Role of TGF-ß1 and C-Kit Mutations in the Development of Hepatocellular Carcinoma in Hepatitis C Virus-Infected Patients: in vitro Study.

Transforming growth factor beta (TGF-ß) acts as a tumor-suppressing cytokine in healthy tissues and non-malignant tumors. Yet, in malignancy, TGF-ß can exert the opposite effects that can promote proliferation of cancer cells. C-Kit plays a prominent role in stem cell activation and liver regeneration after injury. However, little is known about the cross-talk between TGF-ß and C-Kit and its role in the progression of hepatocellular carcinoma (HCC). Here, we studied the effect of increasing doses of TGF-ß1 on CD44+CD90+ liver stem cells (LSCs) and C-Kit gene expression in malignant and adjacent non-malignant liver tissues excised from 32 HCC patients. The percentage of LSCs in malignant tumors was two times higher compared to their counterparts from the non-malignant tissues. When treated with increasing doses of TGF-ß1, proliferation of both malignant and non-malignant LSCs was progressively suppressed, but low TGF-ß1 dose failed to suppress the growth of malignant LSCs. Moreover, C-Kit exons 9 and 11 were expressed in malignant LSCs, but not in their non-malignant counterparts. Analysis of C-Kit detected mutations in exon 9 (but not in exon 11) in some malignant liver cells resulting in the changes in the amino acid sequence and dysregulation of protein structure and function. Interestingly, in malignant liver cells, mutations in exon 9 were associated with high-viremia hepatitis C virus (HCV), and expression of this exon was not suppressed by the TGF-ß1 treatment at all doses. To our knowledge, this is the first report that mutations in the C-Kit gene in HCC patients are associated with high- viremia HCV. Our study emphasizes the need for investigation of the TGF-ß1 level and C-Kit mutations in patients with chronic HCV for HCC prevention and better therapy management.

Adolescence alcohol exposure impairs fear extinction and alters medial prefrontal cortex plasticity.

After experiencing a traumatic event people often turn to alcohol to cope with symptoms. In those with post-traumatic stress disorder (PTSD) and a co-occurring alcohol use disorder (AUD), PTSD symptoms can worsen, suggesting that alcohol changes how traumatic memory is expressed. The objective of this series of experiments is to identify how alcohol drinking (EtOH), following cued fear conditioning and extinction, impacts fear expression in mice. Molecular (activity-regulated cytoskeleton-associated protein, Arc/arg3.1) and structural (dendrite and spine morphometry) markers of neuronal plasticity were measured following remote extinction retrieval. Mouse age (adolescent and adult) and sex were included as interacting variables in a full factorial design. Females drank more EtOH than males and adolescents drank more EtOH than adults. Adolescent females escalated EtOH intake across drinking days. Adolescent drinkers exhibited more conditioned freezing during extinction retrieval, an effect that persisted for at least 20 days. Heightened cued freezing in the adolescent group was associated with greater Arc/arg3.1 expression in layer (L) 2/3 prelimbic (PL) cortex, greater spine density, and reduced basal dendrite complexity. In adults, drinking was associated with reduced L2/3 infralimbic (IL) Arc expression but no behavioral differences. Few sex interactions were uncovered throughout. Overall, these data identify prolonged age-related differences in alcohol-induced fear extinction impairment and medial prefrontal cortex neuroadaptations.

Serum amyloid A is elevated in the serum of lung cancer patients with poor prognosis.

BACKGROUND: Lung cancer is known as the top cancer killer in most developed countries. However, there is currently no promising diagnostic or prognostic biomarker for lung cancer. This study aims to discover non-invasive differential markers in the serum of lung cancer patients, to determine the protein identity of the candidate biomarker(s), and to investigate any clinical implication of the biomarker(s) concerned. METHODS: Blood specimens were collected from 154 pre-operative patients with lung cancer and 35 healthy blood donors with no evidence of lung cancer. Fractionated serum samples were processed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (MS). Candidate biomarker was identified using sodium dodecyl sulphate polyacrylamide gel electrophoresis and tryptic digestion followed by tandem MS fragmentation analysis, which was subsequently validated with immunoassay. RESULTS: A differential protein with m/z 11.6 kDa was detected and identified as an isoform of human serum amyloid A (SAA). It was significantly increased by 1822% in lung cancer patients when compared with the healthy controls, which gave an area under the receiver operator characteristic curve of 0.88. In addition, the protein was also significantly elevated by 77% in lung cancer patients with survival <5 years when compared with patients with survival > or =5 years. CONCLUSION: There are several functions of the SAA protein, described in the context of inflammation, that are compatible with the mechanism of tumour invasion and metastasis. Our study not only detected increased SAA level in the serum of lung cancer patients but also identified that elevated SAA level may be a non-invasive biomarker useful for the prediction of lung cancer prognosis.

Detection of the mesenchymal-to-epithelial transition of invasive non-small cell lung cancer cells by their membrane undulation spectra.

A cancer cell changes its state from being epithelial- to mesenchymal-like in a dynamic manner during tumor progression. For example, it is well known that mesenchymal-to-epithelial transition (MET) is essential for cancer cells to regain the capability of seeding on and then invading secondary/tertiary regions. However, there is no fast yet reliable method for detecting this transition. Here, we showed that membrane undulation of invasive cancer cells could be used as a novel marker for MET detection, both in invasive model cell lines and repopulated circulating tumor cells (rCTCs) from non-small cell lung cancer (NSCLC) patients. Specifically, using atomic force microscopy (AFM), it was found that the surface oscillation spectra of different cancer cells, after undergoing MET, all exhibited two distinct peaks from 0.001 to 0.007 Hz that are absent in the spectra before MET. In addition, by adopting the long short-term memory (LSTM) based recurrent neural network learning algorithm, we showed that the positions of recorded membrane undulation peaks can be used to predict the occurrence of MET in invasive NSCLC cells with high accuracy (>90% for model cell lines and >80% for rCTCs when benchmarking against the conventional bio-marker vimentin). These findings demonstrate the potential of our approach in achieving rapid MET detection with a much reduced cell sample size as well as quantifying changes in the mesenchymal level of tumor cells.

An electro-osmotic microfluidic system to characterize cancer cell migration under confinement.

We have developed a novel electro-osmotic microfluidic system to apply precisely controlled osmolarity gradients to cancer cells in micro-channels. We observed that albeit adhesion is not required for cells to migrate in such a confined microenvironment, the migrating velocity of cells is strongly influenced by the interactions between the cells and the channel wall, with a stronger adhesion leading to diminished cell motility. Furthermore, through examining more than 20 different types of cancer cells, we found a linear positive correlation between the protein concentration of the aquaporin-4 (AQP4) and the cell migrating speed. Knockdown of AQP4 in invasive re-populated cancer stem cells reduced their migration capability down to the level that is comparable to their parental cancer cells. Interestingly, these observations can all be quantitatively explained by the osmotic engine model where the cell movement is assumed to be driven by cross-membrane ion/water transport, while adhesion acts as a frictional resistance against the cell motility. By providing versatile and controllable features in regulating and characterizing the migration capability of cells, our system may serve as a useful tool in quantifying how cell motility is influenced by different physical and biochemical factors, as well as elucidating the mechanisms behind, in the future.

A future of cancer prevention and cures: highlights of the Centennial Meeting of the American Association for Cancer Research.

The Centennial Meeting of the American Association for Cancer Research (AACR) was held from 14-18 April 2007 at the Los Angeles Convention Center. This meeting brought together a diverse group of over 18 000 researchers working in the fields of basic and applied cancer sciences, and explored how cancer research could be used most effectively to prevent and cure cancer at the earliest possible stage. The goal of the AACR Annual Meeting was to stimulate the dialog between basic and clinical researchers so that the translation of new discoveries might be speeded up for the benefit of cancer patients. Advances in the clinical application of genomics, epigenomics and proteomics to diagnose, monitor and prognosticate cancer development led to a dramatic increase in the number of presentations with a translational focus at this year's meeting. Several remarkable areas were particularly highlighted in this report, including The Cancer Genome Atlas, cancer stem cells, microRNA and siRNA, targeted therapy and individualized treatment. This article tries to bring attention to some hot topics in the program that are both new and noteworthy. For those who did not attend the meeting, this report may serve as a highlight of this important international cancer research meeting.

Cadherin- and Rigidity-Dependent Growth of Lung Cancer Cells in a Partially Confined Microenvironment.

During tumor development, cancer cells constantly confront different types of extracellular barriers. However, fundamental questions like whether tumor cells will continue to grow against confinement or away from it and what key factors govern this process remain poorly understood. To address these issues, here we examined the growth dynamics of human lung epithelial carcinoma A549 cells partially confined in micrometer-sized cylindrical pores with precisely controlled wall stiffness. It was found that, after reaching confluency, the cell monolayer enclosed by a compliant wall was able to keep growing and pushing the boundary, eventually leading to a markedly enlarged pore. In contrast, a much reduced in-plane growth and elevated strain level among cells were observed when the confining wall becomes stiff. Furthermore, under such circumstance, cells switched their growth from within the monolayer to along the out-of-plane direction, resulting in cell stacking. We showed that these observations can be well explained by a simple model taking into account the deformability of the wall and the threshold stress for inhibiting cell growth. Interestingly, cadherins were found to play an important role in the proliferation and stress buildup within the cell monolayer by aggregating at cell-cell junctions. The stiff confinement led to an elevated expression level of cadherins. Furthermore, inhibition of N-cadherin resulted in a significantly suppressed cell growth under the same confining conditions.

Quantitative profile of estrogen receptor variants/isoforms in Taiwanese women with breast cancer.

AIM: To quantify ER variants/isoforms for women with breast cancer in Taiwan, an area with remarkably low breast cancer rates, but with an early onset and poor prognosis. METHODS: Real-time PCR technology was exploited to quantify mRNAs of ERalpha, ERalphaE3Delta, ERalphaE5Delta, ERalphaE7Delta and ERbeta, in 49 breast cancer and paired adjacent normal tissues. Clinical parameters were assembled and tested for assocation with the ER expression. RESULTS: Comparison of cancer and matched normal samples showed significantly decreased ERbeta (p < 0.001) in cancer tissues, and constant amounts of ERalpha and their variants. The results revealed significantly lower ERalphaE7Delta/ERalpha (p = 0.030) and ERbeta/ERalpha (p = 0.035) ratios in patients with lymph node (LN) metastasis than in those without LN metastasis. CONCLUSION: Our data suggests that ERalphaE7Delta and ERbeta may regulate ERalpha in normal human breast tissue.

DDX3 modulates cell adhesion and motility and cancer cell metastasis via Rac1-mediated signaling pathway.

The DEAD-box RNA helicase DDX3 is a versatile protein involved in multiple steps of gene expression and various cellular signaling pathways. DDX3 mutations have been implicated in the wingless (Wnt) type of medulloblastoma. We show here that small interfering RNA-mediated DDX3 knockdown in various cell lines increased cell-cell adhesion but decreased cell-extracellular matrix adhesion. Moreover, DDX3 depletion suppressed cell motility and impaired directional migration in the wound-healing assay. Accordingly, DDX3-depleted cells exhibited reduced invasive capacities in vitro as well as reduced metastatic potential in mice. We also examined the mechanism underlying DDX3-regulated cell migration. DDX3 knockdown reduced the levels of both Rac1 and ß-catenin proteins, and consequentially downregulated the expression of several ß-catenin target genes. Moreover, we demonstrated that DDX3-regulated Rac1 mRNA translation, possibly through an interaction with its 5'-untranslated region, and affected ß-catenin protein stability in an Rac1-dependent manner. Taken together, our results indicate the DDX3-Rac1-ß-catenin regulatory axis in modulating the expression of Wnt/ß-catenin target genes. Therefore, this report provides a mechanistic context for the role of DDX3 in Wnt-type tumors.

Remarkable application of serum EBV EBER-1 in monitoring response of nasopharyngeal cancer patients to salvage chemotherapy.

Nineteen consecutive patients with metastatic or recurrent nasopharyngeal cancer (NPC) receiving combination chemotherapy were monitored for EBV DNA in their serum. EBV DNA (EBER-1) concentration in serum was measured before, during, and after chemotherapy. Thirteen patients had additional multiple prechemotherapy readings. There was a significant lead time from first detection of serum EBER-1 to clinical recurrence in 62% of patients by a mean of 17.4 weeks (range: 8-74.5 weeks; mean = 28.2 weeks if confined to the 8 patients with significant lead time). The median EBER-1 concentration was significantly higher in those with distant metastasis as compared to those with loco-regional recurrence only (17,468 vs. 684 pg/mL serum; p = 0.046, Mann-Whitney U test). Among the 13 patients who responded to chemotherapy, 4 exhibited clinical complete remission (CR) who were only found in the group with EBER-1 DNA drop to background level, while the magnitude of EBER-1 drop did not discriminate partial remission (PR) and stable disease (SD) patients clearly. Subsequent profile of EBER-1 DNA showed concordance with clinical course of either continuous remission or later progression. EBER-1 DNA in serum can become a useful adjunctive surrogate marker to monitor chemotherapeutic response in NPC patients with distant metastasis or advanced local recurrence.

Pilot fragile X screening in normal population of Taiwan.

Fragile X syndrome (FXS) is the most common hereditary form of mental retardation. Molecular analysis of the FMR1 gene has now been applied to diagnosis and carrier detection. Because treatment is not feasible, prevention of FXS by prenatal diagnosis of carrier women early during pregnancy is important. The aim of this pilot study was to ascertain the prevalence of mutant FMR1 gene in normal population of Taiwan and to evaluate the efficacy of a betaine-based polymerase chain reaction (PCR) and nonradioactive Southern blot assays. The DNA was randomly and anonymously collected from 100 women and 100 men. The results showed 62% of the women were heterozygous for the CGG-repeat size in FMR1 gene. One of 300 X chromosomes in this study showed premutation, with 95 CGG repeats. All other chromosomes have CGG repeats ranging from 19 to 52, with eight chromosomes (3%) having more than 40 CGG repeats. The most prevalent allele has 29 repeats (48.1%), followed by 30 (24.0%) and 36 (9.5%), respectively. The results of this study reconfirmed previous reports that the prevalent FMR1 CGG repeat alleles in Chinese population are different from that of other populations. However, the prevalence of premutation gene seems to be comparable among them. The betaine-based PCR could minimize the intrinsic problem of preferential amplification and may reliably determine the different allele repeats in heterozygous females. This nonradioactive Southern blot protocol is safe, efficient, and inexpensive. However, further technical improvement may be needed to be more cost-effective for a wide screening of all pregnant women.

Evidence of paralytic shellfish poisoning toxin in milkfish in South Taiwan.

Natural phytoplankton blooms of the dinoflagellate Alexandrium minutum, milkfish (Chanos chanos) exposed to natural blooms, sediment and mangrove crab (Scylla serrata) were analysed for paralytic shellfish poisoning toxins by high-performance liquid chromatography. The toxin profiles of milkfish and mangrove crab were similar to that of A. minutum collected from blooming fishponds. In a laboratory A. minutum-blooming environment, the stomach and intestine of milkfish accumulated paralytic shellfish poisoning toxins during the exposure period. The non-visceral tissues were non-toxic. However, milkfish lost their entire body burden of toxin on the first day of transferring to a toxic algae-free environment. The result shows that milkfish concentrate paralytic shellfish poisoning toxins in digestive organs and did not retain toxins.