Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma and its prognostic significance.

MicroRNAs are a class of non-coding molecules found to regulate a variety of cellular functions in health and disease. Dysregulation of microRNAs is involved in liver disease, especially hepatocarcinogenesis. Since primary hepatic malignancies are typically characterized by late diagnosis, frequent recurrence, and poor response to adjuvant therapy, there is a need for the discovery of novel biomarkers in order to achieve earlier diagnosis, predict tumor aggressiveness and response to adjuvant therapy. The purpose of this study is to evaluate the expression of certain microRNAs (miR-21, -31, -122, -145, -146a, - 200c, -221, -222 and -223) in patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), as well as to assess their prognostic significance. Micro-RNA expression was assessed by reverse transcription and real-time PCR (RT-PCR). Clinicopathological data and survival rates were retrieved and analyzed. According to our results, miR-21, miR-31, miR-122, miR-221, miR-222 were significantly up-regulated in HCC tissues, whereas miR-145, miR-146a, miR-200c, and miR-223 were found to be down-regulated. Concerning ICC samples, miR-21, miR-31, and miR-223 were found to be over-expressed, whereas miR-122, miR-145, miR-200c, miR-221, and miR-222 were down-regulated. Additionally, expression of miR-21, miR-31, miR-122, and miR-221 in HCC correlated with cirrhosis, while miR-21 and miR-221 associated with tumor stage and poor prognosis. In ICC tissues, miR-21, miR-31, and miR-223 were found to be over-expressed, but no correlation with clinicopathological features was found.

Quantum dots hold promise for early cancer imaging and detection.

Despite all major breakthroughs in recent years of research concerning the complex events that lead to cancer expression and metastasis, we are not yet able to effectively treat cancer that has spread to vital organs. The various clinical phases originating from cancer diagnosis through treatment and prognosis require a comprehensive understanding of these events, to utilise pre-symptomatic, minimally invasive and targeted cancer management techniques. Current imaging modalities such as ultrasound, computed tomography, magnetic resonance imaging and gamma scintigraphy facilitate the pre-operative study of tumours, but they have been rendered unable to visualise cancer in early stages, due to their intrinsic limitations. The semiconductor nanocrystal quantum dots (QDs) have excellent photo-physical properties, and the QDs-based probes have achieved encouraging developments in cellular (in vitro) and in vivo molecular imaging. However, the same unique physical and chemical properties which renowned QDs attractive may be associated with their potentially catastrophic effects on living cells and tissues. There are critical issues that need to be further examined to properly assess the risks associated with the manufacturing and use of QDs in cancer management. In this review, we aim to describe the current utilisation of QDs as well as their future prospective to decipher and confront cancer.

In vitro effect of hyperthermic Ag and Au Fe3O4 nanoparticles in cancer cells.

PURPOSE: To investigate the anti-cancer efficacy of hyperthermic Ag and Au Fe3O4 core nanoparticles via cytotoxicity study (MTT assay) and the underlying molecular mechanism of action (changes in gene expression via quantitive real time PCR (qRT-PCR). METHODS: HEK293, HCT116, 4T1 and HUH7 human cell lines and 4T1 musculus mammary gland cell line were incubated with Fe3O4 core Ag(Au) shell nanoparticles (NPs) prior to a hyperthermia session. MTT assay was performed to estimate the cytotoxic effects of these NPs. RNA extraction and cDNA synthesis followed so as to quantify mRNA fold change of hsp-70, p53, bcl-2 and casp-3 via qRT-PCR. RESULTS: Fe3O4 core Au shell (concentrations of 400 and 600µg/mL) produced the greatest reduction of viability on HCT116 and 4T1 cells while Fe3O4 core Ag shell (200, 400 and 600µg/mL) reduce viability on HUH7 cells. Hsp-70, p53 and casp-3 were up-regulated while bcl-2 was downregulated in most cases. CONCLUSIONS: Fe3O4 core Ag (Au) shell induced apoptosis on cancer cells (HCT116 and HUH7) via the p53/bcl-2/casp-3 pathway. 4T1 cells also underwent apoptosis via a p53-independent pathway.

Ag/Au Bimetallic Nanoparticles Inhibit Tumor Growth and Prevent Metastasis in a Mouse Model.

PURPOSE: To evaluate the antitumor efficacy of Ag3Au1Trp1:2NPs in a SCID mouse cancer model, with respect to their effect on tumor growth, on tumor's metastatic potential and the underlying molecular mechanism. SUBJECTS AND METHODS: Ag3Au1Trp1:2NPs were radiolabeled with Gallium-68 and the biodistribution was studied in Swiss mice without tumors and in SCID mice bearing tumors. SCID mice received intratumoral Ag3Au1Trp1:2NPs and tumor size was measured using calipers. Lung and liver tissues were extracted and studied microscopically for the detection of any metastatic sites. Changes in the Caspase-3 and TNF-related apoptosis-inducing ligand (TRAIL) were also investigated using real-time PCR and Western blot techniques, respectively. RESULTS: In the 4T1 tumor-bearing SCID mice, Ag3Au1Trp1:2NPs showed quick passive accumulation at tumor sites at 30 mins post-injection. Mice that received the highest dose of NPs (5.6mg/mL) demonstrated a 1.9-fold lower tumor volume compared to that of the control group at 11 days post-injection, while mice that did not receive NPs showed metastatic sites in liver and lung. Extracted tumor tissue of treated mice revealed increased Casp-3 mRNA levels as well as elevated TRAIL protein levels. CONCLUSION: Based on our results, Ag3Au1Trp1:2NPs express anti-tumor and anti-metastatic effects in vivo. Ag3Au1Trp1:2NPs also reach tumor site via the enhancement and retention effect which results in the apoptotic death of cancerous cells selectively via the extrinsic TRAIL-dependent pathway.

Ag/Au bimetallic nanoparticles induce apoptosis in human cancer cell lines via P53, CASPASE-3 and BAX/BCL-2 pathways.

Au/Ag bimetallic nanoparticles (BNPs) exhibit a wide range of excellent electronic, chemical, biological, mechanical and thermal properties due to synergistic effects. However, critical questions regarding stability, biocompatibility and their cytotoxic effects remain to be answered. In this study, Ag/Au BNPs have been synthesized as "alloy" via a chemical reduction method using double molar excess of tryptophan [ν(M):ν(Trp) = 1:2]. We then estimated their toxicity in HCT116, 4T1, HUH7 and HEK293 cell lines in monocellular and spheroid cultures. Ag/Au nanoparticles with metal ratio 3:1, had the maximal antitumor effect in cancer cell lines, while the toxicity was found significantly decreased in non-cancerous cell lines. Our results were also compared to previous data regarding Ag/Au using single molar excess of tryptophan [ν(M):ν(Trp) = 1:1], suggesting that tryptophan has a protective effect on HEK293 and not in cancer cells. Aiming to investigate the molecular mechanism behind nanopartricles cytotoxicity, we studied the expression of cell cycle and apoptosis related genes on HCT116, 4T1, and HUH7 monocellular culture. Hence, we showed that bimetallic cytotoxicity is mediated via the caspase and the p53/Bax/Bcl-2 apoptotic pathway. In conclusion, our study suggests tryptophan ratio along with metal ratio used in Ag/Au BNPs as a successful way to control the toxicity in cancer cells towards non-cancerous cells, underlying the potency of bimetallic nanoparticles as selective anti-tumor agents.

Identification of Methylation Profiles of Cancer-related Genes in Circulating Tumor Cells Population.

BACKGROUND: We performed an epigenetic analysis of the first exon of the hVIM gene and the SFRP2 in circulating tumor cells (CTCs) and correlation with the corresponding primary colorectal cancer (CRC) tissue. PATIENTS AND METHODS: CTCs detection in 52 colorectal cancer patients was managed by a multi-marker immunomagnetic method with the use of quantum dots (QDs). To determine methylation levels we used high-resolution melting (HRM) technology. RESULTS: In the case of VIM we found 76.9% methylated samples, compared to 53.8% in tissue samples. Regarding SFRP2 promoter methylation levels in tissue and CTCs samples, 67.3% and 73.1%, were found methylated respectively. Correlation analysis of methylation levels with KRAS and BRAF mutations (performed in our previous study) demonstrates that high-methylation epigenotype strongly correlates to BRAF mutation. CONCLUSION: CTCs is a promising diagnostic tool. The combination of genetic mutations and epigenetic aberrations specifically in CTCs, will ameliorate CRC diagnosis in the future.

Gallium-68 Labeled Iron Oxide Nanoparticles Coated with 2,3-Dicarboxypropane-1,1-diphosphonic Acid as a Potential PET/MR Imaging Agent: A Proof-of-Concept Study.

The aim of this study was to develop a dual-modality PET/MR imaging probe by radiolabeling iron oxide magnetic nanoparticles (IONPs), surface functionalized with water soluble stabilizer 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), with the positron emitter Gallium-68. Magnetite nanoparticles (Fe3O4 MNPs) were synthesized via coprecipitation method and were stabilized with DPD. The Fe3O4-DPD MNPs were characterized based on their structure, morphology, size, surface charge, and magnetic properties. In vitro cytotoxicity studies showed reduced toxicity in normal cells, compared to cancer cells. Fe3O4-DPD MNPs were successfully labeled with Gallium-68 at high radiochemical purity (>91%) and their stability in human serum and in PBS was demonstrated, along with their further characterization on size and magnetic properties. The ex vivo biodistribution studies in normal Swiss mice showed high uptake in the liver followed by spleen. The acquired PET images were in accordance with the ex vivo biodistribution results. Our findings indicate that 68Ga-Fe3O4-DPD MNPs could serve as an important diagnostic tool for biomedical imaging.

Mutational analysis of circulating tumor cells from colorectal cancer patients and correlation with primary tumor tissue.

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.

Quantum dots-bevacizumab complexes for in vivo imaging of tumors.

BACKGROUND/AIM: The basic role of vascular endothelial growth factor (VEGF) in cancer is underscored by the approval of bevacizumab for first-line treatment of cancer patients. Recent anticancer therapeutics based on active tumor targeting by conjugating tumor-specific antibodies has become of great interest in oncology. Current progress in nanomedicine has exploited the possibility of designing tumor-targeted nanocarriers able to deliver specific molecule payloads in a selective manner to improve the efficacy and safety of cancer imaging and therapy. We herein aimed to determine the targeting ability of bevacizumab-conjugated quantum dots (QDs) in vitro and in vivo. MATERIALS AND METHODS: We used QDs labeled with bevacizumab, in various in vitro experiments using cell lines derived from colorectal cancer (CRC) and breast cancer (BC). For a competition study of QD-bevacizumab complex and bevacizumab, the cells were pre-treated with bevacizumab (100 nmol/L) for 24 h before exposure to the QD-bevacizumab complex. The breast cancer cells (MDA-MB-231) were injected to 9 nude mice to make the xenograft tumor model. The QD-bevacizumab complex was injected into the tumor model and fluorescence measurements were performed at 1, 12, and 24 h post-injection. RESULTS: Immunocytochemical data confirmed strong and specific binding of the QD-bevacizumab complex to the cell lines. The cells pre-treated with an excess of bevacizumab showed absence of QD binding. The in vivo fluorescence image disclosed that there was an increased signal of tumor after the injection of QDs. Ex vivo analysis showed 3.1 ± 0.8%, 28.6 ± 5.4% and 30.8 ± 4.2% injected dose/g accumulated in the tumors at 1, 12 and 24 h respectively. Tumor uptake was significantly decreased in the animals pretreated with excess of bevacizumab (p=0.001). CONCLUSION: In conclusion, we could successfully detect the VEGF-expressing tumors using QDs-bevacizumab nanoprobes in vitro and in vivo, opening new perspectives for VEGF-targeted non-invasive imaging in clinical practice.

Expression of microRNAs in patients with pancreatic cancer and its prognostic significance.

OBJECTIVES: Investigation of expression profile of well-established microRNAs in pancreatic adenocarcinoma, and its correlation with clinicopathological factors. METHODS: Eighty-eight samples of ductal pancreatic adenocarcinoma and 98 control samples were analyzed by real-time polymerase chain reaction for miR-21, miR-31, miR-122, miR-145, miR-146a, miR-155, miR-210, and miR-222 expressions. The results were normalized and then statistically analyzed using nonparametric statistical tests. RESULTS: According to our results, miR-21, miR-155, miR-210, miR-221, and miR-222, were overexpressed in diseased tissues than in the control samples, whereas miR-31, miR-122, miR-145, and miR-146a were underexpressed. Additionally, the expressions of miR-21 and miR-155 were associated with tumor stage and poor prognosis. CONCLUSIONS: The tumorigenic role of miR-21 and miR-155 was confirmed, whereas down-regulation of miR-31, miR-145, and miR-146a, in dispute with current literature, renders necessary the revision of use of microRNAs as biological markers.

Development of a quantum-dot-labelled magnetic immunoassay method for circulating colorectal cancer cell detection.

AIM: To detect of colorectal cancer (CRC) circulating tumour cells (CTCs) surface antigens, we present an assay incorporating cadmium selenide quantum dots (QDs) in these paper. METHODS: The principle of the assay is the immunomagnetic separation of CTCs from body fluids in conjunction with QDs, using specific antibody biomarkers: epithelial cell adhesion molecule antibody, and monoclonal cytokeratin 19 antibody. The detection signal was acquired from the fluorescence signal of QDs. For the evaluation of the performance, the method under study was used to isolate the human colon adenocarcinoma cell line (DLD-1) and CTCs from CRC patients' peripheral blood. RESULTS: The minimum detection limit of the assay was defined to 10 DLD-1 CRC cells/mL as fluorescence was measured with a spectrofluorometer. Fluorescence-activated cell sorting analysis and Real Time RT-PCR, they both have also been used to evaluate the performance of the described method. In conclusion, we developed a simple, sensitive, efficient and of lower cost (than the existing ones) method for the detection of CRC CTCs in human samples. We have accomplished these results by using magnetic bead isolation and subsequent QD fluorescence detection. CONCLUSION: The method described here can be easily adjusted for any other protein target of either the CTC or the host.