A Licorice Roots Extract Induces Apoptosis and Cell Cycle Arrest and Improves Metabolism via Regulating MiRNAs in Liver Cancer Cells.

Targeting altered metabolism in cancer provides a promising preventive and therapeutic approach. Natural products interplay between gene expression and metabolism either by targeting altered metabolic enzymes and/or affecting the regulating miRNAs. Licorice is a widely known product used as flavoring agent. Glycyrrhizin and other metabolites were reported to exert several metabolic benefits. Here, we investigated the effect of licorice roots extract on some metabolic pathways and their regulating miRNAs in hepatocellular carcinoma cells. Our data showed various beneficial effects of licorice roots extract including induction of apoptosis and cell cycle arrest. Second, upregulating tumor suppressor miRNAs; let7a-3p, miR-34c-5p, miR-122-5p, miR-126-3p, miR195-5p, miR-199a-5p, miR-206, and miR-326-5p. Third, inhibiting HIF1α, PI3K and C-Myc and activating AMPK and p53. Fourth, inhibiting enzymes of glycolysis; HK-2, LDH-A and PK-M2; pentose phosphate pathway; G6PD and glutaminolysis; glutaminase. However, such an extract upregulated oncogenic miRNAs; miR-21, miR-221, and miR-222. Although the present data highlights the ability of licorice roots extract to enhance apoptosis and cell cycle arrest and correct altered metabolism, it warns against its unfavorable effects, hence, its use for prevention and therapy should proceed with caution. Further experiments are required to investigate whether a specific bioactive ingredient is responsible for upregulating the oncogenic miRNAs.

Identification of circulating protein biomarkers in patients with hepatocellular carcinoma concomitantly infected with chronic hepatitis C virus.

CONTEXT: The incidence rate of hepatocellular carcinoma (HCC) is higher in developing countries, and most cases are associated with chronic hepatitis C virus (HCV) infection. OBJECTIVE: To evaluate the circulating proteins as liver biomarkers for the identification of HCC associated with HCV infection in Egyptian patients using LC-MS/MS analysis. METHODS: Blood sera were collected from 31 HCC patients and the fractionated proteins were subjected to LC-MS/MS analysis. Protein candidates were validated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-three proteins were significantly identified in the sera of HCC patients with persistent HCV infection. These proteins are involved in several biological processes including acute phase response, complement activation, hemostasis process and lipid metabolism. The level of lectin galactoside-binding soluble 3 binding protein (LGALS3BP), Kininogen-1 (KNG1), serum amyloid A2 (SAA2) and paraoxonase 1 (PON1) and alpha-fetoprtoein (AFP) were elevated in serum. CONCLUSION: In HCC patients with chronic HCV infection, we identified a group of differentially expressed circulating proteins involved in regulating different cellular mechanisms.

Promoter SNPs rs116896264 and rs73933062 form a distinct haplotype and are associated with galectin-4 overexpression in colorectal cancer.

Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Urinary high molecular weight matrix metalloproteinases as non-invasive biomarker for detection of bladder cancer.

BACKGROUND: Matrix Metalloproteinases (MMPs) are key molecules for tumor growth, invasion and metastasis. Over-expression of different MMPs in tumor tissues can disturb the homeostasis and increase the level of various body fluids. Many MMPs including high molecular weights (HMWs) were detected in the urine of prostate and bladder cancer patients. Our aim here is to assess the usefulness of HMW MMPs as non invasive biomarkers in bilharzial bladder cancer in Egyptian patients. METHODS: The activity of different MMPs including HMW species was determined using zymographic analysis technique in the urine samples procured from sixty six bladder cancer patients (bilharzial and non-bilharzial) as well as hundred healthy control subjects. Also, the correlation between these HMW MMPs activities and different clinico-pathological parameters was investigated. RESULTS: High frequency of urine MMPs (uMMPs) activity was determined in 63.6% of examined tumor cases, however, none of the control cases showed any uMMPs activity. MMP-9 had the highest activity (62%) followed by MMP9/NGAL (60%), MMP-2 (54.5%), MMP-9 dimer (53%), ADAMTS (25.6%), and the lowest one was MMP-9/TIMP-1 (12%) only. There was no correlation between uMMPs and any of clinico-pathological parameters including age, gender, tumor size and type, bilharziasis, grade, lymph node involvement, and invasion to the prostate. A significant correlation was established only between MMP-9/TIMP-1 activities with the tumor size. CONCLUSIONS: This study revealed that the detection of urinary MMPs including HMWs activity might be sensitive biomarkers for prediction of bladder cancer. It is also demonstrate that the detection of these urinary HMW gelatinases could not differentiate between bilharzial and non bilharzial bladder cancer subtypes.

Lymphocyte to monocyte ratio predicts survival and is epigenetically linked to miR-222-3p and miR-26b-5p in diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. 10-20% of the patients present with bone marrow (BM) involvement which predicts a worse survival. This study aimed to determine the prognostic significance of serum miR-222-3p, miR-26b-5p, EBV-miR-BHRF1-2-5p, and EBV-miR-BHRF1-2-3p and correlate their levels to clinical and haematological markers in DLBCL with special emphasis on the lymphocyte-monocyte ratio (LMR) and neutrophil-monocyte ratio. We also studied the role of BM BMI1 and PIM2 proteins in predicting BM infiltration. Serum miRNAs were studied on 40 DLBCL and 18 normal individuals using qRT-PCR. BMI1 and PIM2 proteins were studied on BM biopsies by immunohistochemistry. The results were correlated with clinical and follow-up data. All the studied miRNAs were dysregulated in DLBCL serum samples. BMI1 and PIM2 were expressed in 67% and 77.5% of BM samples, respectively. LMR was significantly associated with disease-free survival (DFS) (P = 0.022), miR-222-3P (P = 0.043), and miR-26b-5p (P = 0.043). EBV-miR-BHRF1-2-3p was significantly correlated to haemoglobin level (P = 0.027). MiR-222-3p, miR-26b-5p, and EBV-miR-BHRF1-2-5p expressions were significantly correlated to each other (P = 0.001). There was no significant correlation between the studied markers and follow-up data. LMR is a simple method for predicting survival in DLBCL. MiR-222-3p and miR-26b-5p may be implicated in an immunological mechanism affecting patients' immunity and accordingly influence LMR. The correlation between miR-222-3p, miR-26b-5p, and EBV-miR-BHRF1-2-5p may indicate a common mechanism among the 3 miRNAs that may explain DLBCL pathogenesis.

Clinical relevance of cyclooxygenase-2 and matrix metalloproteinases (MMP-2 and MT1-MMP) in human breast cancer tissue.

Breast cancer (BC) is the most common neoplasm among women in most developed countries, including Egypt. Elevated levels of certain proteins in human BC are associated with unfavorable prognosis and progressive stages of the disease. The aim of our study was to evaluate the protein expression profile and prognostic significance of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), MMP-9 and membrane type 1-MMP (MT1-MMP) and their interaction in operable BC patients. The protein expression of COX-2, MMP-2 and MT1-MMP were evaluated by western blot technique, whereas enzymatic activity of MMP-2 and MMP-9 was determined by zymography in 47 breast cancer patients as well as normal adjacent tissues. Also, the correlation between these proteins and age, tumor size, LN stage, TNM stage, estrogen receptor, progesterone receptor, disease-free survival, and overall survival (OS) has been investigated. As compared to adjacent normal tissues, COX-2, MMP-2 and MT1-MMP were over-expressed in 43, 64, and 60 % of tumor tissues, respectively. In the same pattern, the activity of MMP-2 (62 %) and MMP-9 (45 %) was elevated in BC tissues. Multivariate analysis showed a positive correlation between the protein expression of COX-2, MMP-2, and MT1-MMP and the activity of MMP-2 and MMP-9 in BC patients. However, the enzymatic activity showed no correlation with clinicopathological features. This study confirms the preclinical evidence that COX-2 increased the expression of MT1-MMP, which in turn activates MMP-2. The lack of correlation with clinicopathological features, OS or disease-free survival ascertains the complexity of tumor progression and metastasis with many pro- and counter regulatory factors.

Possible Use of miR-223-3p as a Prognostic Marker in Transarterial Chemoembolization Treatment of Hepatocellular Carcinoma Patients.

INTRODUCTION AND OBJECTIVES: Transcatheter chemoembolization (TACE) is the recommended therapy for intermediate stage hepatocellular carcinoma patients. Unfortunately, one of the main reasons for its failure is the emergence of multidrug resistance (MDR). Therefore, this study explored the possibility of using MDR-related miRNA as a response biomarker in HCC patients treated with doxorubicin drug-eluting bead TACE (DEB-TACE). PATIENTS AND METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the expression level of 14 MDR-related miRNAs in doxorubicin-resistant HepG2 cells (HepG2/Dox) developed by single-dose of doxorubicin mimicking the situation of liver cells surviving TACE. The sera level of miR-223-3p, which was the most significantly downregulated in the HepG2 cells, was determined in 60 primary HCC patients undergoing TACE. Restoring miR-223-3p in HepG2/Dox cell line was achieved by its mimic transfection. Cell sensitivity was measured by SRB assay. Cell apoptosis and doxorubicin uptake were assessed by flow cytometry. The expression of miR-223-3p target protein, P-glycoprotein, was evaluated using qRT-PCR and western blotting. RESULTS: We detected a significant downregulation of circulating miR-223-3p in patients non-responders to TACE treatment compared with responders. The expression of miR-223-3p was markedly decreased in resistant HepG2/Dox cells compared to the parental control. In addition, the expression of miR-223-3p was found to be inversely correlated with P-glycoprotein expression thus confirming the role of miR-223-3p in MDR. Furthermore, overexpression of miR-223-3p suppressed P-glycoprotein which promoted cellular uptake of doxorubicin and increased apoptosis. CONCLUSIONS: Our data suggest a potential role for miR-233-3p as a prognostic as well as a therapeutic target for HCC.

The Role, Significance, and Association of MicroRNA-10a/b in Physiology of Cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the translation of mRNA and protein, mainly at the posttranscriptional level. Global expression profiling of miRNAs has demonstrated a broad spectrum of aberrations that correlated with several diseases, and miRNA- 10a and miRNA-10b were the first examined miRNAs to be involved in abnormal activities upon dysregulation, including many types of cancers and progressive diseases. It is expected that the same miRNAs behave inconsistently within different types of cancer. This review aims to provide a set of information about our updated understanding of miRNA-10a and miRNA-10b and their clinical significance, molecular targets, current research gaps, and possible future applications of such potent regulators.

Role of let7-g and miR-221 level as potential predictors for overall survival of hepatocellular carcinoma patients.

BACKGROUND AND STUDY AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancer types worldwide. A hallmark of epithelial-mesenchymal transition is the loss of epithelial E-cadherin, which is considered an epithelial differentiation marker. MicroRNAs serve vital roles in various biological processes in the cell via post-transcriptional gene regulation. Therefore, the present study aimed to investigate the involvement of certain miRNAs in the progression of HCC. PATIENTS AND METHODS: A reverse transcription-quantitative PCR assay was conducted to detect the expression levels of 20 EMT-related miRNAs in 36 fresh tissue biopsies from patients with primary HCC compared with healthy controls. Gene expression levels, as well as immunohistochemistry assays, were performed for E-cadherin, ZEB1 and ZEB2 proteins. The correlation between their expression levels and different clinicopathological factors was also assessed. RESULTS: A significant decrease of E-Cadherin and an increase in ZEB1 expression levels were identified in HCC groups compared with controls, while no significant changes for ZEB2 were found. The absence of E-cadherin membranous protein was observed in ∼48% of the cases examined. Moreover, ZEB1 protein was absent in 46% of E-cadherin positive cases. Upregulation of miR-182, miR-221 and miR-222 expression levels, and downregulation of let-7g, miR-9, miR-16, miR29c, miR122, miR-145, miR-148a, miR-193b, miR-194 and miR-215 expression levels were identified. A positive correlation between let7-g with E-Cadherin expression was reported. No significant association was identified between each of E-cadherin, ZEB1, ZEB2 or miRNAs examined with different clinicopathological features of the patients. Furthermore, the low expression of let7-g and high expression of miR-221 were associated with poorer survival. CONCLUSION: Collectively, the present data suggested that let7-g functions as a tumor suppressor in the development of HCC via regulating E-Cadherin. Furthermore, both let7-g and miR-221 may be potential biomarkers for the outcomes of HCC patients.

MicroRNA-520c-3p Modulates Doxorubicin-Chemosensitivity in HepG2 Cells.

BACKGROUND: Doxorubicin (DOX) is one of the most common drugs used in cancer therapy, including Hepatocellular Carcinoma (HCC). Drug resistance is one of chemotherapy's significant problems. Emerging studies have shown that microRNAs (miRNAs) could participate in regulating this mechanism. Nevertheless, the impact of miRNAs on HCC chemoresistance is still enigmatic. OBJECTIVE: Investigating the role of microRNA-520c-3p (miR-520c-3p) in the enhancement of the anti-tumor effect of DOX against HepG2 cells. METHODS: Expression profile for liver-related miRNAs (384 miRNAs) has been analyzed on HepG2 cells treated with DOX using qRT-PCR. miR-520c-3p, the most deregulated miRNA, was selected for combination treatment with DOX. The expression level for LEF1, CDK2, CDH1, VIM, Mcl-1 and p53 was evaluated in miR-520c-3p transfected cells. Cell viability, colony formation, wound healing as well as apoptosis assays have been demonstrated. Furthermore, Mcl-1 protein level was measured using the western blot technique. RESULTS: The present data indicated that miR-520c-3p overexpression could render HepG2 cells chemo-sensitive to DOX through enhancing its suppressive effects on proliferation, migration, and induction of apoptosis. The suppressive effect of miR-520c-3p involved altering the expression levels of some key regulators of cell cycle, proliferation, migration and apoptosis, including LEF1, CDK2, CDH1, VIM, Mcl-1 and p53. Interestingly, Mcl-1 was found to be one of the potential targets of miR-520c-3p, and its protein expression level was down-regulated upon miR-520c-3p overexpression. CONCLUSION: Our data referred to the tumor suppressor function of miR-520c-3p that could modulate the chemosensitivity of HepG2 cells towards DOX treatment, providing a promising therapeutic strategy in HCC.

MicroRNA-372-3p Predicts Response of TACE Patients Treated with Doxorubicin and Enhances Chemosensitivity in Hepatocellular Carcinoma.

BACKGROUND: Identification of factors to detect and improve chemotherapy.response in cancer is the main concern. microRNA-372-3p (miR-372-3p) has been demonstrated to play a crucial role in cellular proliferation, apoptosis and metastasis of various cancers including Hepatocellular Carcinoma (HCC). However, its contribution towards Doxorubicin (Dox) chemosensitivity in HCC has never been studied. OBJECTIVE: This study aims to investigate the potential role of miR-372-3p in enhancing Dox effects on HCC cell line (HepG2). Additionally, the correlation between miR-372-3p and HCC patients who received Transarterial Chemoembolization (TACE) with Dox treatment has been analyzed. METHODS: Different cell processes were elucidated by cell viability, colony formation, apoptosis and wound healing assays after miR-372-3p transfection in HepG2 cells Furthermore, the miR-372-3p level has been estimated in the blood of primary HCC patients treated with TACE/Dox by quantitative real-time PCR assay. Receiver Operating Curve (ROC) analysis for serum miR-372-3p was constructed for its prognostic significance. Finally, the protein level of Mcl-1, the anti-apoptotic player, has been evaluated using western blot. RESULTS: We found a significantly higher level of miR-372-3p in the blood of the responder group of HCC patients who received TACE with Dox than of non-responders. Ectopic expression of miR-372-3p reduced cell proliferation, migration and significantly induced apoptosis in HepG2 cells which was coupled with a decrease of anti-apoptotic protein Mcl-1. CONCLUSION: Our study demonstrated that miR-372-3p acts as a tumor suppressor in HCC and can act as a predictor biomarker for drug response. Furthermore, the data referred for the first time its potential role in drug sensitivity that might be a therapeutic target for HCC.

DNA Methylation of TGFß Target Genes: Epigenetic Control of TGFß Functional Duality in Liver Cancer.

Transforming growth factor beta (TGFß) plays a key role in liver carcinogenesis. However, its action is complex, since TGFß exhibits tumor-suppressive or oncogenic properties, depending on the tumor stage. At an early stage TGFß exhibits cytostatic features, but at a later stage it promotes cell growth and metastasis, as a potent inducer of epithelial to mesenchymal transition (EMT). Here, we evaluated DNA methylation as a possible molecular mechanism switching TGFß activity toward tumor progression in hepatocellular carcinoma (HCC). We report that decitabine, a demethylating agent already used in the clinic for the treatment of several cancers, greatly impairs the transcriptional response of SNU449 HCC cells to TGFß. Importantly, decitabine was shown to induce the expression of EMT-related transcription factors (e.g., SNAI1/2, ZEB1/2). We also report that the promoter of SNAI1 was hypomethylated in poor-prognosis human HCC, i.e., associated with high grade, high AFP level, metastasis and recurrence. Altogether, the data highlight an epigenetic control of several effectors of the TGFß pathway in human HCC possibly involved in switching its action toward EMT and tumor progression. Thus, we conclude that epidrugs should be carefully evaluated for the treatment of HCC, as they may activate tumor promoting pathways.

Induction of Anti-Proliferative and Apoptotic Effects of Sorafenib Using miR-27a Inhibitor in Hepatocellular Carcinoma Cell Lines.

OBJECTIVE: The purpose of the current study was to investigate the possible anti-tumor effect of miR-27a inhibitor in combination with Sorafenib (SOR) on cell proliferation and apoptosis of hepatocellular carcinoma cell lines. METHODS: Transient transfection by oligo-miR27a inhibitor (miR-27ai) was used in this study for targeting the oncogenic miR-27a in HepG2 and Huh7 cells followed by SOR treatment. Cell viability was measured using SRB assay. The cell cycle and apoptosis were assessed by flow cytometry assay. Moreover, the level of oncogenic miR-27a was evaluated in 19 tissues of primary HCC patients as well as cell lines using qRT-PCR assay. Finally, caspase-3 activity was determined using ELISA assay. RESULTS: Significant up-regulation of miR-27a expression was reported in HCC patients confirming its oncogenic role. Treatment of cells with SOR following transfection with miR-27ai declined cell viability significantly compared with either control or single agent treatment (p≤0.05). Highly significant decreasing in the number of cell in S-phase associated with increasing in G0-phase was also observed. Furthermore, apoptotic rate was highly significantly increased for transfected/SOR treated cells (p≤0.01). Finally, combination treatment demonstrated a significant elevation of caspase-3 activity level in both cell lines examined. CONCLUSION: The present data demonstrated targeting miR-27a enhances the anti-tumor effect of SOR in HCC cell lines considering as one of the promising therapeutic targets for advanced HCC management.

Withdrawal Notice: Role, Significance and Association of microRNA-10a/b in Physiology of Cancer

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Circulating miR-26a, miR-106b, miR-107 and miR-133b stratify hepatocellular carcinoma patients according to their response to transarterial chemoembolization.

BACKGROUND: A number of hepatocellular carcinoma (HCC) patients have developed resistance against transcatheter arterial chemoembolization (TACE) treatment. In this study, we aimed to develop a panel of microRNAs (miRs) biomarkers to predict clinical outcomes in HCC patients after TACE treatment. METHODS: The expression level of twenty miRs was evaluated in FFPE tissues collected from 33 HCC patients. We selected four differentially expressed miRs in TACE-responders versus non-responders and re-assessed their expression in 51 serum samples. The expressions of miRs associated with overall survival (OS), progression-free survival (PFS), and treatment outcomes were investigated. The diagnostic accuracy of these miRs in predicting patients' response to TACE was also evaluated. RESULTS: The baseline of miR-106b, miR-107 and miR-133b was significantly elevated (p < .001) in sera of TACE-responders while miR-26a was elevated (p < .001) in non-responders. miR-26a and miR-133b recorded the highest diagnostic performance as individual classifiers in response to TACE (AUC = 1.0 and 100% sensitivity and specificity). Intriguingly, miR-133b distinguished complete responders from partial responders and non-responders (AUC ≥ 0.90). The PFS was improved (p < .05) in the high expression group of miR-31, miR-200b, miR-133b and miR-181a over their low expression group. CONCLUSION: Circulating miR-133b, miR-26a, miR-107 and miR-106 in serum are potential candidates to be utilized as prognostic biomarkers for predication of TACE treatment outcomes in HCC patients.

Novel CYP1B1 mutations and a possible prognostic use for surgical management of congenital glaucoma.

AIM: To identify CYP1B1 gene mutations and evaluate their possible role as a prognostic factor for success rates in the surgical management of Egyptian congenital glaucoma patients. METHODS: Totally 42 eyes of 29 primary congenital glaucoma patients were operated on with combined trabeculotomy/trabeculectomy with mitomycin-C and followed up at 1d, 1wk, 1, 6 and 12mo postoperatively. Genomic DNA was extracted from peripheral blood leukocytes. Coding regions of CYP1B1 gene were amplified using 13 pairs of primers, screened for mutations using single-strand conformation polymorphism followed by sequencing of both strands. Efficacy of the operation was graded as either a success [maintaining intraocular pressure (IOP) less than 21 mm Hg with or without anti-glaucoma medication], or a failure (IOP more than 21 mm Hg with topical antiglaucoma medications). RESULTS: Seven novel mutations out of a total of 15 different mutations were found in the CYP1B1 genes of 14 patients (48.2%). The presence of CYP1B1 gene mutations did not correlate with the failure of the surgery (P=0.156, odds ratio=3.611, 95%CI, 0.56 to 22.89); while the positive consanguinity strongly correlated with failure of the initial procedure (P=0.016, odds ratio=11.25, 95%CI, 1.57 to 80.30). However, the Kaplan-Meier survival analysis revealed a significantly lower time of IOP control in the subgroup with mutations in CYP1B1 versus the congenital primary glaucoma group without mutations (log rank test, P=0.015). CONCLUSION: Seven new CYP1B1 mutations are identified in Egyptian patients. Patients harboring confirmed mutations suffered from early failure of the initial surgery. CYP1B1 mutations could be considered as a prognostic factor for surgery in primary congenital glaucoma.

A microfluidic platform for high-purity separating circulating tumor cells at the single-cell level.

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.

Design, synthesis and screening of 1, 2, 4-triazinone derivatives as potential antitumor agents with apoptosis inducing activity on MCF-7 breast cancer cell line.

Some triazinone derivatives are designed and synthesized as potential antitumor agents. Triazinone derivatives 4c, 5e and 7c show potent anticancer activity over MCF-7 breast cancer cells higher than podophyllotoxin (podo) by approximate 6-fold. DNA flow cytometry analysis for the compounds 3c, 4c, 5e, 6c and 7c show a potent inhibitory activity of cell proliferation and cell cycle arrest at G2/M phase. Compounds 4c, 5e and 7c exhibit low to moderate ß-tubulin polymerization inhibition percentage. Meanwhile, compound 6c displayed excellent ß-tubulin percentage of polymerization inhibition equivalent to that exhibited by podo. In addition, compounds 4c, 5e and 7c show strong topoisomerase (topo) II inhibitory activity in nano-molar concentration, compared to known topo inhibitor as etoposide. Finally, apoptotic inducing activity over MCF-7 of compounds 4c, 5e, 6c and 7c is due to up-regulation of p53, increased Bax/Bcl-2 ratio and caspase3/7 levels 2-fold higher than podo.

Crosstalk between liver-related microRNAs and Wnt/ß-catenin pathway in hepatocellular carcinoma patients.

BACKGROUND AND STUDY AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with highest incidence in Asia and Africa. MicroRNAs (miRNAs), a class of non-coding single stranded RNA, which not only post transcriptionally regulate gene expression but also respond to signaling molecules to affect cell functions such as Wnt/ß-catenin signaling specifically in HCC. The goal of this study is to investigate the crosstalk between Wnt/ß-catenin signaling proteins and microRNAs expression in HCC patients. PATIENTS AND METHODS: Fresh tissue samples of 30 primary HCC patients and 10 control subjects were included. Expression level of 13 different miRNAs (miR-10a- miR-106b- miR-99a- miR-148a- miR-125b- miR-30e- miR-183- miR-155- miR-199a- miR-199a3p- miR-24- miR-122 and miR-215) were examined using real-time PCR assay. Five proteins involved in the Wnt/ß-catenin pathway (ß-catenin, APC, c-myc, survivin and cyclin D1) were analysed by immunohistochemistry technique. The correlation between miRNAs expression levels with protein expressions was assessed. RESULTS: Up-regulation of miR-155 and miR-183 was reported in HCC patients compared to normal controls and this up-regulation was significantly correlated with liver cirrhosis in the case of miR-155 (p<0.05) referring to their oncogenic activity. Down-regulation was observed for 11 miRNAs in HCC indicating their tumour suppression activity. MiRNA-10a, miR-30e, miR-215, miR-125b and miR-148a were significantly correlated with the expression of important players in Wnt/ß-catenin pathway including ß-catenin, APC and c-myc (p<0.05). Detailed analysis revealed that miR-215 is associated with the grade of the disease and miR-125b is associated with HCV infection. CONCLUSION: Collectively, our data showed potential role of miR-10a, miR-30e, miR-215, miR-125b and miR-148a as important mediators in HCC progression. Furthermore, their association with Wnt/ß-catenin cascade proteins could be exploited to develop new therapeutic target strategies in HCC.

Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy.

Lung cancer cells show inherent and acquired resistance to chemotherapy. The lack of good predictive markers/novel targets and the incomplete understanding of the mechanisms of resistance limit the success of lung cancer response to chemotherapy. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) to study the role of epigenetics and miRNA in resistance/response of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 and4, DNA methyltransferase, acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells, of these 14 miRNAs, 4 (has-mir-1973, 494, 4286 and 29b-3p) have shown 2.99 - 4.44 fold increase in their expression. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide. Sequential treatment with the epigenetic modifiers trichostatin A or 5-aza-2'-deoxycytidine followed by doxorubicin resulted in: (i) enhanced sensitivity of both cell lines to doxorubicin especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing induction of DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as resistance markers/targets to improve response of resistant cells to doxorubicin and for the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.