A Novel Biomarker Driving Poor-Prognosis Liver Cancer: Overexpression of the Mitochondrial Calcium Gatekeepers.

Several studies have indicated the biological role of mitochondrial Ca2+ uptake in cancer pathophysiology; however, its implications in predicting the prognosis of hepatocellular carcinoma (HCC) are not yet fully understood. Here, we collected tumor specimens and adjacent normal liver tissues from 354 confirmed HCC patients and analyzed the levels of cyclic adenosine monophosphate (cAMP) responsive element binding protein 1 (CREB), mitochondrial calcium uniporter (MCU), mitochondrial calcium uptake 1 and 2 (MICU1, MICU2) using bioinformatics, qRT-PCR, and immunohistochemistry (IHC), and their relationship with clinicopathological characteristics and prognosis. HCC patients with low CREB/MICU1 and high MCU/MICU2 expression exhibited poor survival rate and prognosis in overall survival (OS) and disease-free survival (DFS) analyses. Low CREB/MICU1 and low MICU1 alone indicated poor prognosis in stage I/II and III/IV patients, respectively. In the poor differentiation/undifferentiation group, low expression of MICU1 indicated poor clinical outcomes. Low CREB/MICU1 expression suggested poor outcomes in patients with or without hepatitis B virus (HBV) infection and poor prognosis in the HCV infection group. In the non- hepatitis C virus (HCV) infection group, low MCU1 indicated a poor prognosis. Multivariate analysis demonstrated that CREB and MICU1 expression showed prognostic significance. This study demonstrates the prognostic significance of CREB, MCU, MICU1, and MICU2, in predicting HCC outcomes. Low CREB/MICU1 and high MCU/MICU2 in HCC tissues are associated with poor prognosis, thus offering a novel perspective in the clinical management for HCC patients.

Glycolic acid attenuates UVB-induced aquaporin-3, matrix metalloproteinase-9 expression, and collagen degradation in keratinocytes and mouse skin.

Ultraviolet-B exposure causes an inflammatory response, photoaged skin, and degradation of extracellular matrix proteins including collagen and elastin. The regulation of these genes was suggested as an important mechanism to attenuate skin aging. Glycolic acid (GA) is commonly present in fruits and recently used to treat dermatological diseases. We reported that GA slows down cell inflammation and aging caused by UVB. Little is known about GA retarding the skin premature senescence or how to impede these events. To investigate the potential of GA to regulate the expression of MMPs and collagen, GA was topically applied onto human keratinocytes and the C57BL/6J mice dorsal skin. In the present study, we demonstrated that GA reduced UVB-induced type-I procollagen expression and secretory collagen levels. GA reverted and dose-dependently increased the level of aquaporin-3 (AQP3), the expression of which was down-regulated by UVB. The UV-induced MMP-9 level and activity were reduced by GA pre-treatment. Concomitantly, GA reverted mitogen-activated protein kinase (MMP-9) activation and inhibited the extracellular signal-regulated kinase activation (p38, pERK) triggered by UVB. The animal model also presented that GA attenuated the wrinkles caused by UVB on the mouse dorsal skin. Finally, GA triggers the transient receptor potential vanilloid-1 (TRPV-1) channel to initiate the anti-photoaging mechanism in keratinocytes. These findings clearly indicated that the mechanisms of GA promote skin protection against UVB-induced photoaging and wrinkle formation. GA might be an important reagent and more widely used to prevent UVB-induced skin aging.

Cell Reprogramming in Tumorigenesis and Its Therapeutic Implications for Breast Cancer.

Breast cancer is the most common malignancy in women worldwide and can be categorized into several subtypes according to histopathological parameters or genomic signatures. Such heterogeneity of breast cancer can arise from the reactivation of mammary stem cells in situ during tumorigenesis. Moreover, different breast cancer subtypes exhibit varieties of cancer incidence, therapeutic response, and patient prognosis, suggesting that a specific therapeutic protocol is required for each breast cancer subtype. Recent studies using molecular and cellular assays identified a link between specific genetic/epigenetic alterations and distinct cells of origin of breast cancer subtypes. These alterations include oncogenes, tumor suppressor genes, and cell-lineage determinants, which can induce cell reprogramming (dedifferentiation and transdifferentiation) among two lineage-committed mammary epithelial cells, namely basal and luminal cells. The interconversion of cell states through cell reprogramming into the intermediates of mammary stem cells can give rise to heterogeneous breast cancers that complicate effective therapies of breast cancer. A better understanding of mechanisms underlying cell reprogramming in breast cancer can help in not only elucidating tumorigenesis but also developing therapeutics for breast cancer. This review introduces recent findings on cancer gene-mediated cell reprogramming in breast cancer and discusses the therapeutic potential of targeting cell reprogramming.

Synergistic effect of platelet-rich plasma injections and scalp lifting in androgenetic alopecia.

Androgenetic alopecia (AGA) is a common hair loss disorder, especially in men and the elderly. In this study, we analyzed the therapeutic effects of platelet-rich plasma (PRP) injections and embedded sutures in patients with AGA. In each participant, we administered different treatments in one area of hair loss that was divided into four sections. Each section received one of the following treatments: No treatment, PRP injection, suture embedding, and combined PRP injection/suture-embedded areas. The thickness of the scalp, and scalp perfusion were measured using an ultrasound imaging system and Moor FLPI full-field laser perfusion imaging system, respectively. The diameters of the hair were measured using optical microscopy. Our results show that PRP injection treatments increased the diameter of the hair (P = 0.034), and the combined PRP injection/suture-embedded treatments had a significant effect on the thickness of the scalp (P = 0.002), the blood flow (P = 0.014) through the scalp, and the diameter of the hair (P= 0.013). This study has demonstrated that there is a synergistic effect between PRP injections and suture embedding for increasing the thickness and blood flow of the scalp, and diameter of the hair. Combined PRP injection/suture-embedded PRP injections might have therapeutic benefit for patients with AGA.

Evaluating the Prognostic Value of ERCC1 and Thymidylate Synthase Expression and the Epidermal Growth Factor Receptor Mutation Status in Adenocarcinoma Non-Small-Cell Lung Cancer.

The present study evaluated the prognostic value of the epidermal growth factor receptor (EGFR) mutation status, and excision repair cross-complementation group 1 (ERCC1) and thymidylate synthase (TS) expression following intercalated tyrosine kinase inhibitor (TKI) therapy and platinum- and pemetrexed-based chemotherapies (subsequent second-line treatment) for patients with adenocarcinoma non-small-cell lung cancer (AC-NSCLC). In total, 131 patients with AC-NSCLC were enrolled. The EGFR mutation status and ERCC1 and TS expression were evaluated through direct DNA sequencing and immunohistochemical analyses, respectively. The EGFR mutation status and ERCC1 and TS expression were the significant predictors of clinical outcomes. The EGFR mutation status was the main outcome predictor for overall survival (OS) benefits in the overall population. Further exploratory ERCC1 and TS expression analyses were conducted to provide additional insights. Low TS expression was predictive of improved OS of patients with negative EGFR-mutated advanced AC-NSCLC, whereas high ERCC1 expression resulted in poor OS in patients with positive EGFR-mutated advanced AC-NSCLC. TS and ERCC1 expression levels were effective prognostic factors for negative and positive EGFR-mutated AC-NSCLC, respectively. In conclusion, the present results indicate that the EGFR mutation status and TS and ERCC1 expression can be used as the predictors of OS after subsequent second-line treatments for AC-NSCLC.

Tricetin suppresses human oral cancer cell migration by reducing matrix metalloproteinase-9 expression through the mitogen-activated protein kinase signaling pathway.

Tricetin is a flavonoid derivative and a potent anti-inflammatory and anticancer agent. However, the molecular mechanism underlying the effects of tricetin on human oral cancer cell migration remains unclear. The cell migration and invasion abilities of three oral cancer cell lines (SCC-9, HSC-3, and OECM-1) were analyzed using Boyden chamber migration assays. Our results demonstrated that tricetin attenuates 12-O-tetradecanoylphorbol-13-acetate-induced SCC-9, HSC-3, and OECM-1 cell invasiveness and migration by reducing matrix metalloproteinase (MMP)-9 enzyme activity. The reverse transcription polymerase chain reaction and luciferase reporter assay revealed that tricetin downregulates the mRNA expression and promoter activity of MMP-9. In addition, Western blot analysis revealed that tricetin significantly reduced the levels of phosphorylated c-Jun N-terminal kinase (JNK) 1/2 and p38 levels but not those of extracellular signal-regulated kinase 1/2. In conclusion, this study demonstrated that tricetin suppresses MMP-9 enzymatic activity by downregulating the p38/JNK1/2 pathway and might be a beneficial chemopreventive agent.

Multiplex Immunoassays Utilizing Differential Affinity Using Aptamers Generated by MARAS.

Disease diagnosis typically requires to determine concentration of multiple biomarkers in patient serums. Here, a novel method for multiplex immunoassays is proposed and the feasibility is demonstrated. The method utilizes the differential affinity between aptamers and multiple analytes for multiplex immunoassays. During the selection, aptamers capable of binding to multiple analytes with different affinities are screened from a random oligonucleotide library using the MARAS procedure with different magnetic field conditions for different target analytes. During the detection, the same magnetic field conditions are applied to differentiate different target analytes in blind serums. The results show that the recovery rates of the spiked targets in BD buffer and blind serums are similar. Moreover, there is a minimal interference resulting from non-specific binding of molecules in serums other than the target molecules. Therefore, the use of differential affinities between aptamers and different analytes for multiplex immunoassays is proved to be feasible.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection.

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Rapidly Developed Multiple Face and Neck Skin Cancers in a Patient with Sjögren's Syndrome: A Case Report.

BACKGROUND Sjögren's syndrome is a chronic, systemic disorder of an autoimmune nature, and its primary etiopathogenetic events are not known. Previous studies have found elevated incidence of malignancies in patients with primary Sjögren's syndrome. However, there are few reports regarding the association of Sjögren's syndrome with skin cancers, especially with multiple skin cancers developed within a short time. CASE REPORT We reported an unusual case of a patient with primary Sjögren's syndrome who suffered from rapidly developed facial and neck skin cancers within two years. CONCLUSIONS Sjögren's syndrome associated with skin cancer is rare. Our case report suggests that Sjögren's syndrome patients require continuous follow-up with conventional cancer examination, including skin biopsy for suspected skin lesions.

Topical application of glycolic acid suppresses the UVB induced IL-6, IL-8, MCP-1 and COX-2 inflammation by modulating NF-κB signaling pathway in keratinocytes and mice skin.

BACKGROUND: Glycolic acid (GA), commonly present in fruits, has been used to treat dermatological diseases. Extensive exposure to solar ultraviolet B (UVB) irradiation plays a crucial role in the induction of skin inflammation. The development of photo prevention from natural materials represents an effective strategy for skin keratinocytes. OBJECTIVE: The aim of this study was to investigate the molecular mechanisms underlying the glycolic acid (GA)-induced reduction of UVB-mediated inflammatory responses. METHODS: We determined the effects of different concentrations of GA on the inflammatory response of human keratinocytes HaCaT cells and C57BL/6J mice dorsal skin. After GA was topically applied, HaCaT and mice skin were exposed to UVB irradiation. RESULTS: GA reduced the production of UVB-induced nuclear factor kappa B (NF-κB)-dependent inflammatory mediators [interleukin (IL)-1ß, IL-6, IL-8, cyclooxygenase (COX)-2, tumor necrosis factor-α, and monocyte chemoattractant protein (MCP-1)] at both mRNA and protein levels. GA inhibited the UVB-induced promoter activity of NF-κB in HaCaT cells. GA attenuated the elevation of senescence associated with ß-galactosidase activity but did not affect the wound migration ability. The topical application of GA inhibited the genes expression of IL-1ß, IL-6, IL-8, COX-2, and MCP-1 in UVB-exposed mouse skin. The mice to UVB irradiation after GA was topically applied for 9 consecutive days and reported that 1-1.5% of GA exerted anti-inflammatory effects on mouse skin. CONCLUSION: We clarified the molecular mechanism of GA protection against UVB-induced inflammation by modulating NF-κB signaling pathways and determined the optimal concentration of GA in mice skin exposed to UVB irradiation.

Elevated transglutaminase-2 expression in the epidermis of psoriatic skin and its role in the skin lesion development.

Psoriasis, an inflammatory skin disease triggered by the immune system, presents keratinocyte hyperproliferation, differentiation and angiogenesis. The role of transglutaminase-2 (TG2) in psoriasis has not been fully established yet. In this retrospective, non-randomized and non-blinded study, skin biopsies were collected from 37 psoriatic patients and immunohistochemical staining was performed. TG2 staining was positive in all 37 samples, among which 32 were strong and five weak. The localization of TG2 staining was present in the epidermis and spreading from basal layer to stratum granulosum in decreasing staining intensity. However, TG2 was also expressed in the basal layer in the non-lesional site of psoriasis and the skin of healthy people. The presence of TG2 was not associated with disease duration, stage of disease and subtype of psoriasis. Overexpression of TG2 seems to be an important role in psoriatic development.

Case Report of False-Negative Diffusion-Weighted Image of Brain Maggnetic Resonance Imaging (MRI) in Acute Ischemic Stroke.

BACKGROUND Acute ischemic stroke is a major cause of mortality and morbidity in Taiwan. Diffusion-weighted image (DWI) is a sensitive and common strategy used for imaging acute ischemic stroke. CASE REPORT We present a case of a negative DWI MRI for detecting acute ischemic stroke in a clinical setting. A 75-year-old male had a DWI performed after onset of symptoms suggesting acute ischemic stroke. The initial DWI result was negative at 72 hours of presentation. The neurological symptoms of the patient persisted and DWI was repeated. After 14 days, the DWI data confirmed and demonstrated an acute ischemic stroke. The delay in DWI confirmation, from symptom onset until DWI diagnosis, was 336 hours. CONCLUSIONS DWI may not have 100% sensitivity and accuracy in early stages of acute ischemic stroke. The time course to the development of abnormalities detected by DWI may be longer than anticipated.

Magnetic-assisted rapid aptamer selection (MARAS) for generating high-affinity DNA aptamer using rotating magnetic fields.

A new SELEX protocol for the development of DNA aptamers has been demonstrated, referred to as magnetic-assisted rapid aptamer selection (MARAS). This method uses magnetic beads and an externally applied rotating magnetic field to provide the competitive mechanism for the selection aptamers with different affinities to the molecular target. The MARAS protocol efficiently generated aptamers with high affinity and specificity for C-reactive protein, a common cardiovascular disease indicator. The binding affinities of the selected aptamers could be varied by changing the frequency of the externally applied rotating magnetic field and optimal cases bound with low-nanomolar dissociation constants.

A novel protocol for generating high-affinity ssDNA aptamers by using alternating magnetic fields.

BACKGROUND: Aptamers that have been generated using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) process have been found to have high specificity and affinity with molecular targets, and, as a result, have found use in diagnostic and therapeutic applications. The SELEX protocol generally requires 5 to 15 rounds of selection to generate high-affinity aptamers; however, the standard protocol is labor-intensive and time-consuming. Here, we propose a method for DNA aptamer screening, in which the evolutionary process is completely abandoned. METHODS: The working principle of this new protocol, Magnetic-Assisted Rapid Aptamer Selection (MARAS), is discussed. We used C-reactive protein, a common cardiovascular disease (CVD) indicator, as a molecular target. An externally applied alternating magnetic field, aided by target-bound magnetic nanoparticles, was used to provide the competitive mechanism for the selection of DNA aptamers with different affinities to the target protein. RESULTS: The range of the dissociation constants of the screened aptamers was approximately several nMs, depending on the frequency and the field strength of the externally applied alternating magnetic field. We also compared the diagnostic applicability of the aptamers generated by the proposed MARAS protocol with an enzyme-linked immunosorbent assay (ELISA), using antibodies. CONCLUSIONS: The proposed MARAS protocol efficiently generates aptamers that have high affinity and specificity with molecular targets. In addition, the proposed protocol represents significant time savings, as it can be completed in less than an hour. Furthermore, due to the simplicity of the MARAS protocol, we suggest that the proposed process could be easily automated.

O6-Methylguanine-DNA methyltransferase hypermethylation modulated by 17beta-estradiol in lung cancer cells.

BACKGROUND: Our recent report indicated that MGMT hypermethylation is more common in squamous cell carcinomas (SCC) in males, and smokers than in adenocarcinomas (ADC) in females, and nonsmokers. More interestingly, MGMT hypermethylation in SCC and ADC was pronouncedly influenced by gender factor, not by smoking status. We questioned whether 17beta-estradiol could modulate the machinery of promoter methylation to cause the gender difference of MGMT hypermethylation in lung cancer. MATERIALS AND METHODS: Two MGMT hypermethylated Ch27 and H1355 lung cancer cell lines were treated with or without 17beta-estradiol and the status of hypermethylation was examined by methylated specific methylation (MSP) as compared with both cells treated with demethylating agents, 5-AZA-dC (AZA) or TSA. RESULTS: Our data showed that 17beta-estradiol, similar to AZA, diminished the MGMT hypermethylation and restored MGMT mRNA expression, which was not observed in the case of TSA. Western blotting showed that 17beta-estradiol markedly reduced DNMT1 expression in Ch27 and H1355 cells, but slightly reduced HDAC1 expression. Consequently, acetylated H3 and H4 histone levels were slightly increased by 17beta-estradiol in both cell types. In addition, ChIP analysis revealed that 17beta-estradiol simultaneously diminished the binding activity of both proteins on the MGMT promoter of both cell lines. CONCLUSION: 17beta-Estradiol decreased DNMT1 and HDAC1 protein expressions and their binding activity on MGMT promoter, and this may partially contribute to the gender difference of MGMT hypermethylation in lung cancer.

Association of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation with p53 mutation occurrence in non-small cell lung cancer with different histology, gender, and smoking status.

BACKGROUND: O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been demonstrated to associate with the G:C-->A:T transition mutation in the p53 gene of lung tumors. The purpose of this study is to clarify whether MGMT promoter methylation is not only associated with the shift from the G:C-->A:T mutation in the p53 gene but also whether MGMT increases other mutation patterns in lung tumors. MATERIALS AND METHODS: To further verify whether a different prevalence of MGMT promoter methylation is observed in lung tumors with a different tumor histology, gender, and smoking status, 220 lung tumors were collected to evaluate the status of MGMT promoter methylation and p53 mutation using methylation-specific PCR (MSP) and direct sequencing, respectively. RESULTS: The data shows that a higher prevalence of MGMT promoter methylation was observed in tumors with the G:C-->A:T transition or other p53 mutation patterns compared with those with p53 wild-type (P < 0.001 for G:C-->A:T; P = 0.015 for other mutation patterns), and this prevalence was more pronounced in tumors from male than from female patients. MGMT promoter methylation in p53 mutation patterns had a different effect on squamous cell carcinomas (SCC) and adenocarcinomas (ADC). Interestingly, the highest prevalence of MGMT promoter methylation was found in male nonsmokers followed by male smokers and female nonsmokers. This may be a partial explanation for the reason why male nonsmokers had a higher p53 mutation occurrence than female nonsmokers. CONCLUSIONS: MGMT promoter methylation may associate with increased occurrence of p53 mutation including the G:C-->A:T transition and other p53 mutation patterns in lung cancer, especially among male nonsmokers.

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53.

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.

Accumulation of chromium and nickel metals in lung tumors from lung cancer patients in Taiwan.

Metallic carcinogenicity is generally thought to generate of free radicals, and thus some metals were reported to play a role in lung tumorigenesis. In order to verify the role of heavy metals in the development of Taiwanese lung cancer, a case-control study was conducted to compare heavy metal contents between 60 tumor and 42 normal lung tissues surgically resected from lung cancer and noncancer patients. The tissue concentration of heavy metals, including cadmium (Cd), chromium (Cr), cobalt (Co), lead (Pb), and nickel (Ni), was measured using by atomic absorption spectrometry (AAS). Our results indicated that Cr and Ni contents in lung tumors of lung cancer patients were significantly higher than those in normal lung tissue of noncancer controls, but Co content was markedly lower in lung tumors. Additionally, Pb content in lung tumors was associated with nodal involvement, and Co amounts in squamous-cell carcinomas were relatively higher than those in adenocarcinomas. Data suggest that accumulation of Cr and Ni in lung tumors may play a role, at least in part, in the development of lung cancer in Taiwan.