Anti-Atopic Dermatitis Effect of TPS240, a Novel Therapeutic Peptide, via Suppression of NF-κB and STAT3 Activation.

Atopic dermatitis (AD) is a relapsing skin disease with persistent inflammation as a causal factor for symptoms and disease progression. Current therapies provide only temporary relief and require long-term usage accompanied by side effects due to persistent relapses. A short peptide, TPS240, has been tested for its potential to subside AD. In this study, we confirmed the anti-atopic effect of TPS240 in vivo and in vitro using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical treatment with TPS240 diminished AD-like skin lesions and symptoms such as epidermal thickening and mast cell infiltration induced by DNCB, similar to the existing treatment, dexamethasone (Dex). Furthermore, skin atrophy, weight loss, and abnormal organ weight changes observed in the Dex-treated group were not detected in the TPS240-treated group. In TNF-α/IFN-γ-stimulated HaCaT cells, TPS240 reduced the expression of the inflammatory chemokines CCL17 and CCL22 and the pruritic cytokines TSLP and IL-31 by inhibiting NF-κB and STAT3 activation. These results suggest that TPS240 has an anti-atopic effect through immunomodulation of AD-specific cytokines and chemokines and can be used as a candidate drug for the prevention and treatment of AD that can solve the safety problems of existing treatments.

Parnassin, a Novel Therapeutic Peptide, Alleviates Skin Lesions in a DNCB-Induced Atopic Dermatitis Mouse Model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease which requires continuous treatment due to its relapsing nature. The current treatment includes steroids and nonsteroidal agents targeting inflammation but long-term administration causes various side effects such as skin atrophy, hirsutism, hypertension and diarrhea. Thus, there is an unmet need for safer and effective therapeutic agents in the treatment of AD. Peptides are small biomolecule drugs which are highly potent and remarkably have less side effects. Parnassin is a tetrapeptide with predicted anti-microbial activity curated from Parnassius bremeri transcriptome data. In this study, we confirmed the effect of parnassin on AD using a DNCB-induced AD mouse model and TNF-α/IFN-γ-stimulated HaCaT cells. In the AD mouse model, topical administration of parnassin improved skin lesions and symptoms in AD mice, such as epidermal thickening and mast cell infiltration, similar to the existing treatment, dexamethasone, and did not affect body weight, or the size and weight of spleen. In TNF-α/IFN-γ-stimulated HaCaT cells, parnassin inhibited the expression of Th2-type chemokine CCL17 and CCL22 genes by suppressing JAK2 and p38 MAPK signaling kinases and their downstream transcription factor STAT1. Parnassin also significantly reduced the gene expression of TSLP and IL-31, which are pruritus-inducing cytokines. These findings suggested that parnassin alleviates AD-like lesions via its immunomodulatory effects and can be used as a candidate drug for the prevention and treatment of AD because it is safer than existing treatments.

Elevated CO2 Alters the Physiological and Transcriptome Responses of Pinus densiflora to Long-Term CO2 Exposure.

Physiological response and transcriptome changes were observed to investigate the effects on the growth, metabolism and genetic changes of Pinus densiflora grown for a long time in an environment with an elevated atmospheric CO2 concentration. Pine trees were grown at ambient (400 ppm) and elevated (560 ppm and 720 ppm) CO2 concentrations for 10 years in open-top chambers. The content of nonstructural carbohydrates was significantly increased in elevated CO2. It was notable that the contents of chlorophylls significantly decreased at an elevated CO2. The activities of antioxidants were significantly increased at an elevated CO2 concentration of 720 ppm. We analyzed the differences in the transcriptomes of Pinus densiflora at ambient and elevated CO2 concentrations and elucidated the functions of the differentially expressed genes (DEGs). RNA-Seq analysis identified 2415 and 4462 DEGs between an ambient and elevated CO2 concentrations of 560 ppm and 720 ppm, respectively. Genes related to glycolysis/gluconeogenesis and starch/sucrose metabolism were unchanged or decreased at an elevated CO2 concentration of 560 ppm and tended to increase at an elevated CO2 concentration of 720 ppm. It was confirmed that the expression levels of genes related to photosynthesis and antioxidants were increased at an elevated CO2 concentration of 720 ppm.

Developmental Transcriptome Analysis of Red-Spotted Apollo Butterfly, Parnassius bremeri.

Parnassius bremeri (P. bremeri), a member of the genus Snow Apollo in the swallowtail family (Papilionidae), is a high alpine butterfly that lives in Russia, Korea, and China. It is an endangered wildlife (Class I) in South Korea and is a globally endangered species. The lack of transcriptomic and genomic resources of P. bremeri significantly hinders the study of its population genetics and conservation. The detailed information of the developmental stage-specific gene expression patterns of P. bremeri is of great demand for its conservation. However, the molecular mechanism underlying the metamorphic development of P. bremeri is still unknown. In the present study, the differentially expressed genes (DEGs) across the metamorphic developmental stages were compared using high-throughput transcriptome sequencing. We identified a total of 72,161 DEGs from eight comparisons. GO enrichment analysis showed that a range of DEGs were responsible for cuticle development and the melanin biosynthetic pathway during larval development. Pathway analysis suggested that the signaling pathways, such as the Wnt signaling pathway, hedgehog signaling pathway and Notch signaling pathway, are regulated during the developmental stages of P. bremeri. Furthermore, sensory receptors were also activated, especially during the larval to adult transition stage. Collectively, the results of this study provide a preliminary foundation and understanding of the molecular mechanism in their transcriptomes for further research on the metamorphic development of P. bremeri.

Ulmus macrocarpa Hance extract modulates intestinal microbiota in healthy adults: a randomized, placebo-controlled clinical trial.

The stem and root bark of Ulmus macrocarpa Hance has been used as traditional pharmacological agent against inflammation related disorders. The objective of this study was to explore the impact of Ulmus macrocarpa Hance extract (UME) on human gut microbiota. A randomized placebo-controlled clinical study was conducted in healthy adults. The study subjects were given 500 mg/day of UME or placebo orally for 4 weeks. Eighty fecal samples were collected at baseline and 4 weeks of UME or placebo intervention. The gut microbiota variation was evaluated by 16S rRNA profiling. The microbial response was highly personalized, and no statistically significant differences was observed in both species richness and abundance. The number of bacterial species identified in study subjects ranged from 86 to 182 species. The analysis for taxonomical changes revealed an increase in Eubacterium ventriosum, Blautia faecis, Ruminococcus gnavus in the UME group. Functional enrichment of bacterial genes showed an increase in primary and secondary bile acid biosynthesis in UME group. Having known from previous studies Eubacterium regulated bile acid homeostasis in protecting gut microbial architecture and immunity, we suggest that UME supplementation might enhance host immunity by modulating gut microbiota. This is the first stage study and forthcoming clinical studies with larger participants are needed to confirm these findings.

Molecular Insights of Fruit Quality Traits in Peaches, Prunus persica.

Fleshy fruits are the most demanded fruits because of their organoleptic qualities and nutritional values. The genus Prunus is a rich source of diversified stone/drupe fruits such as almonds, apricots, plums, sweet cherries, peaches, and nectarines. The fruit-ripening process in Prunus involves coordinated biochemical and physiological changes resulting in changes in fruit texture, aroma gain, color change in the pericarp, sugar/organic acid balance, fruit growth, and weight gain. There are different varieties of peaches with unique palatable qualities and gaining knowledge in the genetics behind these quality traits helps in seedling selection for breeding programs. In addition, peaches have shorter post-harvest life due to excessive softening, resulting in fruit quality reduction and market loss. Many studies have been executed to understand the softening process at the molecular level to find the genetic basis. To summarize, this review focused on the molecular aspects of peach fruit quality attributes and their related genetics to understand the underlying mechanisms.

Utilizing Red Spotted Apollo Butterfly Transcriptome to Identify Antimicrobial Peptide Candidates against Porphyromonas gingivalis.

Classical antibiotics are the foremost treatment strategy against microbial infections. Overuse of this has led to the evolution of antimicrobial resistance. Antimicrobial peptides (AMPs) are natural defense elements present across many species including humans, insects, bacteria, and plants. Insect AMPs are our area of interest, because of their stronger abilities in host defense. We have deciphered AMPs from an endangered species Parnassius bremeri, commonly known as the red spotted apollo butterfly. It belongs to the second largest insect order Lepidoptera, comprised of butterflies and moths, and lives in the high altitudes of Russia, China, and Korea. We aimed at identifying the AMPs from the larvae stages. The rationale of choosing this stage is that the P. bremeri larvae development occurs at extremely low temperature conditions, which might serve as external stimuli for AMP production. RNA was isolated from larvae (L1 to L5) instar stages and subjected to next generation sequencing. The transcriptomes obtained were curated in in-silico pipelines. The peptides obtained were screened for requisite AMP physicochemical properties and in vitro antimicrobial activity. With the sequential screening and validation, we obtained fifteen candidate AMPs. One peptide TPS-032 showed promising antimicrobial activity against Porphyromonas gingivalis, a primary causative organism of periodontitis.

Deciphering Novel Antimicrobial Peptides from the Transcriptome of Papilio xuthus.

An insect's innate immune system is the front line of defense against many invading microorganisms. One of the important components of this defense system is antimicrobial peptides (AMPs). Papiliocin is a well-studied antimicrobial peptide (AMP) isolated from the swallowtail butterfly, Papilio xuthus, and it was previously reported to be effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, particularly in drug resistant Gram-negative bacteria. Hence, we aimed to identify novel AMPs from Papilio xuthus using its transcriptome. We immunized the swallowtail butterfly with Escherichia coli, Staphylococcus aureus, Candida albicans, and the total RNA was isolated. De novo transcriptome assembly and functional annotations were conducted, and AMPs were predicted using an in-silico pipeline. The obtained 344,804,442 raw reads were then pre-processed to retrieve 312,509,806 (90.6%) total clean reads. A total of 38,272 unigenes were assembled with the average length of 1010 bp. Differential gene expression analysis identified 584 and 1409 upregulated and downregulated genes, respectively. The physicochemical, aggregation, and allergen propensity were used as filtration criteria. A total of 248 peptides were predicted using our in-house pipeline and the known AMPs were removed, resulting in 193 novel peptides. Finally, seven peptides were tested in vitro and three peptides (Px 5, 6, and 7) showed stronger antimicrobial activity against Gram-negative bacteria and yeast. All the tested peptides were non-allergens. The identified novel AMPs may serve as potential candidates for future antimicrobial studies.

Transcriptome Analysis of Psacothea hilaris: De Novo Assembly and Antimicrobial Peptide Prediction.

Antimicrobial peptides (AMPs) are the frontline innate defense system evolutionarily preserved in insects to combat invading pathogens. These AMPs could serve as an alternative to classical antibiotics to overcome the burden of treating multidrug resistant bacteria. Psacotheasin, a knottin type AMP was isolated from Psacothea hilaris and shown to exhibit antimicrobial activity, especially against fungi through apoptosis mediated cell death. In this study, we aimed to identify novel probable AMPs from Psacothea hilaris, the yellow spotted longicorn beetle. The beetle was immunized with the two bacterial strains (E. coli and S. aureus), and the yeast strain C. albicans. After immunization, total RNA was isolated and sequenced in Illumina platform. Then, beetle transcriptome was de novo assembled and searched for putative AMPs with the known physiochemical features of the AMPs. A selection of AMP candidates were synthesized and tested for antimicrobial activity. Four peptides showed stronger activity against E. coli than the control AMP, melittin while one peptide showed similar activity against S. aureus. Moreover, four peptides and two peptides showed antifungal activity stronger than and similar to melittin, respectively. Collectively one peptide showed both antibacterial and antifungal activity superior to melittin; thus, it provides a potent antimicrobial peptide. All the peptides showed no hemolysis in all the tested concentrations. These results suggest that in silico mining of insects' transcriptome could be a promising tool to obtain and optimize novel AMPs for human needs.

Citral Induced Apoptosis through Modulation of Key Genes Involved in Fatty Acid Biosynthesis in Human Prostate Cancer Cells: In Silico and In Vitro Study.

The isomers of citral (cis-citral and trans-citral) were isolated from the Cymbopogon citratus (DC.) Stapf oil demonstrates many therapeutic properties including anticancer properties. However, the effects of citral on suppressing human prostate cancer and its underlying molecular mechanism have yet to be elucidated. The citral was isolated from lemongrass oil using various spectroscopic analyses, such as electron ionized mass spectrometry (EI-MS) and nuclear magnetic resonance (NMR) spectroscopy respectively. We carried out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cell viability of citral in prostate cancer cells (PC-3 and PC3M). Furthermore, to confirm that PC3 undergoes apoptosis by inhibiting lipogenesis, we used several detection methods including flow cytometry, DNA fragmentation, Hoechst staining, PI staining, oil staining, qPCR, and Western blotting. Citral impaired the clonogenic property of the cancer cells and altered the morphology of cancer cells. Molecular interaction studies and the PASS biological program predicted that citral isomers tend to interact with proteins involved in lipogenesis and the apoptosis pathway. Furthermore, citral suppressed lipogenesis of prostate cancer cells through the activation of AMPK phosphorylation and downregulation of fatty acid synthase (FASN), acetyl coA carboxylase (ACC), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), and sterol regulatory element-binding protein (SREBP1) and apoptosis of PC3 cells by upregulating BAX and downregulating Bcl-2 expression. In addition, in silico studies such as ADMET predicted that citral can be used as a safe potent drug for the treatment of prostate cancer. Our results indicate that citral may serve as a potential candidate against human prostate cancer and warrants in vivo studies.

Phyllanthus emblica fruit extract attenuates lipid metabolism in 3T3-L1 adipocytes via activating apoptosis mediated cell death.

BACKGROUND: Phyllanthus emblica L. (Indian gooseberry) is widely used in the Ayurveda for thousands of years to treat health complications including disorders of the immune system, diabetes, and obesity. PURPOSE: For the first time, our study aims to demonstrate the molecular mechanisms of the fruit extract of Phyllanthus emblica (PEFE) involved in the promotion of fat cell apoptosis and alleviation of adipogenesis. METHODS: The active constituents from PEFE were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS). We carried out the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effects of PEFE using 3T3-L1 pre-adipocytes. The colonogenic assay was carried out to determine the inhibitory effect of 3T3-L1 adipocytes after PEFE treatment. In addition, inhibition of pancreatic lipase activity was performed and the lipolytic activity of PEFE and digallic acid was compared with the well-known standard drug orlistat. Besides, the molecular interaction and ligand optimization between digallic and adipogenesis/apoptosis markers were also carried out. Furthermore, to confirm fat cell apoptosis we have used several detection methods that includes Hoechst staining, PI staining, Oil staining and qPCR respectively. RESULTS: Digallic acid was identified as a major component in the PEFE. The IC50 values of digallic acid and PEFE were found to be 3.82 µg/ml and 21.85 µg/ml respectively. PEFE and digallic acid showed significant anti-lipolytic activity compared to the standard drug orlistat. In the mature adipocytes, PEFE significantly decreased triglyceride accumulation by downregulating adiponectin, PPARγ, cEBPα, and FABP4 respectively. We further analyzed the expression of apoptosis related genes upon PEFE treatment. Apoptotic process initiated through upregulation of BAX and downregulation of BCL2 resulting in an increased caspase-3 activity. In addition, we have also confirmed the apoptosis and DNA fragmentation in 3T3-L1 cells using Hoechst, PI and TUNEL assays. CONCLUSION: PEFE negatively regulates adipogenesis by initiating fat cell apoptosis and therefore it can be considered as a potential herbal medicinal product for treating obesity.

In silico and in vitro analysis of coumarin derivative induced anticancer effects by undergoing intrinsic pathway mediated apoptosis in human stomach cancer.

BACKGROUND: Coumarin plays a vital role in drug discovery process due to its diverse biologically active components. Recently, coumarin derivatives are paying attention to treat various diseases including cancer. The effect of coumarin derivatives on gastric cancer is not well established although gastric cancer being the fourth leading cancer. Therefore, we attempt to study the effect of styrene substituted biscoumarin (SSBC) to induce apoptosis and inhibit cancer proliferation using in silico and in vitro approaches. METHODS: We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to identify the anti-proliferative activity of SSBC in stomach cancer cell lines (AGS) and toxicity of the compared was also assessed using lung normal cell lines (L-132 and MRC-5). A docking study was carried out between anti-apoptotic protein (BCL2) and SSBC compound. Furthermore, we analyzed the drug likeliness by screening pharmacological properties (ADME) and biological activity of SSBC by performing spectrum prediction analysis (PASS). The apoptotic effect of SSBC in AGS cell lines were detected using flow cytometry (FACS), Hoechst staining and DAPI/PI staining. Later, the regulation of apoptotic pathway by SSBC was also confirmed by qRT-PCR and western blotting analysis. RESULTS: The inhibition concentration (IC50) of SSBC was assayed against AGS and lung normal cell lines (L-132 and MRC-5). The IC50 value of SSBC toward AGS, L-132 and MRC-5 was 4.56, 268 and 285 µg/ml, respectively. In silico analysis predicted SSBC could bind to the active site of BH3 domain of anti-apoptotic protein and thus resulted in apoptotic mediated cell death. ADME prediction of SSBC exhibit strong binding capacity of 99.08% and showed absorption rate about 95.57% in the intestine. In addition, biological activity of SSBC was also predicted using PASS program and we found SSBC exhibit high activity for various cancer related protein expression including apoptosis pathway proteins such as caspase 3 stimulant, apoptosis agonist. Furthermore, apoptosis of AGS was also assessed using Hoechst staining, DAPI/PI analysis, flow-cytometric analysis, qRT-PCR and western blot analysis. CONCLUSION: Our study denotes that SSBC could be very effective against AGS by inducing apoptosis through intrinsic pathway and recommended for in vivo and human trials.

Inhibition of IKKß by celastrol and its analogues - an in silico and in vitro approach.

CONTEXT: Alzheimer's disease (AD) is the most common form of dementia affecting the aged population and neuroinflammation is one of the most observed AD pathologies. NF-κB is the central regulator of inflammation and inhibitor κB kinase (IKK) is the converging point in NF-κB activation. Celastrol is a natural triterpene used as a treatment for inflammatory conditions. OBJECTIVE: This study determines the neuroprotective and inhibitory effect of celastrol on amyloid beta1-42 (Aß1-42) induced cytotoxicity and IKKß activity, respectively. MATERIALS AND METHODS: Retinoic acid differentiated IMR-32 cells were treated with celastrol (1 µM) before treatment with Aß1-42 (IC30 10 µM) for 24 h. The cytotoxicity and IKK phosphorylation were measured by MTT and western blotting analysis, respectively. We screened 36 celastrol analogues for the IKKß inhibition by molecular docking and evaluated their drug like properties to delineate the neuroprotective effects. RESULTS: Celastrol (1 µM) inhibited Aß1-42 (10 µM) induced IκBα phosphorylation and protected IMR-32 cells from cell death. Celastrol and 25 analogues showed strong binding affinity with IKKß as evidenced by strong hydrogen-bonding interactions with critical active site residues. All the 25 analogues displayed strong anti-inflammatory properties but only 11 analogues showed drug-likeness. Collectively, molecule 15 has highest binding affinity, CNS activity and more drug likeness than parent compound celastrol. DISCUSSION AND CONCLUSION: The decreased expression of pIκBα in celastrol pretreated cells affirms the functional representation of inhibited IKKß activity in these cells. The neuroprotective potentials of celastrol and its analogues may be related to IKK inhibition.

Interferon ß improves the efficacy of low dose cisplatin by inhibiting NF-κB/p-Akt signaling on HeLa cells.

The purpose of this study was to evaluate the anticancer efficacy of interferon ß in combination with low dose of cisplatin on human cervical cancer progression, as well as its principal action mechanism. The combination treatment synergistically potentiated the effect of interferon ß on cell growth inhibition and DNA damage on HeLa cells by repressing NF-κB/p-Akt signaling. Synergistic targeting of these pathways has a therapeutic potential. Further, the combination treatment ameliorated the expression of pro-apoptotic Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Additionally, the expression of active PARP was significantly increased and MMP-9 level was decreased in combination group as compared to the expression seen for the treatment with interferon ß or cisplatin alone. Results demonstrate that the synergistic inhibitory effects of interferon ß and low dose of cisplatin on human cervical cancer cells and also suggest that the inhibition of NF-κB/p-Akt signaling pathway plays a critical role in the anticancer effects of combination treatment along with the induction of PARP. Therefore, the combination of interferon ß and cisplatin may be a useful treatment for human cervical cancer, with a greater effectiveness than other treatments.

Whole Genome Re-Sequencing and Characterization of Powdery Mildew Disease-Associated Allelic Variation in Melon.

Powdery mildew is one of the most common fungal diseases in the world. This disease frequently affects melon (Cucumis melo L.) and other Cucurbitaceous family crops in both open field and greenhouse cultivation. One of the goals of genomics is to identify the polymorphic loci responsible for variation in phenotypic traits. In this study, powdery mildew disease assessment scores were calculated for four melon accessions, 'SCNU1154', 'Edisto47', 'MR-1', and 'PMR5'. To investigate the genetic variation of these accessions, whole genome re-sequencing using the Illumina HiSeq 2000 platform was performed. A total of 754,759,704 quality-filtered reads were generated, with an average of 82.64% coverage relative to the reference genome. Comparisons of the sequences for the melon accessions revealed around 7.4 million single nucleotide polymorphisms (SNPs), 1.9 million InDels, and 182,398 putative structural variations (SVs). Functional enrichment analysis of detected variations classified them into biological process, cellular component and molecular function categories. Further, a disease-associated QTL map was constructed for 390 SNPs and 45 InDels identified as related to defense-response genes. Among them 112 SNPs and 12 InDels were observed in powdery mildew responsive chromosomes. Accordingly, this whole genome re-sequencing study identified SNPs and InDels associated with defense genes that will serve as candidate polymorphisms in the search for sources of resistance against powdery mildew disease and could accelerate marker-assisted breeding in melon.

Inhibitory effects of interferon-ß on hepatocellular carcinoma HepG2 via Akt/STAT phosphorylation.

Conventional chemotherapy fails to cure metastatic hepatoma mainly due to its high hepatotoxicity. Currently, doxorubicin is the most widely used drug against liver cancer either as single agent or in combination with other chemotherapeutics such as cisplatin. It is limited due to their severe toxicity on normal hepatocytes. Therefore, alternative therapeutic agents without or with low hepatotoxicity are highly desirable. Interferons are a family of cytokines that potently demonstrate antiviral, immunomodulatory, and antiproliferative activities. It also exerts direct cytotoxic effects on tumor cells. The purpose of this study was to examine the in vitro cytotoxicity of interferon-ß on HepG2 cells. We revealed the presence of binding receptor of interferon-ß in HepG2 cells. The dose-dependent inhibition on cell proliferation was observed. We demonstrated that IFN-ß exhibited significant cytotoxicity in HepG2 cells mainly through phosphorylation of signal transducers and activators of transcription 2. The activation of Akt was suppressed. The stimulation of pro-apoptotic protein expression of Bax, inhibition of anti-apoptotic protein expression of Bcl-2, activation of cleaved caspases 9 and 3 was found at increasing concentrations. In conclusion, our results suggest that interferon-ß has potential to inhibit cell proliferation dose dependently. Increased concentrations of interferon-ß influenced apoptosis via mitochondrial pathway through inhibition of p-Akt.

Synergistic anti-carcinogenic effect of interferon-ß with cisplatin on human breast adenocarcinoma MDA MB231 cells.

Cisplatin is one of the most commonly used chemotherapeutic agents for breast cancer treatment. However, its efficacy is greatly limited by its toxic side effects. The present study investigated the synergistic effect of interferon ß with cisplatin on MDA MB231 cells. The antiproliferative effect was measured by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) was calculated using the method of Chou and Talalay. Cytotoxicity was determined by trypan blue and clonogenic assay. Genotoxic and cytostatic effects were studied using micronucleus assay and nuclear division index (NDI). Protein expression was analyzed using immunoblotting. Interferon ß (100-2500 IU/mL) and Cisplatin (0.01-100 µM) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 1500 IU/mL and 20 µM for interferon ß and cisplatin, respectively. Western blot analysis revealed expression of interferon ß binding receptor in MDA MB231 cells. More interestingly, synergistic, cytotoxic and genotoxic effects were observed after treatment with a combination of interferon ß with reduced dosage of cisplatin. Decreased expression of Bcl-2 and increased expression of Bax stimulated the cytochrome c release, which triggers caspase-9 and -3 activation significantly increased in the combinational group. In conclusion the combination of interferon ß with reduced dose of cisplatin results synergistically improved growth-inhibition and apoptosis-inducing effect on MDA MB231 cells.

Stimulative effect of ginsenosides Rg5:Rk1 on murine osteoblastic MC3T3-E1 cells.

Panax ginseng C.A. Meyer (P. ginseng), hereafter referred to as P. ginseng, is known to exert a wide range of pharmacological effects both in vitro and in vivo; however, few studies have investigated the effects of ginseng on bone metabolism. We therefore investigated the potential antiosteoporotic properties of ginseng on the growth and differentiation of murine MC3T3-E1 cells. Rg5:Rk1 is a mixture of protopanaxadiol-type ginsenosides, isolated from fresh P. ginseng root, via a repetitive steaming and drying process. In this study, we examined the stimulatory effects of Rg5:Rk1 on the differentiation and mineralization of MC3T3-E1 cells. Undifferentiated cells were treated with a range of concentrations of Rg5:Rk1 (1-50 µg/mL), and cell viability was measured with the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Treatment with Rg5:Rk1 significantly increased cell viability in a dose-dependent manner. To investigate the possible mechanisms by which Rg5:Rk1 affects the early differentiation phase of MC3T3-E1 cells, the cells were treated with Rg5:Rk1 for 14-24 days before assessing the levels of multiple osteoblastic markers. The markers examined included alkaline phosphatase (ALP) activity type I collagen content (Coll-I), calcium deposition (by Alizarin Red S staining), extracellular mRNA expression of bone morphogenetic protein-2 (BMP-2), and the level of Runt-related transcription factor 2 (Runx2). Rg5:Rk1 treatment also increased the activities of proteins associated with osteoblast growth and differentiation in a dose-dependent manner. Overall, we found that the Rg5:Rk1 mixture of ginsenosides improved the osteoblastic function of MC3T3-E1 cells by increasing their proliferative capacity. This improvement is due to the action of Rg5:Rk1 on BMP-2, which is mediated by Runx2-dependent pathways.