Exosomal-microRNA transcriptome profiling of Parental and CSC-like MDA-MB-231 cells in response to cisplatin treatment.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with higher risk of metastasis and cancer reoccurrence. Cisplatin is one of the potential anticancer drugs for treating TNBC, where its effectiveness remains challenged by frequent occurrence of cisplatin resistance. Since acquirement of drug resistance often being associated with presence of cancer stem cells (CSCs), investigation has been conducted, suggesting CSC-like subpopulation to be more resistant to cisplatin than their parental counterpart. On the other hand, plethora evidences showed the transmission of exosomal-miRNAs are capable of promoting drug resistance in breast cancers. In this study, we aim to elucidate the differential expression of exosomal-microRNAs profile and reveal the potential target genes in correlation to cisplatin resistance associated with CSC-like subpopulation by using TNBC cell line (MDA-MB-231). Utilizing next generation sequencing and Nanostring techniques, cisplatin-induced dysregulation of exosomal-miRNAs were evaluated in maximal for CSC-like subpopulation as compared to parental cells. Intriguingly, more oncogenic exosomal-miRNAs profile was detected from treated CSC-like subpopulation, which may correlate to enhancement of drug resistance and maintenance of CSCs. In treated CSC-like subpopulation, unique clusters of exosomal-miRNAs namely miR-221-3p, miR-196a-5p, miR-17-5p and miR-126-3p were predicted to target on six genes (ATXN1, LATS1, GSK3ß, ITGA6, JAG1 and MYC), aligned with previous finding which demonstrated dysregulation of these genes in treated CSC-like subpopulation. Our results highlight the potential correlation of exosomal-miRNAs and their target genes as well as novel perspectives of the corresponding pathways that may be essential to contribute to the attenuated cytotoxicity of cisplatin in CSC-like subpopulation.

Regulation of Cellular and Cancer Stem Cell-Related Putative Gene Expression of Parental and CD44+CD24- Sorted MDA-MB-231 Cells by Cisplatin.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that promotes a higher risk of metastasis and cancer reoccurrence. Cisplatin is one of the potential anticancer drugs for treating TNBC. However, the occurrence of cisplatin resistance still remains one of the challenges in fully eradicating TNBC. The presence of cancer stem cells (CSCs) has been proposed as one of the factors contributing to the development of cisplatin resistance. In this study, we aimed to characterize the cellular properties and reveal the corresponding putative target genes involved in cisplatin resistance associated with CSCs using the TNBC cell line (MDA-MB-231). CSC-like cells were isolated from parental cells and the therapeutic effect of cisplatin on CSC-like cells was compared to that of the parental cells via cell characterization bioassays. A PCR array was then conducted to study the expression of cellular mRNA for each subpopulation. As compared to treated parental cells, treated CSCs displayed lower events of late apoptosis/necrosis and G2/M phase cell arrest, with higher mammosphere formation capacity. Furthermore, a distinct set of putative target genes correlated to the Hedgehog pathway and angiogenesis were dysregulated solely in CSC-like cells after cisplatin treatment, which were closely related to the regulation of chemoresistance and self-renewability in breast cancer. In summary, both cellular and gene expression studies suggest the attenuated cytotoxicity of cisplatin in CSC-like cells as compared to parental cells. Understanding the role of dysregulated putative target genes induced by cisplatin in CSCs may aid in the potential development of therapeutic targets for cisplatin-resistant breast cancer.

Phenotypic and microRNA characterization of the neglected CD24+ cell population in MCF-7 breast cancer 3-dimensional spheroid culture.

BACKGROUND: In vitro 3-dimensional (3D) spheroid culture has been widely used as model to enrich CD44CD24 cancer stem cells (CSC) with high aldehyde dehydrogenase 1 (ALDH1) activity. Although CD24 subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. METHODS: In this study, CD24 population from the Michigan Cancer Foundation-7 (MCF-7) spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test, as well as microRNA expression profiling. RESULTS: Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in nonadherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f, and Nanog was dim in MCF-7 CD24 cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared with that of the parental cells. Moreover, microRNA profiling has shown that the absence of CSC properties was consistent with the downregulation of major CSCs-related pathways including Hedgehog, wingless-related integration site (Wnt), and microtubule associated protein kinase (MAPK) signaling pathways. However, the upregulated pathways such as adherens junctions, focal adhesion, and tight junction suggest that CD24 cells were probably at an epithelial-like state of cell transition. CONCLUSION: In conclusion, neglected CD24 cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analyzed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.

Anti-inflammatory, analgesic and acute toxicity effects of fermented soybean.

BACKGROUND: Tempeh is a widely known fermented soybean that contains elevated level of bioactive contents. Our previous study has shown that anaerobic fermented Nutrient Enriched Soybean Tempeh (NESTE) with increase amino acid and antioxidant levels possessed better hepatoprotective effect than raw soybean. METHODS: In this study, the anti-inflammatory effect of the NESTE aqueous extract and raw soybean aqueous extract (SBE) were evaluated by quantifying the inhibition of IL-1ß, TNF-α and nitric oxide (NO) secretion in LPS treated RAW 264.7 cell in vitro. On the other hand, in vivo oral acute toxicity effect of the extract was tested on mice at the dose of 5000 mg/kg body weight. In vivo oral analgesic effect of both aqueous extracts at 200 and 1000 mg/kg body weight was evaluated by the hot plate test. RESULTS: In the in vitro anti-inflammatory study, 5 mg/mL NESTE was able to inhibit 25.50 ± 2.20%, 35.88 ± 3.20% and 28.50 ± 3.50% of NO, IL-1ß and TNF-α production in LPS treated RAW 264.7 cells without inducing cytotoxic effect on the cells. However, this effect was lower than 4 µg/mL of curcumin, which inhibited NO, IL-1ß and TNF-α production by 89.50 ± 5.00%, 78.80 ± 6.20% and 87.30 ± 4.00%, respectively. In addition, 1.5 to 2.5-fold increase of latency period up to 120 min for mice in the hot plate test was achieved by 1000 mg/kg NESTE. The analgesic effect of NESTE was better than 400 mg/kg of acetyl salicylic acid, which only increased ~ 1.7-fold of latency period up to 90 min. Moreover, NESTE did not show acute toxicity (no LD50) up to 5000 mg/kg body weight. CONCLUSION: NESTE is a nutritious food ingredient with potential anti-inflammatory and analgesic effects.

Synthetic curcumin derivative DK1 possessed G2/M arrest and induced apoptosis through accumulation of intracellular ROS in MCF-7 breast cancer cells.

AIMS: Curcumin is a lead compound of the rhizomes of Curcuma longa and possess a broad range of pharmacological activities. Chemically, curcumin is 1,3-dicarbonyl class of compound, which exhibits keto-enol tautomerism. Despite of its strong biological properties, curcumin has yet been recommended as a therapeutic agent because of its poor bioavailability. MAIN METHODS: A curcumin derivative (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (DK1) was synthesized and its cytotoxicity was tested on breast cancer cell MCF-7 and normal cell MCF-10A using MTT assay. Meanwhile, cell cycle regulation and apoptosis on MCF-7 cell were evaluated using flow cytometry. Regulation of cell cycle and apoptosis related genes expression was investigated by quantitative real time polymerase chain reaction (qRT-PCR), western blot and caspases activity analyses. Activation of oxidative stress on MCF-7 were evaluated by measuring ROS and GSH levels. KEY FINDINGS: DK1 was found to possess selective cytotoxicity on breast cancer MCF-7 cell than normal MCF-10A cell. Flow cytometry cell cycle and AnnexinV/PI analyses reported that DK1 effectively arrested MCF-7 at G2/M phase and induced apoptosis after 72 h of incubation than curcumin. Upregulation of p53, p21 and downregulation of PLK-1 subsequently promote phosphorylation of CDC2 which were found contributed to the arrest of G2/M phase. Moreover, increased of reactive oxygen species and reduced of antioxidant glutathione level correlate with apoptosis observed with raised of cytochrome c and active caspase 9. SIGNIFICANCE: DK1 was found to be more effective in inducing cell cycle arrest and apoptosis against MCF-7 cell with much higher selectivity index of MCF-10A/MCF-7 than curcumin, which might be contributed by the overexpression of p53 protein.

Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line.

Known as naturally occurring biologically active compounds, flavokawain A and B are the leading chalcones that possess anticancer properties. Another flavokawain derivative, (E)-1-(2'-Hydroxy-4',6'-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one (FLS) was characterized with (1)H-nuclear magnetic resonance, electron-impact mas spectrometry, infrared spectroscopy, and ultraviolet ((1)H NMR, EI-MS, IR, and UV) spectroscopic techniques. FLS cytotoxic efficacy against human cancer cells (MCF-7, MDA-MB-231, and MCF-10A) resulted in the reduction of IC50 values in a time- and dose-dependent mode with high specificity on MCF-7 (IC50 of 36 µM at 48 hours) against normal breast cell MCF-10A (no IC50 detected up to 180 µM at 72 hours). Light, scanning electron, and fluorescent microscopic analysis of MCF-7 cells treated with 36 µM of FLS displayed cell shrinkage, apoptotic body, and DNA fragmentation. Additionally, induction of G2/M cell arrest within 24 hours and apoptosis at subsequent time points was discovered via flow cytometry analysis. The roles of PLK-1, Wee-1, and phosphorylation of CDC-2 in G2/M arrest and proapoptotic factors (Bax, caspase 9, and p53) in promotion of apoptosis of FLS against MCF-7 cells were discovered using fluorometric, quantitative real-time polymerase chain reaction, and Western blot analysis. Interestingly, the presence of SCH3 (thiomethyl group) on ring B structure contributed to the selective cytotoxicity against MCF-7 cells compared to other chalcones, flavokawain A and B. Overall, our data suggest potential therapeutic value for flavokawain derivative FLS to be further developed as a new anticancer drug.

MiRNA Transcriptome Profiling of Spheroid-Enriched Cells with Cancer Stem Cell Properties in Human Breast MCF-7 Cell Line.

Breast cancer is the second leading cause of cancer-related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome in spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p<0.05) differentially expressed miRNAs were identified between spheroids and parental cells using miRNA-NGS. MiRNA-NGS analysis revealed 25 up-regulated and 109 down-regulated miRNAs which includes some miRNAs previously reported in the regulation of breast CSCs. A number of miRNAs (miR-4492, miR-4532, miR-381, miR-4508, miR-4448, miR-1296, and miR-365a) which have not been previously reported in breast cancer were found to show potential association with breast cancer chemoresistance and self-renewal capability. The gene ontology (GO) analysis showed that the predicted genes were enriched in the regulation of metabolic processes, gene expression, DNA binding, and hormone receptor binding. The corresponding pathway analyses inferred from the GO results were closely related to the function of signalling pathway, self-renewability, chemoresistance, tumorigenesis, cytoskeletal proteins, and metastasis in breast cancer. Based on these results, we proposed that certain miRNAs identified in this study could be used as new potential biomarkers for breast cancer stem cell diagnosis and targeted therapy.

Comparison of free amino acids, antioxidants, soluble phenolic acids, cytotoxicity and immunomodulation of fermented mung bean and soybean.

BACKGROUND: Mung bean and soybean have been individually reported previously to have antioxidant, cytotoxic and immunomodulatory effects, while fermentation is a well-known process to enhance the bioactive compounds that contribute to higher antioxidant, cytotoxic and immunomodulation effects. In this study, the free amino acids profile, soluble phenolic acids content, antioxidants, cytotoxic and immunomodulatory effects of fermented and non-fermented mung bean and soybean were compared. RESULTS: Fermented mung bean was recorded to have the highest level of free amino acids, soluble phenolic acids (especially protocatechuic acid) and antioxidant activities among all the tested products. Both fermented mung bean and soybean possessed cytotoxicity activities against breast cancer MCF-7 cells by arresting the G0/G1 phase followed by apoptosis. Moreover, fermented mung bean and soybean also induced splenocyte proliferation and enhanced the levels of serum interleukin-2 and interferon-γ. CONCLUSION: Augmented amounts of free amino acids and phenolic acids content after fermentation enhanced the antioxidants, cytotoxicity and immunomodulation effects of mung bean and soybean. More specifically, fermented mung bean showed the best effects among all the tested products. This study revealed the potential of fermented mung bean and soybean as functional foods for maintenance of good health.

In Vivo Antioxidant and Hypolipidemic Effects of Fermented Mung Bean on Hypercholesterolemic Mice.

Legumes have previously been reported with hypolipidemic effect caused by the presence of flavonoid. This study was carried out to evaluate the antioxidant and hypolipidemic effects of fermented mung bean on hypercholesterolemic mice. Blood from all mice was collected and subjected to serum lipid and liver profiles biochemical analysis and quantitative RT-PCR for atherosclerosis related gene expressions. Besides, livers were collected for antioxidant assays and histopathology evaluation. Fermented mung bean was found to reduce the level of serum lipid and liver enzyme profiles of hypercholesterolemic mice. Furthermore, liver antioxidant and nitric oxide levels were also significantly restored by fermented mung bean in a dosage dependent manner. The gene expression study indicated that Apoe and Bcl2a1a were upregulated while Npy and Vwf expressions were downregulated after the treatment. The effects of fermented mung bean were greater than nonfermented mung bean. These results indicated that fermented mung bean possessed antioxidants that lead to its hypolipidemic effect on hypercholesterolemic mice.

Antistress and antioxidant effects of virgin coconut oil in vivo.

Virgin coconut oil (VCO) has been consumed worldwide for various health-related reasons and some of its benefits have been scientifically evaluated. Medium-chain fatty acids were found to be a potential antidepressant functional food; however, this effect had not been evaluated in VCO, which is rich in polyphenols and medium-chain fatty acids. The aim of this study was to evaluate the antistress and antioxidant effects of VCO in vivo, using mice with stress-induced injury. The antistress effect of VCO (administered per os, at a dose of 10 ml/kg body weight) was evaluated using the forced swim test and chronic cold restraint stress models. VCO was able to reduce immobility time and restore oxidative stress in mice post-swim test. Furthermore, mice treated with VCO were found to exhibit higher levels of brain antioxidants, lower levels of brain 5-hydroxytryptamine and reduced weight of the adrenal glands. Consequently, the serum cholesterol, triglyceride, glucose and corticosterone levels were also lower in VCO-treated mice. These results suggest the potential value of VCO as an antistress functional oil.

Anti-inflammatory and antinociceptive activities of untreated, germinated, and fermented mung bean aqueous extract.

Evaluation of anti-inflammatory and antinociceptive activities of untreated mung bean (MB), germinated mung bean (GMB), and fermented mung bean (FMB) was performed on both in vitro (inhibition of inflammatory mediator, nitric oxide(NO)) and in vivo (inhibition of ear oedema and reduction of response to pain stimulus) studies. Results showed that both GMB and FMB aqueous extract exhibited potent anti-inflammatory and antinociceptive activities in a dose-dependent manner. In vitro results showed that GMB and FMB were potent inflammatory mediator (NO) inhibitors at both 2.5 and 5 mg/mL. Further in vivo studies showed that GMB and FMB aqueous extract at 1000 mg/kg can significantly reduce ear oedema in mice caused by arachidonic acid. Besides, both 200 mg/kg and 1000 mg/kg concentrations of GMB and FMB were found to exhibit potent antinociceptive effects towards hotplate induced pain. With these, it can be concluded that GMB and FMB aqueous extract exhibited potential anti-inflammatory and antinociceptive effects.

In vivo antistress and antioxidant effects of fermented and germinated mung bean.

Mung bean has been traditionally used to alleviate heat stress. This effect may be contributed by the presence of flavonoids and γ-aminobutyric acid (GABA). On the other hand, fermentation and germination have been practised to enhance the nutritional and antioxidant properties of certain food products. The main focus of current study was to compare the antistress effect of none-process, fermented and germinated mung bean extracts. Acute and chronic restraint stresses were observed to promote the elevation of serum biochemical markers including cholesterol, triglyceride, total protein, liver enzymes, and glucose. Chronic cold restraint stress was observed to increase the adrenal gland weight, brain 5-hydroxytryptamine (5-HT), and malondialdehyde (MDA) level while reducing brain antioxidant enzyme level. However, these parameters were found reverted in mice treated with diazepam, high concentration of fermented mung bean and high concentration of germinated mung bean. Moreover, enhanced level of antioxidant on the chronic stress mice was observed in fermented and germinated mung bean treated groups. In comparison between germinated and fermented mung bean, fermented mung bean always showed better antistress and antioxidant effects throughout this study.

Damnacanthal: a promising compound as a medicinal anthraquinone.

The Noni fruit, or scientifically known as Morinda citrifolia can be found in various parts of the world, especially in the pacific region. It is a small evergreen bushy-like tree originated from the Rubiaceae family. The plant has been used by polynesians as a medicinal herb for more than 2000 years. A substantial amount of phytochemicals can be found in the roots of this plant. Among all, damnacanthal has been found to be the most interesting, versatile and potent compound. Damnacanthal or chemically known as,3- hydroxy-1-methoxyanthraquinone-2-caboxaldehyde (C16H10O5), appears as pale yellow crystals with a melting point of 210-211 °C. This compound is of particular interest due to its striking pharmacological properties. Damnacanthal was shown to inhibit the oncogene Ras, p56lck tyrosine kinase, NF-KB pathway and induce apoptosis in vitro. This review aims to discuss the biological properties of damnacanthal, specifically on its anti-cancer activity that has been reported.

Hepatoprotective Effect of Fermented Soybean (Nutrient Enriched Soybean Tempeh) against Alcohol-Induced Liver Damage in Mice.

Recently, soybean tempeh has received great attention due to many advantages such as higher nutritional value, lower production cost, and shorter fermentation time. In this study, the in vivo hepatoprotective and antioxidant effects of nutrient enriched soybean tempeh (NESTE) were determined. NESTE fermentation process which involved anaerobic incubation was previously proclaimed to increase the content of amino acids and antioxidant properties remarkably. The evaluation of histological sections, serum biochemical markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cholesterol and triglycerides (TG)), liver immune response level (nitric oxide (NO)) and liver antioxidant level (superoxide dismutase (SOD), ferric reducing antioxidant power (FRAP), and malondialdehyde (MDA)) was conducted in order to compare the effects of nonfermented soybean extract (SBE) and fermented soybean extract (NESTE) on alcohol-induced liver damage in mice. Results demonstrated that 1000 mg/kg of NESTE can significantly reduce the levels of AST, ALT, cholesterol, TG, MDA, and NO. On the other hand, it also raised the level of SOD and FRAP. Furthermore, the histological examination on 1000 mg/kg NESTE treatment group showed that this extract was capable of recovering the damaged hepatocytes to their normal structures. Thus, it can be concluded that NESTE produced through fermentation process was able to enhance hepatoprotective and antioxidant effects in vivo.

In Vivo Immunomodulation and Lipid Peroxidation Activities Contributed to Chemoprevention Effects of Fermented Mung Bean against Breast Cancer.

Mung bean has been reported to have antioxidant, cytotoxic, and immunomodulatory effects in vitro. Fermented products are reported to have enhanced immunomodulation and cancer chemopreventive effects. In this study, fermented mung bean treatments in vivo were studied by monitoring tumor development, spleen immunity, serum cytokine (interleukin 2 and interferon gamma) levels, and spleen/tumor antioxidant levels after injection with low and high risk 4T1 breast cancer cells. Pretreatment with fermented mung bean was associated with delayed tumor formation in low risk mice. Furthermore, this treatment was connected with higher serum anticancer cytokine levels, spleen T cell populations, splenocyte cytotoxicity, and spleen/tumor antioxidant levels. Histopathological evaluation of fermented mung bean treated tumor revealed lower event of mitotic division. On the other hand, antioxidant and nitric oxide levels that were significantly increased in the untreated mice were inhibited in the fermented mung bean treated groups. These results suggested that fermented mung bean has potential cancer chemoprevention effects through the stimulation of immunity, lipid peroxidation, and anti-inflammation.