Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers.

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Lack of correlation between X-ray repair cross-complementing group 1 gene polymorphisms and the susceptibility to colorectal cancer in a Malaysian cohort.

X-ray repair cross-complementing group 1 (XRCC1) is one of the key components in the base excision repair pathway that repairs erroneous DNA lesions and removes nonbulky base adducts for the maintenance of genome integrity. Studies have revealed that differences in individual DNA repair capacity can impact the interindividual variation in cancer susceptibility, tumour aggressiveness and treatment response. The relationship between XRCC1 and sporadic colorectal cancer (CRC) susceptibility, which is hitherto inconclusive, has been explored in many association studies of different populations. In view of the conflicting findings generated, we aimed to investigate the association between XRCC1 and genetic predisposition to CRC among Malaysians. The present case-control association study was conducted on 130 CRC patients and 212 age-matched healthy controls. The genotyping of XRCC1 Arg194Trp, Arg280His and Arg399Gln single nucleotide polymorphisms was performed with allele-specific real-time PCR approach. This was followed by basic statistical analysis on the single nucleotide polymorphisms and haplotype data obtained. No significant difference in the allele and genotype frequencies was observed between CRC patients and healthy controls (P>0.05). There was also no association observed between XRCC1 haplotypes and CRC (P>0.05). In conclusion, a positive association between XRCC1 gene polymorphisms and CRC risk was not established in our Malaysian population.

Correction: Assessment of Telomere Length in Archived Formalin-Fixed, Paraffinized Human Tissue Is Confounded by Chronological Age and Storage Duration.

[This corrects the article DOI: 10.1371/journal.pone.0161720.].

Assessment of Telomere Length in Archived Formalin-Fixed, Paraffinized Human Tissue Is Confounded by Chronological Age and Storage Duration.

Telomeres shorten with physiological aging but undergo substantial restoration during cancer immortalization. Increasingly, cancer studies utilize the archive of formalin-fixed, paraffin-embedded (FFPE) tissues in diagnostic pathology departments. Conceptually, such studies would be confounded by physiological telomere attrition and loss of DNA integrity from prolonged tissue storage. Our study aimed to investigate these two confounding factors. 145 FFPE tissues of surgically-resected, non-diseased appendixes were retrieved from our pathology archive, from years 2008 to 2014. Cases from 2013 to 2014 were categorized by patient chronological age (0-20 years, 21-40 years, 41-60 years, > 60 years). Telomere lengths of age categories were depicted by telomere/chromosome 2 centromere intensity ratio (TCR) revealed by quantitative fluorescence in situ hybridization. Material from individuals aged 0-20 years from years 2013/2014, 2011/2012, 2009/2010, and 2008 were compared for storage effect. Telomere integrity was assessed by telomere fluorescence intensity (TFI). Epithelial TCRs (mean ± SD) for the respective age groups were 4.84 ± 2.08, 3.64 ± 1.21, 2.03 ± 0.37, and 1.93 ± 0.45, whereas corresponding stromal TCRs were 5.16 ± 2.55, 3.84 ± 1.36, 2.49 ± 1.20, and 2.93 ± 1.24. A trend of inverse correlation with age in both epithelial and stromal tissues is supported by r = -0.69, p < 0.001 and r = -0.42, p < 0.001 respectively. Epithelial TFIs (mean ± SD) of years 2013/2014, 2011/2012, 2009/2010 and 2008 were 852.60 ± 432.46, 353.04 ± 127.12, 209.24 ± 55.57 and 429.22 ± 188.75 respectively. Generally, TFIs reduced with storage duration (r = -0.42, p < 0.001). Our findings agree that age-related telomere attrition occurs in normal somatic tissues, and suggest that an age-based reference can be established for telomere studies on FFPE tissues. We also showed that FFPE tissues archived beyond 2 years are suboptimal for telomere analysis.

Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria.

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNex(TM), a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1-100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNex(TM). This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNex(TM). The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.

Association between PDCD1 Gene Polymorphisms and Risk of Systemic Lupus Erythematosus in Three Main Ethnic Groups of the Malaysian Population.

The programmed cell death 1 (PDCD1) gene encodes for the PD-1 (programmed death 1) molecule, which negatively regulates self-reactive T- and B-cells in the maintenance of peripheral tolerance. A previous report had shown the development of lupus-like phenotypes in PD-1-deficient C57BL/6 mice, was suggestive to the role of PDCD1 in predisposing to systemic lupus erythematosus (SLE). Hence, we aimed to investigate the association between PDCD1 and SLE susceptibility in the Malaysian population. A TaqMan-based real-time PCR was employed to screen for PD1.1, PD1.3, PD1.5 and PD1.6 in both SLE and healthy control groups of 200 samples each. The observed frequency for PD1.5C/C genotype was significantly higher in Indian SLE patients and Malay controls (p < 0.01). On the other hand, the PD1.5C/T genotype might predispose the Malays to SLE, but confer a protective effect among the Indians (p < 0.01). The PD1.1, PD1.3 and PD1.6 were, however, not correlated to genetic predisposition of SLE in our Malaysian population. In conclusion, PD1.5 variant was significantly associated to SLE susceptibility in our Malaysian cohort. Our failure in replicating the association between other investigated PDCD1 variants and risk of getting SLE might due to ethnic and geographic variations in the distribution of these genetic variants.

Pair-wise comparison analysis of differential expression of mRNAs in early and advanced stage primary colorectal adenocarcinomas.

OBJECTIVES: To characterise the mRNA expression patterns of early and advanced stage colorectal adenocarcinomas of Malaysian patients. DESIGN: Comparative expression analysis. SETTING AND PARTICIPANTS: We performed a combination of annealing control primer (ACP)-based PCR and reverse transcription-quantitative real-time PCR for the identification of differentially expressed genes (DEGs) associated with early and advanced stage primary colorectal tumours. We recruited four paired samples from patients with colorectal cancer (CRC) of Dukes' A and B for the preliminary differential expression study, and a total of 27 paired samples, ranging from CRC stages I to IV, for subsequent confirmatory test. The tumouric samples were obtained from the patients with CRC undergoing curative surgical resection without preoperative chemoradiotherapy. The recruited patients with CRC were newly diagnosed with CRC, and were not associated with any hereditary syndromes, previously diagnosed cancer or positive family history of CRC. The paired non-cancerous tissue specimens were excised from macroscopically normal colonic mucosa distally located from the colorectal tumours. PRIMARY AND SECONDARY OUTCOME MEASURES: The differential mRNA expression patterns of early and advanced stage colorectal adenocarcinomas compared with macroscopically normal colonic mucosa were characterised by ACP-based PCR and reverse transcription-quantitative real-time PCR. RESULTS: The RPL35, RPS23 and TIMP1 genes were found to be overexpressed in both early and advanced stage colorectal adenocarcinomas (p<0.05). However, the ARPC2 gene was significantly underexpressed in early colorectal adenocarcinomas, while the advanced stage primary colorectal tumours exhibited an additional overexpression of the C6orf173 gene (p<0.05). CONCLUSIONS: We characterised two distinctive gene expression patterns to aid in the stratification of primary colorectal neoplasms among Malaysian patients with CRC. Further work can be done to assess and compare the mRNA expression levels of these identified DEGs between each CRC stage group, stages I-IV.