• Asian Pacific Journal of Cancer Prevention
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• v.16 no.10
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• pp.4369-4376
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• 2015

Background: To investigate in-vitro antagonistic effect of low-dose liquiritigenin on gemcitabine-induced capillary leak syndrome (CLS) in pancreatic adenocarcinoma via inhibiting reactive oxygen species (ROS)-mediated signalling pathways. Materials and Methods: Human pancreatic adenocarcinoma Panc-1 cells and human umbilical vein endothelial cells (HUVECs) were pre-treated using low-dose liquiritigenin for 24 h, then added into gemcitabine and incubated for 48 h. Cell viability, apoptosis rate and ROS levels of Panc-1 cells and HUVECs were respectively detected through methylthiazolyldiphenyl-tetrazoliumbromide (MTT) and flow cytometry. For HUVECs, transendothelial electrical resistance (TEER) and transcellular and paracellular leak were measured using transwell assays, then poly (ADP-ribose) polymerase 1 (PARP-1) and metal matrix proteinase-9 (MMP9) activity were assayed via kits, mRNA expressions of p53 and Rac-1 were determined through quantitative polymerase chain reaction (qPCR); The expressions of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and PARP-1 were measured via western blotting. Results: Low-dose liquiritigenin exerted no effect on gemcitabine-induced changes of cell viability, apoptosis rate and ROS levels in Panc-1 cells, but for HUVECs, liquiritigenin ($3{\mu}M$) could remarkably elevate gemcitabine-induced decrease of cell viability, transepithelial electrical resistance (TEER), pro-MMP9 level and expression of ICAM-1 and VCAM-1 (p<0.01). Meanwhile, it could also significantly decrease gemcitabine-induced increase of transcellular and paracellular leak, ROS level, PARP-1 activity, Act-MMP9 level, mRNA expressions of p53 and Rac-1, expression of PARP-1 and apoptosis rate (p<0.01). Conclusions: Low-dose liquiritigenin exerts an antagonistic effect on gemcitabine-induced leak across HUVECs via inhibiting ROS-mediated signalling pathways, but without affecting gemcitabine-induced Panc-1 cell apoptosis. Therefore, low-dose liquiritigenin might be beneficial to prevent the occurrence of gemcitabine-induced CLS in pancreatic adenocarcinoma.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.1901-1906
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• 2012

Background: The discovery that microRNA (miRNA) regulates metastasis provide a principal molecular basis for tumor heterogeneity. A characteristic of solid tumors is their heterogenous distribution of blood vessels, with significant hypoxia occurring in regions (centers of tumor) of low blood flow. It is necessary to discover the mechanism of breast cancer metastasis in relation to the fact that there is a differential distribution of crucial microRNA in tumors from centers to edges. Methods: Breast tissues from 48 patients (32 patients with breast cancer) were classified into the high invasive and metastatic group (HIMG), low invasive and metastatic group (LIMG), and normal group. Samples were collected from both the centers and edges of all tumors. The first six specimens were detected by microRNA array, and the second ten specimens were detected by real-time qRT-PCR and Western blot analyses. Correlation analysis was performed between the miRNAs and target proteins. Results: The relative content of miR-20a and miR-20b was lower in the center of the tumor than at the edge in the LIMG, lower at the edge of the tumor than in the center in the HIMG, and lower in breast cancer tissues than in normal tissues. VEGF-A and HIF-1alpha mRNA levels were higher in the HIMG than in the LIMG, and levels were higher in both groups than in the normal group; there was no difference in mRNA levels between the edge and center of the tumor. VEGF-A and HIF-1alpha protein levels were higher in the HIMG than in the LIMG, and protein levels in both groups were higher than in the normal group; there was a significant difference in protein expression between the edge and center of the tumor. Correlation analysis showed that the key miRNAs (miR-20a and miR-20b) negatively correlated with the target proteins (VEGF-A and HIF-1alpha). Conclusions: Our data suggest that miR-20a and miR-20b are differentially distributed in breast cancer, while VEGF-A and HIF-1alpha mRNA had coincident distributions, and VEGF-A and HIF-1alpha proteins had uneven and opposing distributions to the miRNAs. It appears that one of the most important facets underlying metastatic heterogeneity is the differential distribution of miR-20a and miR-20b and their regulation of target proteins.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.651-656
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• 2014

Background: Cervical cancer is the third commonest type of cancer among women in Malaysia. Our aim was to determine the distribution of human papilloma virus (HPV) genotypes in cervical cancer in our multi-ethnic population. Materials and Methods: This was a multicentre study with a total of 280 cases of cervical cancer from 4 referral centres in Malaysia, studied using real-time polymerase chain reaction (qPCR) detection of 12 high risk-HPV genotypes. Results: Overall HPV was detected in 92.5% of cases, in 95.9% of squamous cell carcinomas and 84.3%of adenocarcinomas. The five most prevalent high-risk HPV genotypes were HPV 16 (68.2%), 18 (40%), 58 (10.7%), 33 (10.4%) and 52 (10.4%). Multiple HPV infections were more prevalent (55.7%) than single HPV infections (36.8%). The percentage of HPV positive cases in Chinese, Malays and Indians were 95.5%, 91.9% and 80.0%, respectively. HPV 16 and 18 genotypes were the commonest in all ethnic groups. We found that the percentage of HPV 16 infection was significantly higher in Chinese (75.9%) compared to Malays (63.7%) and Indians (52.0%) (p<0.05), while HPV 18 was significantly higher in Malays (52.6%) compared to Chinese (25.0%) and Indians (28%) (p<0.05). Meanwhile, HPV 33 (17.9%) and 52 (15.2%) were also more commonly detected in the Chinese (p<0.05). Conclusions: This study showed that the distribution of HPV genotype in Malaysia is similar to other Asian countries. Importantly, we found that different ethnic groups in Malaysia have different HPV genotype infection rates, which is a point to consider during the implementation of HPV vaccination.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3431-3436
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• 2016

Background: Carcinogenesis is a multifaceted intricate cellular mechanism of transformation of the normal functions of a cell into neoplastic alterations. Metastasis may result in failure of conventional treatment and death Hence, research on metastatic suppressors in cancer is a high priority. The metastatic suppressor gene CD82, also known as KAI1, is a member of the transmembrane 4 superfamily which was first identified in carcinoma of prostate. Little work has been done on this gene in breast cancer. Herein, we aimed to determine the gene and protein level expression of CD82/KAI1 in breast cancer and its role as a prognosticator. Materials and Methods: In this study, 83 histologically proven cases of breast cancer and a similar number of controls were included. Patient age ranged from 18-70 years. Quantitative Real Time Polymerase Chain Reaction (q-RT PCR) and immunohistochemistry (IHC) were used to investigate KAI1 expression at gene and protein levels, respectively. Statistical analysis was done to correlate expression of KAI1 and clinicopathological parameters. Results: It was revealed that: (i) KAI1 was remarkably diminished in metastatic vs non metastatic breast cancer both at the gene and the protein levels (P < .05); (ii) KAI1 expression levels were strongly correlated with TNM staging, histological grade and advanced stage (p<0.001) and no association was found with any other studied parameter; (iii) Lastly, a significant correlation was observed between expression of KAI1 and overall median survival of BC patients (P = 0.04). Conclusions: Our results suggest that lack of expression of the KAI1 might indicate a more aggressive form of breast cancer. Loss of KAI1 may be considered a significant prognostic marker in predicting metastatic manifestation. When evaluated along with the clinical and pathological factors, KAI1 expression may be beneficial to tailor aggressive therapeutic strategies for such patients.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3437-3445
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• 2016

Background: There is an increasing concern in the role of microRNA (miRNA) in the pathogenesis of bone metastasis (BM) secondary to prostate cancer (CaP). In this exploratory study, we hypothesized that the expression of vinculin (VCL) and chemokine X3C ligand 1 (CX3CL1) might be down-regulated in clinical samples, most likely due to the post-transcriptional modification by microRNAs. Targeted genes would be up-regulated upon transfection of the bone metastatic prostate cancer cell line, PC3, with specific microRNA inhibitors. Materials and Methods: MicroRNA software predicted that miR-21 targets VCL while miR-29a targets CX3CL1. Twenty benign prostatic hyperplasia (BPH) and 16 high grade CaP formalin-fixed paraffin embedded (FFPE) specimens were analysed. From the bone scan results, high grade CaP samples were further classified into CaP with no BM and CaP with BM. Transient transfection with respective microRNA inhibitors was done in both RWPE-1 (normal) and PC3 cell lines. QPCR was performed in all FFPE samples and transfected cell lines to measure VCL and CX3CL1 levels. Results: QPCR confirmed that VCL messenger RNA (mRNA) was significantly down-regulated while CX3CL1 was up-regulated in all FFPE specimens. Transient transfection with microRNA inhibitors in PC3 cells followed by qPCR of the targeted genes showed that VCL mRNA was significantly upregulated while CX3CL1 mRNA was significantly down-regulated compared to the RWPE-1 case. Conclusions: The down-regulation of VCL in FFPE specimens is most likely regulated by miR-21 based on the in vitro evidence but the exact mechanism of how miR-21 can regulate VCL is unclear. Up-regulated in CaP, CX3CL1 was found not regulated by miR-29a. More microRNA screening is required to understand the regulation of this chemokine in CaP with bone metastasis. Understanding miRNA-mRNA interactions may provide additional knowledge for individualized study of cancers.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3213-3222
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• 2016

Background: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyl-donor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. Materials and Methods: The human cervical cancer cell line, C4-II, was grown in complete medium and medium depleted of folate (F-M+) and folate and methionine (F-M-). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT-PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. Results: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant down-regulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. Conclusions: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.943-954
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• 2009

Diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the final and rate-limiting step of triglyceride synthesis. Both DGAT1 and DGAT2 genes code proteins with DGAT activity. Studies have shown DGAT1 polymorphisms associate with intramuscular fat deposition in beef cattle, but fewer associations between DGAT2 and beef cattle economic traits have been reported. The objective of this study was to investigate single nucleotide polymorphism (SNP) in intron3 of bovine DGAT2 and evaluate the associations of that with carcass, meat quality, and fat yield traits. Test animals were 157 commercial feedlot steers belonging to 3 Chinese native breeds (22 for Luxi, 24 for Jinnan, and 23 for Qinchuan), 3 cross populations (20 for Charolais${\times}$Fuzhou, 18 for Limousin ${\times}$Luxi, and 17 for Simmental${\times}$Jinan) and 1 Taurus pure breed population (16 Angus steers). In the current study, 15 SNP were discovered in intron3 and exon4 of DGAT2 at positions 65, 128, 178, 210, 241, 255, 270, 312, 328, 334, 365, 366, 371, 415, and 437 (named as their positions in PCR amplified fragments). Only 7 of them (128, 178, 241, 270, 312, 328, and 371) were analyzed, because SNP in three groups (65-128-255, 178-210-365 and 241-334-366) were in complete linkage disequilibrium within the group, and SNP 415 was a deletion and 437 was a null mutation. Frequencies for rare alleles in the 3 native breed populations were higher than in the 3 cross populations for 178 (p = 0.04), 270 (p = 0.001), 312 (p = 0.03) and 371 (p = 0.002). A general linear model was used to evaluate the associations between either SNP genotypes or allele substitutions and the measured traits. Results showed that SNP 270 had a significant association with the fat yield associated with kidney, pelvic cavity, heart, intestine, and stomach (KPHISY). Animals with genotype CC and CT for 270 had less (CC: -7.71${\pm}$3.3 kg and CT: -5.34${\pm}$2.5 kg) KPHISY than animals with genotype TT (p = 0.02). Allele C for 270 was associated with an increase of -4.26${\pm}$1.52 kg KPHISY (p = 0.006) and $-0.92{\pm}0.45%$ of retail cuts weight percentage (NMP, Retail cuts weight/slaughter body weight) (p = 0.045); allele G for 312 was associated with an increase of -5.45${\pm}$2.41 kg KPHISY (p = 0.026). An initial conclusion was that associations do exist between DGAT2 gene and carcass fat traits. Because of the small sample size of this study, it is proposed that further effort is required to validate these findings in larger populations.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5199-5205
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• 2013

Background/Aim: The Hippo signaling pathway is a newly discovered and conserved signaling cascade, which regulates organ size control by governing cell proliferation and apoptosis. This study aimed to investigate its effects in human gastric cancer. Methods: Tumor tissues (n=60), adjacent non-tumor tissues (n=60) and normal tissues (n=60) were obtained from the same patients with primary gastric cancer (GC). In addition, 70 samples of chronic atrophic gastritis (CAG) tissues were obtained from patients with intestinal metaplasia (IM) by endoscopic biopsy. Hippo signaling molecules, including Mst1, Lats1, YAP1, TAZ, TEAD1, Oct4 and CDX2, were determined by quantitative polymerase chain reaction (qPCR). Protein expression of Mst1, Lats1, YAP1, TEAD1 and CDX2 was assessed by immunohistochemistry and Western blotting. Results: Mst1, Lats1 and Oct4 mRNA expression showed an increasing tendency from GC tissues to normal gastric tissues, while the mRNA expression of YAP1, TAZ and TEAD1 was up-regulated (all P<0.01). Mst1 and Lats1 protein expression presented a similar trend with their mRNA expression. In addition, YAP1 and TEAD1 protein expression in GC was significantly higher than in the other groups (all P<0.01). CDX2 mRNA and protein expression in the CAG group were higher than in the other groups (all P<0.01). In GC, mRNA expression of Mst1, Lats1, Oct4, YAP1, TAZ, TEAD1 and CDX2 had a close correlation with lymphatic metastasis and tumor TNM stage (all P<0.01). Furthermore, protein expression of Mst1, Lats1, YAP1, TAZ, TEAD1 and CDX2 had a close correlation between each other (P<0.05). Conclusion: The Hippo signaling pathway is involved in the development, progression and metastasis of human gastric cancer. Therefore, manipulation of Hippo signaling molecules may be a potential therapeutic strategy for gastric cancer.

• Fisheries and Aquatic Sciences
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• v.22 no.11
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• pp.25.1-25.9
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• 2019

Background: Due to the continuous demand for fish coupled with decline in capture fisheries, there is the need to increase aquaculture production to meet the demand. Aquaculture is faced with high cost of feeding since fish oil and fish meal are expensive. In view of this, there are calls to explore alternatives that are cheap and reliable. Objectives: This study on Oreochromis niloticus was conducted to evaluate the effects of replacing fish oil (FO) with palm oil (PO) at 0%, 25%, 50%, 75%, and 100% on muscle fatty acid and proximate composition as well as growthrelated enzyme activities and mRNA expression. Methods: Oreochromis niloticus were fed five experimental diets (33% crude protein and 10% crude lipid) for 8 weeks. Feed had variation in fish oil and palm oil contents. After the 8 weeks feeding trial, five fish were sampled from each tank (15 from each treatment) and euthanized using an excess dose of tricaine methane sulfonate (MS-222 at 200 mg/L). Fatty acid and enzyme activities were analyzed using standard protocols. Also, RT-qPCR was used to quantify the expression levels of selected growth-related genes. Results: Fish fed 25% PO recorded the least muscle protein content and was significantly lower than the group fed 100% PO. Paired box protein 7 (Pax-7) enzyme activity was significantly higher in the group fed 50% PO compared to the groups fed 25% PO and 100% PO, while caplain-3 (Capn-3) was significantly lower in the group fed 0% PO compared to all other groups. There was a significant difference among treatments with respect to mRNA expression of Pax-7 and Capn-3. Group fed 25% PO had significantly lower mRNA expression of Pax-7, while the group fed 75% PO recorded significantly higher mRNA expression of Capn-3 compared to groups fed 0% PO, 25% PO, and 100% PO. Pearson's correlation analysis revealed that Igf-I and Igf-II mRNA expression have significant correlation with n-3 polyunsaturated fatty acids content in muscle. Conclusion: The results suggest muscle protein content could be modified if FO is replaced with PO. Also, mRNA expression of Pax-7 and Capn-3 is affected by replacing FO with PO.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.296-302
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• 2017

Rice (Oryza sativa) cultivars show an impairment of growth and development in response to abiotic stresses such as drought, salinity, heat and cold at the early seedling stage. The tolerance to heat stress in plants has been genetically modulated by the overexpression of heat shock transcription factor genes or proteins. In addition to a high temperature-tolerance that has also been altered by elevating levels of osmolytes, increasing levels of cell detoxification enzymes and through altering membrane fluidity. To examine the heat tolerance in transgenic rice plants, three OsOPT10 overexpressing lines were characterized through a physiological analysis, which examined factors such as the electrolyte leakage (EL), soluble sugar and proline contents. We further functionally characterized the OsOPT10 gene and found that heat induced the expression of OsOPT10 and P5CS gene related proline biosynthesis. It has been suggested that the expression of OsOPT10 led to elevated heat tolerance in transgenic lines.