background

CD26, dipeptidyl peptidase IV, was discovered firstly as a membrane-associated peptidase on the surface of leukocyte. We previously demonstrated that a subpopulation of CD26⁺ cells were associated with the development of distant metastasis, enhanced invasiveness and chemoresistance in colorectal cancer (CRC). In order to understand the clinical impact of CD26, the expression was investigated in CRC patient''s specimens. This study investigated the prognostic significance of tumour CD26 expression in patients with CRC. Examination of CD26⁺ cells has significant clinical impact for the prediction of distant metastasis development in colorectal cancer, and could be used as a selection criterion for further therapy.

Methods

Tumour CD26 expression levels were studied by immunohistochemistry using Formalin-fixed paraffin embedded (FFPE) tissues in 143 patients with CRC. Tumour CD26 expression levels were correlated with clinicopathological features of the CRC patients. The prognostic significance of tumour tissue CD26 expression levels was assessed by univariate and multivariate analyses.

Result

CD26 expression levels in CRC patients with distant metastasis were significantly higher than those in non-metastatic. High expression levels of CD26 were significantly associated with advanced tumour staging. Patients with a high CD26 expression level had significantly worse overall survival than those with a lower level (p<0.001).

Conclusions

The expression of CD26 was positively associated with clinicopathological correlation such as TNM staging, degree of differentiation and development of metastasis. A high CD26 expression level is a predictor of poor outcome after resection of CRC. CD26 may be a useful prognostic marker in patients with CRC.     

Gene Expression Profiling Reveals Epithelial Mesenchymal Transition (EMT) Genes Can Selectively Differentiate Eribulin Sensitive Breast Cancer Cells

Objectives

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a mechanistically unique inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with eribulin activity compared to paclitaxel, which stabilizes microtubules, based on gene expression profiling of cell line panels of breast, endometrial, and ovarian cancer in vitro.

Results

We determined the sets of genes that were differentially altered between eribulin and paclitaxel treatment in breast, endometrial, and ovarian cancer cell line panels. Our unsupervised clustering analyses revealed that expression profiles of gene sets altered with treatments were correlated with the in vitro antiproliferative activities of the drugs. Several tubulin isotypes had significantly lower expression in cell lines treated with eribulin compared to paclitaxel. Pathway enrichment analyses of gene sets revealed that the common pathways altered between treatments in the 3 cancer panels were related to cytoskeleton remodeling and cell cycle regulation. The epithelial-mesenchymal transition (EMT) pathway was enriched in genes with significantly altered expression between the two drugs for breast and endometrial cancers, but not for ovarian cancer. Expression of genes from the EMT pathway correlated with eribulin sensitivity in breast cancer and with paclitaxel sensitivity in endometrial cancer. Alteration of expression profiles of EMT genes between sensitive and resistant cell lines allowed us to predict drug sensitivity for breast and endometrial cancers.

Conclusion

Gene expression analysis showed that gene sets that were altered between eribulin and paclitaxel correlated with drug in vitro antiproliferative activities in breast and endometrial cancer cell line panels. Among the panels, breast cancer provided the strongest differentiation between eribulin and paclitaxel sensitivities based on gene expression. In addition, EMT genes were predictive of eribulin sensitivity in the breast and endometrial cancer panels.     

Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate,LynxTag-CQGREEN,as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQ_GREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQ_GREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQ_GREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC₅₀s were determined. These data were compared with LynxTag-CQ_GREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC₅₀ of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.     

Testing Commercial Sex Workers for Sexually Transmitted Infections in Victoria,Australia: An Evaluation of the Impact of Reducing the Frequency of Testing

Background

The frequency of testing sex workers for sexually transmitted infections (STIs) in Victoria, Australia, was changed from monthly to quarterly on 6 October 2012. Our aim was to determine the impact of this change to the clients seen at the Melbourne Sexual Health Centre (MHSC).

Methods

Computerised medical records of all clients attending at MHSC from 7 October 2011 to 7 October 2013 were analysed.

Results

Comparing between the monthly and quarterly testing periods, the number of consultations at MSHC with female sex workers (FSW) halved from 6146 to 3453 (p<0.001) and the consultation time spent on FSW reduced by 40.6% (1942 h to 1153 h). More heterosexual men (p<0.001), and women (p<0.001) were seen in the quarterly testing period. The number of STIs diagnosed in the clinic increased from 2243 to 2589 from the monthly to quarterly period, respectively [15.4% increase (p<0.001)]. Up to AU$247,000 was saved on FSW testing after the shift to quarterly testing.

Conclusions

The change to STIs screening frequency for sex workers from monthly to quarterly resulted in a 15% increase in STI diagnoses in the clinic and approximate a quarter of a million dollars was diverted from FSW testing to other clients. Overall the change in frequency is likely to have had a beneficial effect on STI control in Victoria.     

Pterostilbene Simultaneously Induced G0/G1-Phase Arrest and MAPK-Mediated Mitochondrial-Derived Apoptosis in Human Acute Myeloid Leukemia Cell Lines

Background

Pterostilbene (PTER) is a dimethylated analog of the phenolic phytoalexin, resveratrol, with higher anticancer activity in various tumors. Herein, the molecular mechanisms by which PTER exerts its anticancer effects against acute myeloid leukemia (AML) cells were investigated.

Methodology and Principal Findings

Results showed that PTER suppressed cell proliferation in various AML cell lines. PTER-induced G0/G1-phase arrest occurred when expressions of cyclin D3 and cyclin-dependent kinase (CDK)2/6 were inhibited. PTER-induced cell apoptosis occurred through activation of caspases-8-9/-3, and a mitochondrial membrane permeabilization (MMP)-dependent pathway. Moreover, treatment of HL-60 cells with PTER induced sustained activation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2, and inhibition of both MAPKs by their specific inhibitors significantly abolished the PTER-induced activation of caspases-8/-9/-3. Of note, PTER-induced cell growth inhibition was only partially reversed by the caspase-3-specific inhibitor, Z-DEVE-FMK, suggesting that this compound may also act through a caspase-independent pathway. Interestingly, we also found that PTER promoted disruption of lysosomal membrane permeabilization (LMP) and release of activated cathepsin B.

Conclusion

Taken together, our results suggest that PTER induced HL-60 cell death via MAPKs-mediated mitochondria apoptosis pathway and loss of LMP might be another cause for cell apoptosis induced by PTER.     

Investigation on Natural Diets of Larval Marine Animals Using Peptide Nucleic Acid-Directed Polymerase Chain Reaction Clamping

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.     

Draft genome sequence of Penicillium marneffei strain PM1

Penicillium marneffei is the most important thermal dimorphic, pathogenic fungus endemic in China and Southeast Asia and is particularly important in HIV-positive patients. We report the 28,887,485-bp draft genome sequence of P. marneffei, which contains its complete mitochondrial genome, sexual cycle genes, a high diversity of Mp1p homologues, and polyketide synthase genes.     

Functional genomics reveals diverse cellular processes that modulate tumor cell response to oxaliplatin

Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin.     

A validated enantioselective assay for the simultaneous quantitation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantitation of (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine in ovine plasma. The analytes were extracted from ovine plasma at a basic pH using a single-step liquid-liquid extraction with methyl-tert-butyl ether. Chromatographic separation of all enantiomers was achieved using an AGP-chiral column with a run time of 10 min. (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine were quantitated at the total ion current (TIC) of multiple reaction monitoring (MRM) transitions of m/z 310.2→44.1, m/z 310.2→147.7 for (R)-, (S)-fluoxetine, and m/z 296.2→30.3, m/z 296.2→133.9 for (R)-, (S)-norfluoxetine. This method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), selectivity, recovery, dilution integrity, matrix effect, and evaluation of carry-over. Observed accuracy ranges were as follows: (R)-fluoxetine -8.82 to 3.75%; (S)-fluoxetine -10.8 to 1.46%; (R)-norfluoxetine -7.50 to 0.37% and (S)-norfluoxetine -8.77% to -1.33%. Observed precision ranges were as follows: (R)-fluoxetine 5.29-11.5%; (S)-fluoxetine 3.91-11.1%; (R)-norfluoxetine 4.32-7.67% and (S)-norfluoxetine -8.77% to -1.33%. The calibration curves were weighted (1/X(2), n=4) and observed to be linear for all analytes with the following r(2) values: (R)-fluoxetine ≥ 0.997; (S)-fluoxetine ≥ 0.996; (R)-norfluoxetine ≥ 0.989 and (S)-norfluoxetine ≥ 0.994. The analytical range of the method was 1-500 ng/ml with an LOQ of 1 ng/ml for all analytes, using a sample volume of 300 μL.