Aim: Study the correlation of expression of genes involved in cytarabine metabolism to leukemia-free (LFS) and overall survival (OS) in AML. Introduction: Cytarabine is the backbone of AML therapy. Understanding the roles and polymorphisms of genes involved in cytarabine metabolism and resistance in AML will facilitate development of novel therapeutics. Methods: Adults less than 60 years with non M3 AML were included. Archived diagnostic marrow samples were studied for expression of 10 genes involved in cytarabine metabolism by RT-qPCR; gene expression normalized to GAPDH was compared using the unpaired t-test with Welch correction. SNP rs4694362 in deoxycytidine kinase (DCK) gene was tested using Taqman assay. Median time to relapse and survival was calculated by Kaplan Meier method. Results: 21 Han Chinese patients (median age: 50) were identified; 15 were male; 16 had intermediate risk cytogenetics; 5 had a blast count of over 100×109/L at diagnosis. 17 patients achieved CR; 12 after first induction. No difference in gene expression was seen between CR (n=12) versus non CR (n=9) with first induction. 17 CR patients were followed for a median duration of 67.5 months; median time to relapse was 15 months. 1 patient who underwent allogeneic transplant in CR1 was excluded. Higher mean DCK expression was seen in patients with LFS longer than (n=7) versus less than 2 years (n=9) (1.91±0.67, p: 0.01) and in those with OS longer than (n=8) versus less than 3 years (n=8) (2.02±0.69, p: 0.01). DCK rs4694362 TT genotype was less prevalent than CT in patients with >2 year LFS; but not statistically significant. (49% vs 60%, p: 0.6).1,2 Conclusion: DCK phosphorylates cytarabine to its active metabolite. Our work shows that higher DCK expression is correlated to LFS and OS in AML. The role of DCK SNP rs4694362 should be explored further in the Chinese.
The cancer stem cell hypothesis suggests that tumors are initiated and maintained by cancer stem cells capable of self-renewal and differentiation into mature tumor cells. Recent studies have demonstrated the existence of cancer initiating cells in different solid tumors. In this study, we identified a subpopulation of CD44+ cells within the tumor of gastric cancer patients, which, upon treatment by chemotherapeutic agent 5-fluorouracil (5-FU), were markedly enriched. In vitro culture of isolated CD44+ subpopulation from gastric tumors by magnetic beads sorting led to formation of gastric spheroid colonies. These colonies retained CD44+ surface marker expression during culture, and were undifferentiated in nature. Subcutaneous injections of CD44+ gastric cancer cells conferred tumorigenicity in SCID mice. Moreover, implantation of CD44+ cells from these established tumors remained tumorigenic in successive passages. Using CD44+ cells isolated from the gastric cell lines AGS and SGC7901, similar results were obtained. Upon enrichment by 5-FU, CD44+ cells harbored increased ALDH expression as compared with CD44- cells. Our results demonstrated for the first time the existence of CD44+ cells within the tumors of gastric cancer patients that are endowed with stem cells properties, and also provide a plausible explanation for chemo-resistance frequently observed in gastric cancer patients. Such findings provide a basis for further studies on targeting this tumorigenic subpopulation for better treatment of gastric cancer patients.
Introduction:The elementary steps leading to the development of metastasis is a complex process involving cell spreading, lamellipodia formation, and cell migration.Actopaxin, a focal adhesion and cytoskeleton-associated protein, is a member of a multi-gene family of which its phosphorylation at Ser/Thr-Pro motifs is required for such processes.The objective of this study was to examine the role of Actopaxin in hepatocellular carcinoma (HCC) cell migration, invasion and development of metastasis. Methods:The expression of Actopaxin in primary and metastastic liver cancer cell lines was examined by western blot and RT-PCR.The functional effects of enforced expression or down-regulation of Actopaxin was investigated by cell migration, cell invasion, and wound healing assays.The expression of downstream signalling targets of Actpaxin and markers for epithelial mesenchymal transition was studied.Immunofluorescence staining was used to examine the effect of Actopaxin expression on cell shape, stress fibre organisation, and focal adhesion. Results:The expression of Actopaxin was found to be highly expressed in the metastatic HCC cell lines H2M, MHCC-97L and MHCC-97H as compared with other primary HCC cell lines.Expression of a shorter form of Actopaxin (SF-Actopaxin) was also detected by western blot in some of these cell lines, suggesting the presence of more than one form of Actopaxin in human cells.The SF-Actopaxin lacks a fragment in the C-terminal, resulting in an incomplete second CH domain which consists of binding sites for its downstream activation targets.Enforced expression of long-form Actopaxin (LF-Actopaxin) in PLC, but not its corresponding short form readily enhanced cell migration in wound healing assays.Elevated protein levels of ILK, PINCH and phosphopaxillin, which are involved in focal adhesion complex formation and integrin signalling pathway, were also observed in LF-Actopaxin transfectants.Morphological changes were also observed in PLC LF-Actopaxin transfectants, showing enhanced stress fibre formation, filopodia and lamellipodia (cell protrusions) on the cell surfaces. Conclusion:This study demonstrated for the first time the pro-migratory effects of Actopaxin in human HCC, and the existence of a short form which lacks a complete CH domain that is critical for cell migration, reorganisation of cytoskeletal events and turnover of focal adhesions.The differential effects of LF-and SF-Actopaxin on ILK, PINCH and phospho-paxillin protein levels might partly explain their influence on cell migration capacity in HCC cells.Further studies are warranted to investigate the potential role of Actopaxin in HCC tumour progression and cell invasioned stress fibre formation, filopodia and lamellipodia (cell protrusions) on the cell surfaces.
// Lui Ng 1, * , Timothy Ming-Hun Wan 1, * , Johnny Hon-Wai Man 1 , Ariel Ka-Man Chow 1 , Deepak Iyer 1 , Guanghua Chen 1 , Thomas Chung-Cheung Yau 2 , Oswens Siu-Hung Lo 1 , Dominic Chi-Chung Foo 1 , Jensen Tung-Chung Poon 1 , Wai-Keung Leung 3 , Roberta Wen-Chi Pang 1, 2 , Wai-Lun Law 1 1 Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China 2 Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China 3 Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China * These authors have contributed equally to this work Correspondence to: Wai-Lun Law, email: lawwl@hku.hk Roberta Wen-Chi Pang, email: luing@hku.hk Keywords: miR-139-3p, miR-622, CRC, miRNA, biomarker Abbreviations: CRC, colorectal cancer Received: May 16, 2016 Accepted: January 16, 2017 Published: March 14, 2017 ABSTRACT Aberrant levels of circulating microRNAs are potential biomarkers for the early detection of colorectal cancer. The aim of this study was to study miR-139-3p and miR-622 in serum as a non-invasive biomarker for colorectal cancer diagnosis. We applied quantitative polymerase chain reaction to determine the levels of miR-139-3p and miR-622 in 42 pairs of tumor and adjacent non-tumor tissues, and in serum samples of 117 patients and 90 control subjects. Our results showed that miR-139-3p was silenced whereas miR-622 was overexpressed in colorectal cancer. Similarly, serum miR-139-3p level was significantly lower in colorectal cancer patients than in control subjects whereas miR-622 was more frequently detectable in patients. ROC analysis showed that AUC of miR-139-3p was 0.9935, with a sensitivity of 96.6% and specificity of 97.8%. Serum miR-139-3p level showed high sensitivity and specificity for both early and late stage CRCs and proximal and distal CRCs. Detectable serum miR-622 showed a sensitivity of 87.5% and specificity of 63.5% for discriminating CRC patients, but the sensitivity dropped for late stage patients (72.7%). We also included analyses of the blood CEA level for comparing the diagnostic performance of these blood-based biomarkers. The median level in CRC patients (3.6 ng/ml) was significantly higher than that in control (1.8 ng/ml). The AUC value of CEA in diagnosing CRC patients was 0.7515. CEA showed a positive correlation with tumor stage and age of patients and its level was higher in male. Collectively, serum miR-139-3p has strong potential as a promising non-invasive biomarker in colorectal cancer detection.
Colorectal cancer (CRC) has been one of the most common cancers worldwide. Without an early diagnosis, the 5-year survival rate is drastically reduced when the tumour reaches advanced stages. In addition to promoting early tumour detection, one strategy for reducing CRC incidences is to develop an effective non-invasive screening method targeting polyp, a potential precursor lesion to CRC. To date, no non-invasive test has been developed for polyp identification with a promising sensitivity. One potential novel biomarker is the microRNAs (miRNA), which their dysregulation has been extensively studied in tumorigenesis of CRC, but relatively less in-depth for the polyp. We hypothesized circulatory miRNAs may serve as potential biomarkers for indicating the presence of the polyp.
Methods
8 miRNA candidates were included in this study, and their expressions in the serum of 170 subjects (86 normal and 84 polyp cases) were detected using real-time PCR. A linear regression model was used to establish panels of miRNA for polyp detection, followed by ROC analysis to evaluate their diagnostic values for the polyp.
Results
Among 8 miRNA candidates, expression of miR16–5p, miR21–5p, miR25–3p, miR106b-5p and miR1246 showed significant variation between normal and polyp serum samples. ROC analysis suggested the panel of 5 miRNA may distinguish polyp patients from normal subjects with the AUC value of 0.69, achieving a sensitivity of 54.7% at 77.1% specificity (p < 0.0001).
Conclusions
Serum miRNA panel may serve as an adequate screening method for polyp identification with one of the best performances among non-invasive tests available. Screening of the polyp alongside other blood tests may promote awareness of polyp incidents and lead to earlier polypectomy, especially in the younger age group where colonoscopy is less prevalent.
Methylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.
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