Hinokitiol Inhibits Breast Cancer Cells In Vitro Stemness-Progression and Self-Renewal with Apoptosis and Autophagy Modulation via the CD44/Nanog/SOX2/Oct4 Pathway.

Breast cancer (BC) represents one of the most prevalent malignant threats to women globally. Tumor relapse or metastasis is facilitated by BC stemness progression, contributing to tumorigenicity. Therefore, comprehending the characteristics of stemness progression and the underlying molecular mechanisms is pivotal for BC advancement. Hinokitiol (ß-thujaplicin), a tropolone-related compound abundant in the heartwood of cupressaceous plants, exhibits antimicrobial activity. In our study, we employed three BC cell lines (MDA-MB-231, MCF-7, and T47D) to assess the expression of stemness-, apoptosis-, and autophagy-related proteins. Hinokitiol significantly reduced the viability of cancer cells in a dose-dependent manner. Furthermore, we observed that hinokitiol enhances apoptosis by increasing the levels of cleaved poly-ADP-ribose polymerase (PARP) and phospho-p53. It also induces dysfunction in autophagy through the upregulation of LC3B and p62 protein expression. Additionally, hinokitiol significantly suppressed the number and diameter of cancer cell line spheres by reducing the expression of cluster of differentiation44 (CD44) and key transcription factors. These findings underscore hinokitiol's potential as a therapeutic agent for breast cancer, particularly as a stemness-progression inhibitor. Further research and clinical studies are warranted to explore the full therapeutic potential of hinokitiol in the treatment of breast cancer.

Quantitative Proteomic Analysis on the Slightly Acidic Electrolyzed Water Triggered Viable but Non-Culturable Listeria monocytogenes.

This study undertakes a comprehensive exploration of the impact of slightly acidic electrolyzed water (SAEW) on Listeria monocytogenes, a common foodborne pathogen, with a particular focus on understanding the molecular mechanisms leading to the viable but nonculturable (VBNC) state. Given the widespread application of SAEW as an effective disinfectant in the food industry, uncovering these molecular pathways is crucial for improving food safety measures. We employed tandem mass tags (TMT), labeling proteomic techniques and LC-MS/MS to identify differentially expressed proteins under two doses of SAEW conditions. We indicated 203 differential expressed proteins (DEPs), including 78 up-regulated and 125 down-regulated DEPs. The functional enrichment analysis of these proteins indicated that ribosomes, biosynthesis of secondary metabolites, and aminoacyl-tRNA biosynthesis were enriched functions affected by SAEW. Further, we delved into the role of protein chlorination, a potential consequence of reactive chlorine species generated during the SAEW production process, by identifying 31 chlorinated peptides from 22 proteins, with a dominant sequence motif of Rxxxxx[cY] and functionally enriched in translation. Our findings suggest that SAEW might prompt alterations in the protein translation process and trigger compensatory ribosome biosynthesis. However, an imbalance in the levels of elongation factors and AARSs could hinder recovery, leading to the VBNC state. This research carries substantial implications for food safety and sanitation, as it adds to our understanding of the SAEW-induced VBNC state in L. monocytogenes and offers potential strategies for its control.

Quantitative Proteome Analysis Reveals Melissa officinalis Extract Targets Mitochondrial Respiration in Colon Cancer Cells.

Melissa officinalis (MO), known as lemon balm, is a popular ingredient blended in herbal tea. In recent decades, the bioactivities of MO have been studied in sub-health and pathological status, highlighting MO possesses multiple pharmacological effects. We previously showed that hot water MO extract exhibited anticancer activity in colorectal cancer (CRC). However, the detailed mechanisms underlying MO-induced cell death remain elusive. To elucidate the anticancer regulation of MO extract in colon cancer, a data-driven analysis by proteomics approaches and bioinformatics analysis was applied. An isobaric tandem mass tags-based quantitative proteome analysis using liquid chromatography-coupled tandem mass spectrometry was performed to acquire proteome-wide expression data. The over-representation analysis and functional class scoring method were implemented to interpret the MO-induced biological regulations. In total, 3465 quantifiable proteoforms were identified from 24,348 peptides, with 67 upregulated and 54 downregulated proteins in the MO-treated group. Mechanistically, MO impeded mitochondrial respiratory electron transport by triggering a reactive oxygen species (ROS)-mediated oxidative stress response. MO hindered the mitochondrial membrane potential by reducing the protein expression in the electron transport chain, specifically the complex I and II, which could be restored by ROS scavenger. The findings comprehensively elucidate how MO hot water extract activates antitumor effects in colorectal cancer (CRC) cells.

Adlay Seed (Coix lacryma-jobi L. var. Ma-yuen Stapf.) Ethanolic Extract Fractions and Subfractions Induce Cell Cycle Arrest and Apoptosis in Human Breast and Cervical Cancer Cell Lines.

The antitumor effects of Coix lacryma-jobi L. var. ma-yuen Stapf. (adlay seed) ethanolic extract have been increasingly shown. This study aimed to investigate the beneficial effects of both the fractions and subfractions of adlay seed ethanolic extract on the human breast (MCF-7) and cervical (HeLa) cancer cell lines, as well as exploring their possible mechanisms of action. The ethanolic extracts were obtained from different parts of adlay seed, including AHE (adlay hull extract), ATE (adlay testa extract), ABE (adlay bran extract) and PAE (polished adlay extract). The results of a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) assay showed that AHE-Ea and ATE-Ea showed significant growth inhibitory effects in a dose-dependent manner. The results also showed that the AHE-Ea-K, AHE-Ea-L, ATE-Ea-E and ATE-Ea-F subfractions inhibited cell proliferation, induced cell cycle arrest in the G0/G1 phase and decreased CDK4/Cyclin D1 protein expression. Finally, the extract activated caspase-3 activity and PARP protein expression, which induced MCF-7 and HeLa cell apoptosis. We then used liquid chromatography-mass spectrometry (LC/MS) to identify the potential active components., Quercetin showed an anticancer capacity. In conclusion, the AHE-Ea-K, AHE-Ea-L, ATE-Ea-E and ATE-Ea-F subfractions showed antitumor effects through the inhibition of MCF-7 and HeLa cell line viability, as well as inducing apoptosis and cell cycle arrest.

Protective Effects of Fucoxanthin on Hydrogen Peroxide-Induced Calcification of Heart Valve Interstitial Cells.

Cardiovascular diseases such as atherosclerosis and aortic valve sclerosis involve inflammatory reactions triggered by various stimuli, causing increased oxidative stress. This increased oxidative stress causes damage to the heart cells, with subsequent cell apoptosis or calcification. Currently, heart valve damage or heart valve diseases are treated by drugs or surgery. Natural antioxidant products are being investigated in related research, such as fucoxanthin (Fx), which is a marine carotenoid extracted from seaweed, with strong antioxidant, anti-inflammatory, and anti-tumor properties. This study aimed to explore the protective effect of Fx on heart valves under high oxidative stress, as well as the underlying mechanism of action. Rat heart valve interstitial cells under H2O2-induced oxidative stress were treated with Fx. Fx improved cell survival and reduced oxidative stress-induced DNA damage, which was assessed by cell viability analysis and staining with propidium iodide. Alizarin Red-S analysis indicated that Fx has a protective effect against calcification. Furthermore, Western blotting revealed that Fx abrogates oxidative stress-induced apoptosis via reducing the expression of apoptosis-related proteins as well as modulate Akt/ERK-related protein expression. Notably, in vivo experiments using 26 dogs treated with 60 mg/kg of Fx in combination with medical treatment for 0.5 to 2 years showed significant recovery in their echocardiographic parameters. Collectively, these in vitro and in vivo results highlight the potential of Fx to protect heart valve cells from high oxidative stress-induced damage.

Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE).

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).

Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation.

The circadian nature of melatonin has a protective effect on the progression of female reproductive cancers, including breast and ovarian cancers. However, the effect of melatonin on the growth of uterine leiomyoma is still unclear. In this study, we found that the growth of uterine leiomyoma ELT3 cells was reduced by treatment with melatonin. Treatment with melatonin increased the distribution of sub-G1 phase and increased DNA condensation in ELT3 cells. Melatonin-induced apoptosis and autophagy cell death progression were observed in ELT3 cells. Melatonin exerts a highly selective effect on primary normal human uterine smooth muscle (UtSMC) cells. The UtSMC cell cycle was arrested by melatonin treatment through up-regulation of p21, p27, and PTEN protein expression, but melatonin did not further promote apoptosis program activation. Melatonin reduced cell proliferation in ELT3 cells underlying the activation of melatonin MT1 and MT2 receptors, which in turn down-regulated the Akt-ERK1/2-NFκB signaling pathway. Melatonin reduced ELT3 tumor growth in both xenograft and orthotopic uterine tumor mice models. The extracellular matrix of the tumor was also reduced by melatonin treatment. Taken together, these results suggest that melatonin potentially plays a role in suppression of uterine leiomyoma growth.

Identification of the Effects of Aspirin and Sulindac Sulfide on the Inhibition of HMGA2-Mediated Oncogenic Capacities in Colorectal Cancer.

Distant metastatic colorectal cancer (CRC) is present in approximately 25% of patients at initial diagnosis, and eventually half of CRC patients will develop metastatic disease. The 5-year survival rate for patients with metastatic CRC is a mere 12.5%; thus, there is an urgent need to investigate the molecular mechanisms of cancer progression in CRC. High expression of human high-mobility group A2 (HMGA2) is related to tumor progression, a poor prognosis, and a poor response to therapy for CRC. Therefore, HMGA2 is an attractive target for cancer therapy. In this study, we identified aspirin and sulindac sulfide as novel potential inhibitors of HMGA2 using a genome-wide mRNA signature-based approach. In addition, aspirin and sulindac sulfide induced cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration. Moreover, a gene set enrichment analysis (GSEA) revealed that gene sets related to inflammation were positively correlated with HMGA2 and that the main molecular function of these genes was categorized as a G-protein-coupled receptor (GPCR) activity event. Collectively, this is the first study to report that aspirin and sulindac sulfide are novel potential inhibitors of HMGA2, which can induce cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration through influencing inflammatory-response genes, the majority of which are involved in GPCR signaling.

Roles of Fc Domain and Exudation in L2 Antibody-Mediated Protection against Human Papillomavirus.

To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab')2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis.IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

The Expression Profile and Prognostic Significance of Metallothionein Genes in Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease resulting from the combined influence of many genetic factors. This complexity has caused the molecular characterization of CRC to remain uncharacterized, with a lack of clear gene markers associated with CRC and the prognosis of this disease. Thus, highly sensitive tumor markers for the detection of CRC are the most essential determinants of survival. In this study, we examined the simultaneous downregulation of the mRNA levels of six metallothionein (MT) genes in CRC cell lines and public CRC datasets for the first time. In addition, we detected downregulation of these six MT mRNAs' levels in 30 pairs of tumor (T) and adjacent non-tumor (N) CRC specimens. In order to understand the potential prognostic relevance of these six MT genes and CRC, we presented a four-gene signature to evaluate the prognosis of CRC patients. Further discovery suggested that the four-gene signature (MT1F, MT1G, MT1L, and MT1X) predicted survival better than any combination of two-, three-, four-, five-, or six-gene models. In conclusion, this study is the first to report that simultaneous downregulation of six MT mRNAs' levels in CRC patients, and their aberrant expression together, accurately predicted CRC patients' outcomes.

Fucoidan Inhibits the Proliferation of Leiomyoma Cells and Decreases Extracellular Matrix-Associated Protein Expression.

BACKGROUND/AIMS: Uterine leiomyomas (ULs) are benign uterine tumors, and the most notable pathophysiologic feature of ULs is excessive accumulation of extracellular matrix (ECM). Fucoidan is a polysaccharide extracted from brown seaweeds that has a wide range of pharmacological properties, including anti-fibrotic effects. We aimed to study the effect of fucoidan on the growth of ULs activated by transforming growth factor beta (TGFß). METHODS: We used ELT-3 (Eker rat leiomyoma tumor-derived cells) and HUtSMC (human uterine smooth muscle cells) as in vitro models. Cell viability was determined by the MTT assay. Cell colony formation was stained using crystal violet. The side population, cell cycle and apoptosis were analyzed using flow cytometry. Protein expression was assayed by western blot analysis. We also conducted in vivo experiments to confirm the inhibitory effects of fucoidan in nude mouse xenograft models. Tumor tissues were assayed by immunohistochemistry analysis. RESULTS: In our study, fucoidan caused a 50% growth inhibition using a dose of 0.5 mg/ml and decreased the stem cell activity after 48 h. In addition, fucoidan induced sub-G1 cell cycle arrest and apoptosis. Fucoidan down-regulated fibronectin, vimentin, α-SMA and the COL1A1 protein levels in TGFß3-induced ELT-3 cells. In the cellular mechanism, fucoidan abrogated TGFß3-induced levels of p-Smad2 and p-ERK1/2, as well as ß-catenin translocation into the nucleus. Furthermore, fucoidan suppressed xenograft tumor growth in vivo. CONCLUSION: Fucoidan displays anti-proliferation and anti-fibrotic effects and exerts protective effects against ULs development.

Spontaneous and Vaccine-Induced Clearance of Mus Musculus Papillomavirus 1 Infection.

Mus musculus papillomavirus 1 (MmuPV1/MusPV1) induces persistent papillomas in immunodeficient mice but not in common laboratory strains. To facilitate the study of immune control, we sought an outbred and immunocompetent laboratory mouse strain in which persistent papillomas could be established. We found that challenge of SKH1 mice (Crl:SKH1-Hrhr) with MmuPV1 by scarification on their tail resulted in three clinical outcomes: (i) persistent (>2-month) papillomas (∼20%); (ii) transient papillomas that spontaneously regress, typically within 2 months (∼15%); and (iii) no visible papillomas and viral clearance (∼65%). SKH1 mice with persistent papillomas were treated by using a candidate preventive/therapeutic naked-DNA vaccine that expresses human calreticulin (hCRT) fused in frame to MmuPV1 E6 (mE6) and mE7 early proteins and residues 11 to 200 of the late protein L2 (hCRTmE6/mE7/mL2). Three intramuscular DNA vaccinations were delivered biweekly via in vivo electroporation, and both humoral and CD8 T cell responses were mapped and measured. Previously persistent papillomas disappeared within 2 months after the final vaccination. Coincident virologic clearance was confirmed by in situ hybridization and a failure of disease to recur after CD3 T cell depletion. Vaccination induced strong mE6 and mE7 CD8+ T cell responses in all mice, although they were significantly weaker in mice that initially presented with persistent warts than in those that spontaneously cleared their infection. A human papillomavirus 16 (HPV16)-targeted version of the DNA vaccine also induced L2 antibodies and protected mice from vaginal challenge with an HPV16 pseudovirus. Thus, MmuPV1 challenge of SKH1 mice is a promising model of spontaneous and immunotherapy-directed clearances of HPV-related disease.IMPORTANCE High-risk-type human papillomaviruses (hrHPVs) cause 5% of all cancer cases worldwide, notably cervical, anogenital, and oropharyngeal cancers. Since preventative HPV vaccines have not been widely used in many countries and do not impact existing infections, there is considerable interest in the development of therapeutic vaccines to address existing disease and infections. The strict tropism of HPV requires the use of animal papillomavirus models for therapeutic vaccine development. However, MmuPV1 failed to grow in common laboratory strains of mice with an intact immune system. We show that MmuPV1 challenge of the outbred immunocompetent SKH1 strain produces both transient and persistent papillomas and that vaccination of the mice with a DNA expressing an MmuPV1 E6E7L2 fusion with calreticulin can rapidly clear persistent papillomas. Furthermore, an HPV16-targeted version of the DNA can protect against vaginal challenge with HPV16, suggesting the promise of this approach to both prevent and treat papillomavirus-related disease.

Manzamine A Exerts Anticancer Activity against Human Colorectal Cancer Cells.

Marine sponges are known to produce numerous bioactive secondary metabolites as defense strategies to avoid predation. Manzamine A is a sponge-derived ß-carboline-fused pentacyclic alkaloid with various bioactivities, including recently reported anticancer activity on pancreatic cancer. However, its cytotoxicity and mode of action against other tumors remain unclear. In this study, we exhibit that manzamine A reduced cell proliferation in several colorectal cancer (CRC) cell lines. To further investigate the manzamine A triggered molecular regulation, we analyzed the gene expression with microarray and revealed that pathways including cell cycle, DNA repair, mRNA metabolism, and apoptosis were dysregulated. We verified that manzamine A induced cell cycle arrest at G0/G1 phase via inhibition of cyclin-dependent kinases by p53/p21/p27 and triggered a caspase-dependent apoptotic cell death through mitochondrial membrane potential depletion. Additionally, we performed bioinformatics analysis and demonstrated that manzamine A abolished epithelial⁻mesenchymal transition process. Several mesenchymal transcriptional factors, such as Snail, Slug, and Twist were suppressed and epithelial marker E-cadherin was induced simultaneously in HCT116 cells by manzamine A, leading to the epithelial-like phenotype and suppression of migration. These findings suggest that manzamine A may serve as a starting point for the development of an anticancer drug for the treatment of metastatic CRC.

Isoliquiritigenin Induces Autophagy and Inhibits Ovarian Cancer Cell Growth.

Ovarian cancer is one of the commonest gynecologic malignancies, which has a poor prognosis for patients at the advanced stage. Isoliquiritigenin (ISL), an active flavonoid component of the licorice plant, previously demonstrated antioxidant, anti-inflammatory, and tumor suppressive effects. In this study, we investigated the antitumor effect of ISL on human ovarian cancer in vitro using the human ovarian cancer cell lines, OVCAR5 and ES-2, as model systems. Our results show that ISL significantly inhibited the viability of cancer cells in a concentration- and time-dependent manner. Flow cytometry analysis indicated that ISL induced G2/M phase arrest. Furthermore, the expression of cleaved PARP, cleaved caspase-3, Bax/Bcl-2 ratio, LC3B-II, and Beclin-1 levels were increased in western blot analysis. To clarify the role of autophagy and apoptosis in the effect of ISL, we used the autophagy inhibitor-3-methyladenine (3-MA) to attenuate the punctate fluorescence staining pattern of the p62/sequestosome 1 (SQSTM1, red fluorescence) and LC3 (green fluorescence) proteins after ISL treatment, and 3-MA inhibited the cytotoxicity of ISL. These findings provide new information about the link between ISL-induced autophagy and apoptosis and suggest that ISL is a candidate agent for the treatment of human ovarian cancer.

Quantitative proteomics reveals middle infrared radiation-interfered networks in breast cancer cells.

Breast cancer is one of the leading cancer-related causes of death worldwide. Treatment of triple-negative breast cancer (TNBC) is complex and challenging, especially when metastasis has developed. In this study, we applied infrared radiation as an alternative approach for the treatment of TNBC. We used middle infrared (MIR) with a wavelength range of 3-5 µm to irradiate breast cancer cells. MIR significantly inhibited cell proliferation in several breast cancer cells but did not affect the growth of normal breast epithelial cells. We performed iTRAQ-coupled LC-MS/MS analysis to investigate the MIR-triggered molecular mechanisms in breast cancer cells. A total of 1749 proteins were identified, quantified, and subjected to functional enrichment analysis. From the constructed functionally enriched network, we confirmed that MIR caused G2/M cell cycle arrest, remodeled the microtubule network to an astral pole arrangement, altered the actin filament formation and focal adhesion molecule localization, and reduced cell migration activity and invasion ability. Our results reveal the coordinative effects of MIR-regulated physiological responses in concentrated networks, demonstrating the potential implementation of infrared radiation in breast cancer therapy.

Proteomic analysis reveals that pardaxin triggers apoptotic signaling pathways in human cervical carcinoma HeLa cells: cross talk among the UPR, c-Jun and ROS.

Pardaxin, an antimicrobial peptide secreted by the Red Sea flatfish Pardachirus marmoratus, inhibits proliferation and induces apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are only partially understood at present. In this study, we used proteomic approaches and network reconstruction to clarify the mechanism of pardaxin-induced apoptosis in human cervical carcinoma HeLa cells. We identified that pardaxin-regulated proteins predominantly function in the unfolded protein response, oxidative stress and cytoskeletal distribution. Molecular examination of signal transduction and cellular localization demonstrated that the activator protein-1 transcription factor was activated, which eventually caused apoptosis via both caspase- and apoptosis-inducing factor-dependent pathways. Scavenging of reactive oxygen species (ROS) alleviated c-Jun activation, and small interfering RNA knockdown of c-Jun abrogated pardaxin-induced caspase activation and cell death, thereby implicating ROS and c-Jun in pardaxin-induced apoptosis signaling. In summary, this study provides the first protein-interacting network maps and novel insights into the biological responses and potential toxicity of pardaxin.

Proteomic and functional analysis of zebrafish after administration of antimicrobial peptide epinecidin-1.

Antimicrobial peptides (AMPs) play important roles in innate immunity. One such AMP, epinecidin-1, exhibits antibacterial effects in zebrafish. In the current study, we aimed to identify the antimicrobial-associated proteins affected by epinecidin-1 treatment, and to unravel the underlying antimicrobial molecular mechanisms of epinecidin-1. We analyzed proteome changes in epinecidin-1-treated zebrafish using two-dimensional electrophoresis (2DE) coupled to mass spectrometry. Several differentially expressed proteins were identified, some of which were validated by real-time quantitative RT-PCR. The differentially expressed proteins were mapped onto Ingenuity Pathway Analysis canonical pathways, to construct a possible protein-protein interacting network regulated by epinecidin-1; this network suggested a potential role of epinecindin-1 in cytoskeletal assembly and organization. Our findings imply that epinecidin-1 may stabilize the cytoskeleton network in host cells, thereby promoting resistance to bacterial infection.

Oxidative stress mediates the inhibitory effects of Manzamine A on uterine leiomyoma cell proliferation and extracellular matrix deposition via SOAT inhibition.

Uterine fibroids, the most common benign tumors of the myometrium in women, are characterized by abnormal extracellular matrix deposition and uterine smooth muscle cell neoplasia, with high recurrence rates. Here, we investigated the potential of the marine natural product manzamine A (Manz A), which has potent anti-cancer effects, as a treatment for uterine fibroids. Manz A inhibited leiomyoma cell proliferation in vitro and in vivo by arresting cell cycle progression and inducing caspase-mediated apoptosis. We performed target prediction analysis and identified sterol o-acyltransferases (SOATs) as potential targets of Manz A. Cholesterol esterification and lipid droplet formation were reduced by Manz A, in line with reduced SOAT expression. As a downstream target of SOAT, Manz A also prevented extracellular matrix deposition by inhibiting the ß-catenin/fibronectin/metalloproteinases axis and enhanced autophagy turnover. Excessive free fatty acid accumulation by SOAT inhibition led to reactive oxygen species to impair mitochondrial oxidative phosphorylation and trigger endoplasmic reticulum stress via PERK/eIF2α/CHOP signaling. The inhibitory effect of ManzA on cell proliferation was partially restored by PERK knockdown and eliminated by tauroursodeoxycholic acid, suggesting oxidative stress plays a critical role in the mechanism of action of Manz A. These findings suggest that targeting SOATs by Manz A may be a promising therapeutic approach for uterine fibroids.

Capsaicin alleviates cisplatin-induced muscle loss and atrophy in vitro and in vivo.

BACKGROUND: Cisplatin (CP) is a widely used chemotherapeutic drug with subsequent adverse effects on different organs and tissues including skeletal muscle loss and atrophy as the most common clinical symptoms. The molecular mechanism of cisplatin-induced muscle atrophy is not clearly understood. However, recent significant advances indicate that it is related to an imbalance in both the protein status and apoptosis. Capsaicin (CAP) is one of the major ingredients in chilli peppers. It is a valuable pharmacological agent with several therapeutic applications in controlling pain and inflammation with particular therapeutic potential in muscle atrophy. However, the mechanisms underlying its protective effects against cisplatin-induced muscle loss and atrophy remain largely unknown. This study aims to investigate capsaicin's beneficial effects on cisplatin-induced muscle loss and atrophy in vitro and in vivo. METHODS: The anti-muscle-atrophic effect of capsaicin on cisplatin-induced muscle loss was investigated using in vivo and in vitro studies. By using the pretreatment model, pretreated capsaicin for 24 h and treated with cisplatin for 48 h, we utilized a C2 C12 myotube formation model where cell viability analysis, immunofluorescence, and protein expression were measured to investigate the effect of capsaicin in hampering cisplatin-induced muscle atrophy. C57BL/6 mice were administered capsaicin (10, 40 mg/kg BW) as a pretreatment for 5 weeks and cisplatin (3 mg/kg BW) for seven consecutively days to assess muscle atrophy in an animal model for protein and oxidative stress examination, and the grip strength was tested to evaluate the muscle strength. RESULTS: Our study results indicated that cisplatin caused lower cell viability and showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in the myotube diameter, repression of Akt, and mTOR protein expression. However, pretreatment with capsaicin could ameliorate cisplatin-induced muscle atrophy by up-regulating the protein synthesis in skeletal muscle as well as down-regulating the markers of protein degradation. Additionally, capsaicin was able to downregulate the protein expression of apoptosis-related markers, activated TRPV1 and autophagy progress modulation and the recovery of lysosome function. In vivo, capsaicin could relieve oxidative stress and cytokine secretion while modulating autophagy-related lysosome fusion, improving grip strength, and alleviating cisplatin-induced body weight loss and gastrocnemius atrophy. CONCLUSIONS: These findings suggest that capsaicin can restore cisplatin-induced imbalance between protein synthesis and protein degradation pathways and it may have protective effects against cisplatin-induced muscle atrophy.

Effect of Vitamin D Supplementation on Primary Dysmenorrhea: A Systematic Review and Meta-Analysis of Randomized Clinical Trials.

Dysmenorrhea causes pain and inconvenience during menstruation. In addition to medication, natural compounds are widely used to relieve various types of pain. In this study, we aimed to assess the effects of vitamin D (vit. D) supplementation in relieving the symptoms of primary dysmenorrhea. A comprehensive systematic database search of randomized controlled trials (RCTs) was performed. Oral forms of vit. D supplementation were included and compared with a placebo or standard care. The degree of dysmenorrhea pain was measured with a visual analogue scale or numerical rating scale. Outcomes were compared using the standardized mean difference (SMD) and 95% confidence intervals (CIs) in a meta-analysis. RCTs were assessed using the Cochrane risk-of-bias v2 (RoB 2) tool. The meta-analysis included 8 randomized controlled trials involving 695 participants. The results of the quantitative analysis showed a significantly lower degree of pain in the vit. D versus placebo in those with dysmenorrhea (SMD: -1.404, 95% CI: -2.078 to -0.731). The results of subgroup analysis revealed that pain lessened when the average weekly dose of vit. D was over 50,000 IU, in which dysmenorrhea was relieved regardless of whether vit. D was administered for more or less than 70 days and in any dose interval. The results revealed that vit. D treatment substantially reduced the pain level in the primary dysmenorrhea population. We concluded that vit. D supplementation is an alternative treatment for relieving the pain symptoms of dysmenorrhea.