Evaluation of Circulating MicroRNAs and Adipokines in Breast Cancer Survivors with Arm Lymphedema.

Breast cancer-related lymphedema (BCRL) is a form of secondary lymphedema that is characterized by abnormal swelling of one or both arms due to the accumulation of lymph fluid in the interstitial tissue spaces, resulting from obstruction of the lymphatic vessels due to surgery insults, radiotherapy, or chemotherapy. Due to the multifactorial nature of this condition, the pathogenesis of secondary lymphedema remains unclear and the search for molecular factors associated with the condition is ongoing. This study aimed to identify serum microRNAs and adipokines associated with BCRL. Blood was collected from 113 breast cancer survivors and processed to obtain serum for small RNA-sequencing (BCRL vs. non-BCRL, n = 7 per group). MicroRNAs that were differentially expressed (fold change >1.5, p < 0.05) between lymphedema cases and those without lymphedema were further quantified in a validation cohort through quantitative reverse transcription PCR (BCRL n = 16, non-BCRL, n = 83). Leptin and adiponectin levels were measured in a combined cohort (BCRL n = 23, non-BCRL n = 90) using enzyme-linked immunosorbent assays. Two of the most significantly upregulated microRNAs, miR-199a-3p and miR-151a-3p, were strongly correlated with the onset of lymphedema and diabetes mellitus in the BCRL group. Leptin levels were higher in the BCRL cohort compared to the non-BCRL cohort (p < 0.05). A metabolic syndrome biomarker, the adiponectin/leptin ratio, was found to be lower in the BCRL group than in the non-BCRL group (median: 0.28 vs. 0.41, p < 0.05). Extensive studies on the mechanisms of the identified microRNAs and association of leptin with arm lymphedema may provide new insights on the potential biomarkers for lymphedema that should be followed up in a prospective cohort study.

In Vivo Anti-Tumor Effects of Citral on 4T1 Breast Cancer Cells via Induction of Apoptosis and Downregulation of Aldehyde Dehydrogenase Activity.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females globally. The tumorigenic activities of cancer cells such as aldehyde dehydrogenase (ALDH) activity and differentiation have contributed to relapse and eventual mortality in breast cancer. Thus, current drug discovery research is focused on targeting breast cancer cells with ALDH activity and their capacity to form secondary tumors. Citral (3,7-dimethyl-2,6-octadienal), from lemon grass (Cymbopogon citrates), has been previously reported to have a cytotoxic effect on breast cancer cells. Hence, this study was conducted to evaluate the in vivo effect of citral in targeting ALDH activity of breast cancer cells. BALB/c mice were challenged with 4T1 breast cancer cells followed by daily oral feeding of 50 mg/kg citral or distilled water for two weeks. The population of ALDH+ tumor cells and their capacity to form secondary tumors in both untreated and citral treated 4T1 challenged mice were assessed by Aldefluor assay and tumor growth upon cell reimplantation in normal mice, respectively. Citral treatment reduced the size and number of cells with ALDH+ activity of the tumors in 4T1-challenged BALB/c mice. Moreover, citral-treated mice were also observed with smaller tumor size and delayed tumorigenicity after reimplantation of the primary tumor cells into normal mice. These findings support the antitumor effect of citral in targeting ALDH+ cells and tumor recurrence in breast cancer cells.

Antioxidant Potential and Angiotensin-Converting Enzyme (ACE) Inhibitory Activity of Orotic Acid-Loaded Gum Arabic Nanoparticles.

Orotic acid (OA) nanoparticles were prepared using the freeze-drying method. The antihypertensive activity and antioxidant capacity of OA and orotic acid-loaded gum arabic nanoparticles (OAGANPs) were examined using the angiotensin-converting enzyme (ACE), 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and ß-carotene assays, as well as the quantification of total phenolic content (TPC). The DPPH and NO scavenging activities of OAGANPs were significantly higher than those of the OA solution. The ß-carotene bleaching assay of OAGANPs showed a dose-dependent trend, while 500 µg/ml was significantly more effective than the other concentrations, which exerted 63.4% of the antioxidant activity. The in vitro antihypertensive assay revealed that the OAGANPs exhibited the most potent ACE inhibition activity, when compared to the OA solution. Hence, results revealed the potential of preparing the OA as a nanoparticle formulation in enhancing the antioxidant and antihypertensive properties compared to the OA solution.

Citral induced apoptosis in MDA-MB-231 spheroid cells.

BACKGROUND: Breast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study. METHODS: The cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis. RESULTS: Citral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/ß-catenin pathway. CONCLUSION: The results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis.

In utero exposure to germinated brown rice and its oryzanol-rich extract attenuated high fat diet-induced insulin resistance in F1 generation of rats.

BACKGROUND: The development of insulin resistance is multifactorial, with maternal pre- and postnatal nutrition having significant influences. In this regard, high fat diet (HFD) feeding in pregnancy has been shown to increase risks of metabolic diseases. Thus, we investigated the effects of supplementation of HFD with germinated brown rice (GBR) and GBR-derived gamma oryzanol-rich extract (OE) on insulin resistance and its epigenetic implications in pregnant rats and their offsprings. METHODS: Pregnant female Sprague dawley rats were fed with HFD alone, HFD + GBR or HFD + OE (100 or 200 mg/kg/day) throughout pregnancy and lactation. Their offsprings were weaned at 4 weeks post-delivery and were followed up until 8 weeks. Serum levels of adipokines were measured in dams and their offsprings, and global DNA methylation and histone acetylation patterns were estimated from the liver. RESULTS: The dams and offsprings of the GBR and OE groups had lower weight gain, glycemic response, 8-Iso prostaglandin, retinol binding protein 4 and fasting insulin, and elevated adiponectin levels compared with the HFD group. Fasting leptin levels were lower only in the GBR groups. Hepatic global DNA methylation was lower in the GBR groups while hepatic H4 acetylation was lower in both GBR and OE dams. In the offsprings, DNA methylation and H4 acetylation were only lower in the OE group. However, dams and offsprings of the GBR and OE groups had higher hepatic H3 acetylation. CONCLUSIONS: GBR and OE can be used as functional ingredients for the amelioration of HFD-induced epigeneticallymediated insulin resistance.

Acute toxicity profiling of the ethyl acetate fraction of Swietenia macrophylla seeds and in-vitro neuroprotection studies.

Swietenia macrophylla (SM) is a medicinally important plant found in tropical and subtropical regions of the world. The ethyl acetate fraction of the seeds of S. macrophylla (SMEAF) is reported to exhibit potent anticancer, antitumor, anti-inflammatory and antifeedant activities. Till date, there have been no studies reported on the acute oral toxicity profile of the ethyl acetate fraction of the seeds of SM. The objective of the present study was to determine the acute toxicity of SMEAF and evaluate the in-vitro neuroprotective activity of SMEAF using primary neuronal cell cultures. In acute oral toxicity study, the SMEAF did not produce any lethal signs of morbidity and mortality. Histo-pathological findings, support the safety of SMEAF, as there were no significant changes observed in any of the parameters studied. Based on the results obtained in MTT assay, we infer that SMEAF has a significant neuroprotective effect, as it increased the cell viability and exhibited protection to the neuronal cells against TBHP induced oxidative stress. Thus, SMEAF can be suggested for use in the development of herbal drug formulations with neuroprotective potential.

Calreticulin mediates an invasive breast cancer phenotype through the transcriptional dysregulation of p53 and MAPK pathways.

BACKGROUND: The introduction of effective novel biomarkers of invasion and metastasis is integral for the advancement of breast cancer management. The present study focused on the identification and evaluation of calreticulin (CRT) as a potential biomarker for breast cancer invasion. METHODS: Two-dimensional gel protein electrophoresis and MALDI-TOF were utilized in the analysis of fresh-frozen invasive intra-ductal carcinoma specimens. Calreticulin-associated expression was analyzed using immunohistochemistry of FFPE non-malignant/malignant breast specimens. A CRT-knockdown model of MCF7 cell line was developed using siRNA and the CRT genotype/phenotype correlations based on migration and trans-well invasion assays were determined. Finally, microarray-based global gene expression profiling was conducted to elucidate the possible calreticulin pro-invasive regulatory pathways. RESULTS: Two-dimensional gel protein electrophoresis and MALDI-TOF analysis showed upregulation of calreticulin expression in tumor tissues as compared to the normal adjacent tissues. Meta-analysis of the immunohistochemical results confirmed significantly higher expression of calreticulin (p < 0.05) in the stromal compartments of malignant tissues as compared to non-malignant tissues. Migration and transwell invasion assays showed significant loss in the migratory and invasive potential of CRT-knockdown cells (p < 0.05). Global gene expression profiling successfully identified various putative gene networks such as p53 and MAPK pathways that are involved in calreticulin breast cancer signaling. CONCLUSION: Besides confirming calreticulin overexpression in invasive breast cancer tissues, this study reveals a calreticulin-dependent pro-invasive potential and suggests possible contributing pathways. Defining the mechanistic role of invasion and characterizing the possible calreticulin-dependent molecular targets will be the focus of future work.

CYP2D6 Genetic Polymorphisms and Phenotypes in Different Ethnicities of Malaysian Breast Cancer Patients.

The cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) is an enzyme that is predominantly involved in the metabolism of tamoxifen. Genetic polymorphisms of the CYP2D6 gene may contribute to inter-individual variability in tamoxifen metabolism, which leads to the differences in clinical response to tamoxifen among breast cancer patients. In Malaysia, the knowledge on CYP2D6 genetic polymorphisms as well as metabolizer status in Malaysian breast cancer patients remains unknown. Hence, this study aimed to comprehensively identify CYP2D6 genetic polymorphisms among 80 Malaysian breast cancer patients. The genetic polymorphisms of all the 9 exons of CYP2D6 gene were identified using high-resolution melting analysis and confirmed by DNA sequencing. Seven CYP2D6 alleles consisting of CYP2D6*1, CYP2D6*2, CYP2D6*4, CYP2D6*10, CYP2D6*39, CYP2D6*49, and CYP2D6*75 were identified in this study. Among these alleles, CYP2D6*10 is the most common allele in both Malaysian Malay (54.8%) and Chinese (71.4%) breast cancer patients, whereas CYP2D6*4 in Malaysian Indian (28.6%) breast cancer patients. In relation to CYP2D6 genotype, CYP2D6*10/*10 is more frequently observed in both Malaysian Malay (28.9%) and Chinese (57.1%) breast cancer patients, whereas CYP2D6*4/*10 is more frequently observed in Malaysian Indian (42.8%) breast cancer patients. In terms of CYP2D6 phenotype, 61.5% of Malaysian Malay breast cancer patients are predicted as extensive metabolizers in which they are most likely to respond well to tamoxifen therapy. However, 57.1% of Chinese as well as Indian breast cancer patients are predicted as intermediate metabolizers and they are less likely to gain optimal benefit from the tamoxifen therapy. This is the first report of CYP2D6 genetic polymorphisms and phenotypes in Malaysian breast cancer patients for different ethnicities. These data may aid clinicians in selecting an optimal drug therapy for Malaysian breast cancer patients, hence improve the clinical outcome of the patients.

Synthesis, characterization and biological activity of Cu(II), Zn(II) and Re(I) complexes derived from S-benzyldithiocarbazate and 3-acetylcoumarin.

Cu(II), Zn(II) and Re(I) complexes have been synthesized with the Schiff base, N'-[1-(2-oxo-2H-chromen-3-yl)-ethylidene]-hydrazinecarbodithioic acid benzyl ester (SBCM-H) which was prepared by condensation of S-benzyldithiocarbazate and 3-acetylcoumarin. The metal complexes were characterized on the basis of various physico-chemical and spectroscopic techniques including elemental analysis and electrochemical studies, and FT-IR, UV-Vis, NMR, EPR and mass spectroscopy. The Schiff base was found to behave as a bidentate ligand coordinating with Cu(II) and Zn(II) in the thiolate form with 1:2 metal to ligand stoichiometry. Crystals suitable for X-ray diffractometry (XRD) were obtained from the reaction of ReCl(CO)5 with SBCM-H forming a centrosymmetric dimeric complex Re2L2(CO)6 linked by Re-S-Re bridges, where S is the thiolate sulfur of the N,S-bidentate ligand. This Re(I) complex is the first metal carbonyl complex with a bidentate dithiocarbazate ligand to have been characterized by XRD. Cytotoxicity assays revealed enhancement of the bioactivity of SBCM-H upon complexation. Both Cu(II) and Re(I) complexes are found to be active against human breast adenocarcinoma cancer cell lines MDA-MB-231 and MCF-7. TOC diagram.

Cloning, expression and purification of squalene synthase from Candida tropicalis in Pichia pastoris.

Squalene synthase (SS) is the key precursor and first committed enzyme of the sterol biosynthesis pathway. In a previous work, SS has been identified as one of the immunogenic proteins that could be a potential diagnostic candidate for the pathogenic fungus Candida tropicalis. In this study, SS from C. tropicalis was cloned and expressed as recombinant protein in Pichia pastoris to investigate its reactivity with serum antibodies. ERG9 gene that encodes for SS was amplified by PCR and cloned in-frame into pPICZB expression vector. The recombinant construct was then transformed into P. pastoris GS115 host strain. Expression of the recombinant protein was confirmed by SDS-PAGE and Western blot analysis using anti-His tag probe. Optimal protein production was achieved by cultivating the culture with 1.0% methanol for 72h. The recombinant protein was purified to approximately 97% pure in a single step immobilized metal affinity chromatography with a yield of 70.3%. Besides, the purified protein exhibited specific reactivity with immune sera on Western blot. This is the first report on heterologous expression of antigenic SS from C. tropicalis in P. pastoris which can be exploited for large-scale production and further research. The results also suggested that the protein might be of great value as antigen candidate for serodiagnosis of Candida infection.

Assessment of probiotic potential and anticancer activity of newly isolated vaginal bacterium Lactobacillus plantarum 5BL.

Numerous bacteria in and on its external parts protect the human body from harmful threats. This study aimed to investigate the potential beneficial effects of the vaginal ecosystem microbiota. A type of bacteria was isolated from vaginal secretions of adolescent and young adult women, cultured on an appropriate specific culture medium, and then molecularly identified through 16S rDNA gene sequencing. Results of 16S rDNA sequencing revealed that the isolate belongs to the Lactobacillus plantarum species. The isolated strain exhibited probiotic properties such as low pH and high bile salt concentration tolerance, antibiotic susceptibility and antimicrobial activity against some pathogenic bacteria. The anticancer effects of the strain on human cancer cell lines (cervical, HeLa; gastric, AGS; colon, HT-29; breast, MCF-7) and on a human normal cell line (human umbilical vein endothelial cells [HUVEC]) were investigated. Toxic side effects were assessed by studying apoptosis in the treated cells. The strain exhibited desirable probiotic properties and remarkable anticancer activity against the tested human cancer cell lines (P ≤ 0.05) with no significant cytotoxic effects on HUVEC normal cells (P ≤ 0.05). Overall, the isolated strain showed favorable potential as a bioactive therapeutic agent. Therefore, this strain should be subjected to the other required tests to prove its suitability for clinical therapeutic application.

Safety profile of dextran-spermine gene delivery vector in mouse lungs.

A nano-sized polymer, dextran-spermine (D-SPM), was shown to have the capacity to deliver gene to the lung of mouse via intranasal route. In this study, assessments on the safety profile of D-SPM were performed to complement the gene expression results. African green monkey kidney fibroblast (COS-7) and human adenocarcinoma breast (MCF-7) cells transfected with D-SPM/pDNA showed massive reduction in the number of viable cells. As for in vivo study, elevated level of neutrophils was observed, despite the minimal level of pro-inflammatory cytokines (TNF-alpha, IL-12, IFN-gamma) detected in the bronchoalveolar lavage fluid (BALF) of mice treated with the D-SPM/pDNA complexes. Histology profile examinations of the lungs showed mild inflammatory responses, with inflamed areas overlap with healthy areas. Although reduction of mice weight was seen at day 1 post administration, the mice did not show any sign of abnormal behavior or physical appearance. Biodistribution study was performed to determine the ability of the D-SPM/pDNA complexes to infiltrate to other non-intended organs. The result showed that the D-SPM/pDNA complexes were only localized at the lung and no gene expression was detected in other organs or blood. In short, these results indicate that the D-SPM/pDNA exhibited mild toxicity in the mouse lungs.

Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 µg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics.

Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells.

Lactobacillus acidophilus is categorized as a probiotic strain because of its beneficial effects in human health and prevention of disease transmission. This study is aimed to characterize the probiotic potential of L. acidophilus 36YL originally isolated from the vagina of healthy and fertile Iranian women. The L. acidophilus 36YL strain was identified using 16S rDNA gene sequencing and characterized by biochemical methodologies, such as antibiotics susceptibility, antimicrobial activity, and acid and bile resistance. The bioactivity of the secretion of this strain on four human cancer cell lines (AGS, HeLa, MCF-7, and HT-29) and one normal cell line (HUVEC) was evaluated by cytotoxicity assay and apoptosis analysis. This newly isolated strain was found to exhibit notable probiotic properties, such as admirable antibiotic susceptibility, good antimicrobial activity, and favorable resistance to acid and bile salt. The results of bioactivity assessment demonstrated acceptable anticancer effects on the four tested cancer cell lines and negligible side effects on the assayed normal cell line. Our findings revealed that the anticancer effect of L. acidophilus 36YL strain secretions depends on the induction of apoptosis in cancer cells. L. acidophilus 36YL strain is considered as a nutraceutical alternative or a topical medication with a potential therapeutic index because of the absence of cytotoxicity to normal cells, but effective toxicity to cancer cell lines.

Neural Commitment of Embryonic Stem Cells through the Formation of Embryoid Bodies (EBs).

An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4-/4+ protocol.

MiR-3099 is Overexpressed in Differentiating 46c Mouse Embryonic Stem Cells upon Neural Induction.

BACKGROUND: MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium. METHODS: 46C mouse embryonic stem cells were subjected to neural induction with N2/B27 medium. At 0, 3, 7, 11, 17, and 22 days after neural induction, the cells were screened for various pluripotent, progenitor, and differentiating/differentiated cells markers by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Stem-loop pulse RT-PCR was performed to determine the expression of miR-3099 at all selected time points after neural induction. RESULTS: Our findings showed that after induction, mouse embryonic stem cells differentiated into heterogeneous pools of cells containing neurons, astrocytes, and oligodendrocytes. Mouse embryonic stem cells and neural progenitor/precursor cells were also present in culture up to day 22 as indicated by RT-PCR analysis. Elucidation of miR-3099 expression during in vitro neural induction revealed that this miRNA was expressed throughout the differentiation process of 46C mouse embryonic stem cells. miR-3099 was expressed at higher levels on day 11, 17, and 22 as compared to day 0, 3 and 7 after neural induction. CONCLUSION: The level of miR-3099 expression was higher in differentiated mouse embryonic stem cells after neural induction. This finding suggested that miR-3099 might play a role in regulating neural stem cell differentiation. However, further characterisation of miR-3099 in a better characterised or optimised differentiated neural stem cell culture would provide increased understanding of the cellular function and molecular targets of miR-3099, especially in neuron development.

The mitochondrial C16069T polymorphism, not mitochondrial D310 (D-loop) mononucleotide sequence variations, is associated with bladder cancer.

BACKGROUND: Bladder cancer is a relatively common and potentially life-threatening neoplasm that ranks ninth in terms of worldwide cancer incidence. The aim of this study was to determine deletions and sequence variations in the mitochondrial displacement loop (D-loop) region from the blood specimens and tumoral tissues of patients with bladder cancer, compared to adjacent non-tumoral tissues. METHODS: The DNA from blood, tumoral tissues and adjacent non-tumoral tissues of twenty-six patients with bladder cancer and DNA from blood of 504 healthy controls from different ethnicities were investigated to determine sequence variation in the mitochondrial D-loop region using multiplex polymerase chain reaction (PCR), DNA sequencing and southern blotting analysis. RESULTS: From a total of 110 variations, 48 were reported as new mutations. No deletions were detected in tumoral tissues, adjacent non-tumoral tissues and blood samples from patients. Although the polymorphisms at loci 16189, 16261 and 16311 were not significantly correlated with bladder cancer, the C16069T variation was significantly present in patient samples compared to control samples (p < 0.05). Interestingly, there was no significant difference (p > 0.05) of C variations, including C7TC6, C8TC6, C9TC6 and C10TC6, in D310 mitochondrial DNA between patients and control samples. CONCLUSION: Our study suggests that 16069 mitochondrial DNA D-Loop mutations may play a significant role in the etiology of bladder cancer and facilitate the definition of carcinogenesis-related mutations in human cancer.

Centella asiatica (L.) Urban. Attenuates Cell Damage in Hydrogen Peroxide-Induced Oxidative Stress in Transgenic Murine Embryonic Stem Cell Line-Derived Neural-Like Cells: A Preliminary Study for Potential Treatment of Alzheimer's Disease.

BACKGROUND: Centella asiatica (L.) (C. asiatica) is commonly known in South East and South East Asia communities for its nutritional and medicinal benefits. Besides being traditionally used to enhance memory and accelerate wound healing, its phytochemicals have been extensively documented for their neuroprotective, neuroregenerative, and antioxidant properties. OBJECTIVE: The present study aims to investigate the effects of a standardized raw extract of C. asiatica (RECA) on hydrogen peroxide (H2O2)-induced oxidative stress and apoptotic death in neural-like cells derived from mouse embryonic stem (ES) cell line. METHODS: A transgenic mouse ES cell (46C) was differentiated into neural-like cells using 4-/4+ protocol with addition of all-trans retinoic acid. These cells were then exposed to H2O2 for 24 h. The effects of RECA on H2O2-induced neural-like cells were assessed through cell viability, apoptosis, and reactive oxygen species (ROS) assays, as well as neurite length measurement. The gene expression levels of neuronal-specific and antioxidant markers were assessed by RT-qPCR analysis. RESULTS: Pre-treatment with H2O2 for 24 hours, in a dose-dependent manner, damaged neural-like cells as marked by a decrease in cell viability, substantial increase in intracellular ROS accumulation, and increase in apoptotic rate compared to untreated cells. These cells were used to treat with RECA. Treatment with RECA for 48 h remarkably restored cell survival and promoted neurite outgrowth in the H2O2- damaged neurons by increasing cell viability and decreasing ROS activity. RT-qPCR analysis revealed that RECA upregulated the level of antioxidant genes such as thioredoxin-1 (Trx-1) and heme oxygenase-1 (HO-1) of treated cells, as well as the expression level of neuronal-specific markers such as Tuj1 and MAP2 genes, suggesting their contribution in neuritogenic effect. CONCLUSION: Our findings indicate that RECA promotes neuroregenerative effects and exhibits antioxidant properties, suggesting a valuable synergistic activity of its phytochemical constituents, thus, making the extract a promising candidate in preventing or treating oxidative stress-associated Alzheimer's disease.

Association of psychological distress with arm morbidity symptoms in breast cancer survivors: outcomes from the use of PHQ-9 and GAD-7 questionnaires.

BACKGROUND: Although higher survival rates of breast cancer are achieved these days, breast cancer survivors are challenged with unwanted side effects from treatment or management that affect physical, functional, and psychological well-being of an individual. This study aimed to assess psychological distress status in Malaysian breast cancer survivors and factors that affected the condition. METHODS: A cross-sectional study design was conducted on 162 breast cancer survivors from various breast cancer support groups in Malaysia. Psychological distress status was assessed based on depression and anxiety scores by applying the Malay version of Patient Health Questionnaire (PHQ-9) and General Anxiety Disorder (GAD-7). Both instruments were self-administered along with a set of questionnaires comprising demographic, medical history, quality of life, and upper extremity function assessment. Outcomes from the PHQ-9 and GAD-7 were analyzed for severity level of psychological distress, and its association with relevant variables, arm morbidity symptoms, as well as the duration of cancer survivorship. RESULTS: The univariate analysis showed that breast cancer survivors with arm morbidities after breast surgery had a higher score of depression (5.0 vs 4.0, p = 0.011) and anxiety (3.0 vs 1.0, p = 0.026) than those who did not. Besides that, receiving fewer post-rehabilitation treatments (p = 0.049) and having a family history of cancer (p = 0.022) were correlated with higher anxiety level. The level of depression and anxiety was inversely proportionate with quality of life and positively correlated with greater disability of the arm function (p < 0.05). Subsequent analysis showed that arm morbidity symptoms including difficulties in finding a t-shirt that fits and pain in the arm area after breast cancer surgery were positively associated with a higher level of psychological distress. CONCLUSION: Our study demonstrated the association between psychological distress with arm morbidities in breast cancer survivors. Given that arm morbidities can affect not only physical, but psychological well-being, continuous or serial assessment on both aspects during cancer treatment may effectively help to address mental health issue experienced by this cancer population.

Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines.

BACKGROUND: Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research. METHODS: Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping. RESULTS: MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER+, PR+, HER2+) and (ER+, PR-, HER2+), respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER-, PR-, HER2+) for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52-58 chromosomes per cell. CONCLUSIONS: Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER+/PR+/HER2+ and ER+/PR-/HER2+ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.