Tandem Mass Tag-10plex (TMT-10plex) phosphoproteomics dataset for comprehensive analysis of active compounds with tyrosine kinase inhibition activity.

Bioactive compounds derived from natural products demonstrate a wide range of beneficial properties in cancer treatment. One popular approach to inhibiting cancer cell growth is by stimulating apoptosis. Interestingly, our research has discovered that traditional mushroom and isolated compounds from traditional herbs can induce apoptosis in A549 cells while inhibiting tyrosine kinase activities. We have identified two extracts from traditional mushrooms, Phallus indusiatus and Fomes rimosus (Berk.) Cooke, which exhibit promising abilities to activate apoptotic events in cells. Additionally, isolated compounds such as Chamuangone, Cannabigerol (CBG), Cannabidiol (CBD), and NP1-cyclic peptide have also demonstrated significant apoptotic activation capabilities. To further our understanding, we analyzed phosphoprotein changes in A549 cells exposed to these extracts and compounds, both with and without epidermal growth factor (EGF) stimulation. Our positive controls were two known drugs, Afatinib and Osimertinib, which are tyrosine kinase inhibitors with apoptotic stimulation abilities. In order to enrich our understanding of the kinase pathway, we conducted phosphoprotein enrichment analysis and identified altered phosphoproteins using LC-MS/MS. Across these testing conditions, we found that 1228 phosphoproteins were altered, providing valuable insights into the biochemical mechanisms underlying cell apoptosis in A549 cells through post-translational modifications of proteins. Furthermore, our findings not only shed light on the mechanisms of cell apoptosis in A549 cells but also offer promising avenues for future research and therapeutic development.

Sansevieria trifasciata's specific metabolite improves tolerance and efficiency for particulate matter and volatile organic compound removal.

Phytoremediation has become famous for removing particulate matter (PM) and volatile organic compounds (VOCs), but the ability is affected by plant health. Lately, the priming technique was a simple approach to studying improving plant tolerance against abiotic stress by specific metabolites that accumulated, known as "memory", but the mechanism underlying this mechanism and how long this "memory" was retained in the plant was a lack of study. Sansevieria trifasciata was primed for one week for PM and VOC stress to improve plant efficiency on PM and VOC. After that, the plant was recovered for two- or five-weeks, then re-exposed to the same stress with similar PM and VOC concentrations from cigarette smoke. Primed S. trifasciata showed improved removal of PMs entirely within 2 h and VOC within 24 h. The primed plant can maintain a malondialdehyde (MDA) level and retain the "memory" for two weeks. Metabolomics analysis showed that an ornithine-related compound was accumulated as a responsive metabolite under exposure to PM and VOC stress. Exogenous ornithine can maintain plant efficiency and prevent stress by increasing proline and antioxidant enzymes. This study is the first to demonstrate plant "memory" mechanisms under PM and VOC stress.

Exploring the apoptotic effects of sericin on HCT116 cells through comprehensive nanostring transcriptomics and proteomics analysis.

Sericin, a silk protein from Bombyx mori (silkworms), has many applications, including cosmetics, anti-inflammation, and anti-cancer. Sericin complexes with nanoparticles have shown promise for breast cancer cell lines. Apoptosis, a programmed cell death mechanism, stops cancer cell growth. This study found that Sericin urea extract significantly affected HCT116 cell viability (IC50 = 42.00 ± 0.002 µg/mL) and caused apoptosis in over 80% of treated cells. S-FTIR analysis showed significant changes in Sericin-treated cells' macromolecule composition, particularly in the lipid and nucleic acid areas, indicating major cellular modifications. A transcriptomics study found upregulation of the apoptotic signaling genes FASLG, TNFSF10, CASP3, CASP7, CASP8, and CASP10. Early apoptotic proteins also showed that BAD, AKT, CASP9, p53, and CASP8 were significantly upregulated. A proteomics study illuminated Sericin-treated cells' altered protein patterns. Our results show that Sericin activated the extrinsic apoptosis pathway via the caspase cascade (CASP8/10 and CASP3/7) and the death receptor pathway, involving TNFSF10 or FASLG, in HCT116 cells. Upregulation of p53 increases CASP8, which activates CASP3 and causes HCT116 cell death. This multi-omics study illuminates the molecular mechanisms of Sericin-induced apoptosis, sheds light on its potential cancer treatment applications, and helps us understand the complex relationship between silk-derived proteins and cellular processes.

Anti-methicillin-resistant Staphylococcus aureus and antibiofilm activity of new peptides produced by a Brevibacillus strain.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a highly prioritized pathogen by the World Health Organization (WHO) to search for effective antimicrobial agents. Previously, we isolated a soil Brevibacillus sp. strain SPR19 from a botanical garden, which showed anti-MRSA activity. However, the active substances were still unknown. Methods: The cell-free supernatant of this bacterium was subjected to salt precipitation, cation exchange, and reversed-phase chromatography. The antimicrobial activity of pure substances was determined by broth microdilution assay. The peptide sequences and secondary structures were characterized by tandem mass spectroscopy and circular dichroism (CD), respectively. The most active anti-MRSA peptide underwent a stability study, and its mechanism was determined through scanning electron microscopy, cell permeability assay, time-killing kinetics, and biofilm inhibition and eradication. Hemolysis was used to evaluate the peptide toxicity. Results: The pure substances (BrSPR19-P1 to BrSPR19-P5) were identified as new peptides. Their minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus and MRSA isolates ranged from 2.00 to 32.00 and 2.00 to 64.00 µg/mL, respectively. The sequence analysis of anti-MRSA peptides revealed a length ranging from 12 to 16 residues accompanied by an amphipathic structure. The physicochemical properties of peptides were predicted such as pI (4.25 to 10.18), net charge at pH 7.4 (-3 to +4), and hydrophobicity (0.12 to 0.96). The CD spectra revealed that all peptides in the water mainly contained random coil structures. The increased proportion of α-helix structure was observed in P2-P5 when incubated with SDS. P2 (NH2-MFLVVKVLKYVV-COOH) showed the highest antimicrobial activity and high stability under stressed conditions such as temperatures up to 100 °C, solution of pH 3 to 10, and proteolytic enzymes. P2 disrupted the cell membrane and caused bacteriolysis, in which its action was dependent on the incubation time and peptide concentration. Antibiofilm activity of P2 was determined by which the half-maximal inhibition of biofilm formation was observed at 2.92 and 4.84 µg/mL for S. aureus TISTR 517 and MRSA isolate 2468, respectively. Biofilm eradication of tested pathogens was found at the P2 concentration of 128 µg/mL. Furthermore, P2 hemolytic activity was less than 10% at concentrations up to 64 µg/mL, which reflected the hemolysis index thresholds of 32. Conclusion: Five novel anti-MRSA peptides were identified from SPR19. P2 was the most active peptide and was demonstrated to cause membrane disruption and cell lysis. The P2 activity was dependent on the peptide concentration and exposure time. This peptide had antibiofilm activity against tested pathogens and was compatible with human erythrocytes, supporting its potential use as an anti-MRSA agent in this post-antibiotic era.

Exploring the Apoptotic-Induced Biochemical Mechanism of Traditional Thai Herb (Kerra™) Extract in HCT116 Cells Using a Label-Free Proteomics Approach.

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.

Schomburginones A‒J, geranylated benzophenones from the leaves of Garcinia schomburgkiana and their cytotoxic and anti-inflammatory activities.

Ten undescribed benzophenones, schomburginones A-J, together with 14 known analogs were isolated from the leaves of Garcinia schomburgkiana, an edible plant native to the Indochina region. The structures of the undescribed compounds were elucidated by NMR combined with HRMS spectroscopy, while their absolute configurations were determined using ECD and single-crystal X-ray diffraction analysis. The isolated metabolites represent benzophenone derivatives containing a modified monoterpene unit, including tri- and tetracyclic skeletons, which are rarely found in genus Garcinia. The cytotoxic evaluation on three cancerous cell lines demonstrated that schomburginone G, schomburginone H, and 3-geranyl-2,4,6-trihydroxybenzophenone were active against HeLa cells with IC50 values in the range of 12.2-15.7 µM, respectively, and selective compared to the non-cancerous L929 cells (SI > 3.5). In addition, the three cytotoxic compounds together with clusiacyclol A showed significant NO inhibitory activity in RAW 264.7 macrophage cells over 85% inhibition without obvious cytotoxicity at a final concentration of 100 µM. The promising activities of these compounds in cytotoxic and anti-inflammatory assays make them attractive for further study in the development of anticancer drugs.

Purification and Characterization of Novel Anti-MRSA Peptides Produced by Brevibacillus sp. SPR-20.

Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high-priority pathogen because its infection is associated with a high mortality rate. It is urgent to search for new agents to treat such an infection. Our previous study isolated a soil bacterium (Brevibacillus sp. SPR-20), showing the highest antimicrobial activity against S. aureus TISTR 517 and MRSA strains. The present study aimed to purify and characterize anti-MRSA substances produced by SPR-20. The result showed that five active substances (P1-P5) were found, and they were identified by LC-MS/MS that provided the peptide sequences of 14-15 residues. Circular dichroism showed that all peptides contained ß-strand and disordered conformations as the major secondary structures. Only P1-P4 adopted more α-helix conformations when incubated with 50 mM SDS. These anti-MRSA peptides could inhibit S. aureus and MRSA in concentrations of 2-32 µg/mL. P1 (NH2-VVVNVLVKVLPPPVV-COOH) had the highest activity and was identified as a novel antimicrobial peptide (AMP). The stability study revealed that P1 was stable in response to temperature, proteolytic enzymes, surfactant, and pH. The electron micrograph showed that P1 induced bacterial membrane damage when treated at 1× MIC in the first hour of incubation. The killing kinetics of P1 was dependent on concentration and time. Mechanisms of P1 on tested pathogens involved membrane permeability, leakage of genetic material, and cell lysis. The P1 peptide at a concentration up to 32 µg/mL showed hemolysis of less than 10%, supporting its safety for human erythrocytes. This study provides promising anti-MRSA peptides that might be developed for effective antibiotics in the post-antibiotic era.

Differential plasma proteomes of the patients with Opisthorchiasis viverrini and cholangiocarcinoma identify a polymeric immunoglobulin receptor as a potential biomarker.

In Southeast Asian countries, nitrosamine compounds and the liver fluke Opisthorchis viverrini have long been identified as carcinogens for cholangiocarcinoma (CHCA). In order to effectively treat O. viverrini infections and prevent the development of CHCA, methods for disease detection are needed. This study aims to identify biomarkers for O. viverrini infection and CHCA. In the discovery phase, technical triplicates of five pooled plasma pools (10 plasma each) of healthy control subjects (noOVCCA), O. viverrini subjects (OV), and cholangiocarcinoma subjects (CCA), underwent solution-based digestion, with the label-free method, using a Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer and UltiMate 300 LC systems. The noOVCCA, OV, and CCA groups demonstrated different profiles and were clustered, as illustrated by PCA and heat map analysis. The STRING and reactome analysis showed that both OV and CCA groups up-regulated proteins targeting immune system-related proteins. Differential proteomic profiles, S100A9, and polymeric immunoglobulin receptor (PIGR) were specifically expressed in the CCA group. During the validation phase, another 50 plasma samples were validated via the PIGR sandwich ELISA. Using PIGR >1.559 ng/ml as a cut-off point, 78.00% sensitivity, 71.00% specificity, and AUC = 0.8216, were obtained. It is sufficient to differentially diagnose cholangiocarcinoma patients from healthy patients and those with Opisthorchiasis viverrini. Hence, in this study, PIGR was identified and validated as a potential biomarker for CHCA. Plasma PIGR is suggested for screening CHCA, especially in an endemic region of O. viverrini infection.

Evaluation of potential anti-metastatic and antioxidative abilities of natural peptides derived from Tecoma stans (L.) Juss. ex Kunth in A549 cells.

Background: Tecoma stans (L.) Juss. ex Kunth is a well-known medicinal plant found in tropical and subtropical regions. It contains a broad range of bioactive compounds that exhibit many biological effects, including antidiabetic, antibacterial, and antioxidative activities. However, the effect of natural peptides from T. stans against cancer progression and free radical production is unknown. This study aims to evaluate the cytotoxic, anti-metastatic, and antioxidative activities of natural peptides from T. stans on A549 cells. Methods: The natural peptides were extracted from the flower of T. stans using the pressurized hot water extraction (PHWE) method, followed by size exclusion chromatography and solid-phase extraction-C18. The cytotoxic and anti-metastatic effects of natural peptides were evaluated using MTT and transwell chamber assays, respectively. The free radical scavenging activity of natural peptides was determined using ABTS, DPPH, and FRAP assays. The cells were pretreated with the IC50 dosage of natural peptides and stimulated with LPS before analyzing intracellular reactive oxygen species (ROS) and proteomics. Results: Natural peptides induced cell toxicity at a concentration of less than 1 ng/ml and markedly reduced cell motility of A549 cells. The cells had a migration rate of less than 10% and lost their invasion ability in the treatment condition. In addition, natural peptides showed free radical scavenging activity similar to standard antioxidants and significantly decreased intracellular ROS in the LPS-induced cells. Proteomic analysis revealed 1,604 differentially expressed proteins. The self-organizing tree algorithm (SOTA) clustered the protein abundances into eleven groups. The volcano plot revealed that the cancer-promoting proteins (NCBP2, AMD, MER34, ENC1, and COA4) were down-regulated, while the secretory glycoprotein (A1BG) and ROS-reducing protein (ASB6) were up-regulated in the treatment group. Conclusion: The anti-proliferative and anti-metastatic activities of natural peptides may be attributed to the suppression of several cancer-promoting proteins. In contrast, their antioxidative activity may result from the up-regulation of ROS-reducing protein. This finding suggests that natural peptides from T. stans are viable for being the new potential anti-cancer and antioxidative agents.

In Silico Target Identification of Galangin, as an Herbal Flavonoid against Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies in the bile duct, which proliferates aggressively. CCA is highly prevalent in Northeastern Thailand wherein it is associated with liver fluke infection, or Opisthorchis viverrini (OV). Most patients are diagnosed in advanced stages, when the cancer has metastasized or severely progressed, thereby limiting treatment options. Several studies investigate the effect of traditional Thai medicinal plants that may be potential therapeutic options in combating CCA. Galangin is one such herbal flavonoid that has medicinal properties and exhibits anti-tumor properties in various cancers. In this study, we investigate the role of Galangin in inhibiting cell proliferation, invasion, and migration in OV-infected CCA cell lines. We discovered that Galangin reduced cell viability and colony formation by inducing apoptosis in CCA cell lines in a dose-dependent manner. Further, Galangin also effectively inhibited invasion and migration in OV-infected CCA cells by reduction of MMP2 and MMP9 enzymatic activity. Additionally, using proteomics, we identified proteins affected post-treatment with Galangin. Enrichment analysis revealed that several kinase pathways were affected by Galangin, and the signature corroborated with that of small molecule kinase inhibitors. Hence, we identified putative targets of Galangin using an in silico approach which highlighted c-Met as candidate target. Galangin effectively inhibited c-Met phosphorylation and subsequent signaling in in vitro CCA cells. In addition, Galangin was able to inhibit HGF, a mediator of c-Met signaling, by suppressing HGF-stimulated invasion, as well as migration and MMP9 activity. This shows that Galangin can be a useful anti-metastatic therapeutic strategy in a subtype of CCA patients.

Discovery of a Multifunctional Octapeptide from Lingzhi with Antioxidant and Tyrosinase Inhibitory Activity.

Ganoderma lucidum or Lingzhi is a fungus species widely known as a traditional medicine. Exploring the beneficial peptides by hydrolysis using pepsin and trypsin has been extensively performed to identify new bioactive natural products. A multifunctional peptide that expresses potential scavenging activity and tyrosinase inhibition is valuable in therapeutic and cosmetic applications. This study aimed to identify and investigate the effects of a novel multifunctional peptide from Lingzhi on the melanogenic enzymes in melanoma cells by a targeted-proteomics approach. The multifunctional peptide was de novo sequenced by LC-MS/MS to be NH2-PVRSSNCA-CO2H (octapeptide). This sequence was chemically synthesized by solid-phase peptide synthesis (SPPS). The antioxidant ability of the synthesized octapeptide was measured by the DPPH, ABTS, and FRAP assays. The results showed that the peptide exhibited an antioxidant activity equal to 0.121 ± 0.01 mg equivalent to ascorbic acid, 0.173 ± 0.03 mg equivalent to gallic acid, and 2.21 ± 0.23 mM equivalent to FeSO4, respectively, which is comparable to these well-known antioxidants. The proteomics approach identified a total of 5804 proteins and several pathways involved in the effects of the octapeptide in melanoma cells. Targeted proteomics revealed three specific proteins associated with pigmentation including Rab29, Dct, and Tyrp1. The Rab29 and Dct were upregulated whereas Tyrp1 was downregulated in the octapeptide treatment group. These findings could be used in the understanding of the molecular functions of the multifunctional octapeptide on melanogenic enzymes, supporting its potential as a therapeutic and cosmetic ingredient.

A novel nanobody as therapeutics target for EGFR-positive colorectal cancer therapy: exploring the effects of the nanobody on SW480 cells using proteomics approach.

BACKGROUND: The epidermal growth factor receptor (EGFR) overexpression is found in metastatic colorectal cancer (mCRC). Targeted molecular therapies such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKI) are becoming more precise, targeting specifically for cancer therapeutics. However, there are adverse effects of currently available anti-EGFR drugs, including drug-resistant and side effects. Nanobodies can overcome these limitations. Our previous study has found that cell-penetrable nanobodies targeted at EGFR-tyrosine kinase were significantly reduced EGFR-positive lung cancer cells viability and proliferation. The aim of the present study was to determine the effect of cell-penetrable nanobody (R9VH36) on cell viability and proteomic profile in EGFR-positive human colorectal cancer cell lines. METHODS: The human colorectal carcinoma cell line (SW480) was treated with R9VH36, compared with gefitinib. Cell viability was monitored using the MTT cell viability assay. The proteomic profiling was analyzed by LC-MS/MS . RESULTS: The half-maximal inhibitory concentration (IC50) values determined for R9VH36 and gefitinib against SW480 were 527 ± 0.03 nM and 13.31 ± 0.02 µM, respectively. Moreover, both the gefitinib-treated group and nanobody-treated group had completely different proteome profiles. A total 6626 differentially expressed proteins were identified. PCA analysis revealed different proteome profiling in R9VH36 experiment. There were 8 proteins in R9VH36 that significantly exhibited opposite expression directions when compared to gefitinib. These proteins are involved in DNA-damage checkpoint processes. CONCLUSION: The proteomics explored those 6,626 proteins had different expressions between R9VH36 and gefitinib. There were 8 proteins in R9VH36 exhibited opposite expression direction when comparing to gefitinib. Our findings suggest that R9VH36 has the potential to be an alternative remedy for treating EGFR-positive colon cancer.

Induction of apoptosis in human colorectal cancer cells by nanovesicles from fingerroot (Boesenbergia rotunda (L.) Mansf.).

Colorectal cancer is the leading cause of cancer-related deaths worldwide, warranting the urgent need for a new treatment option. Plant-derived nanovesicles containing bioactive compounds represent new therapeutic avenues due to their unique characteristics as natural nanocarriers for bioactive molecules with therapeutic effects. Recent evidence has revealed potential anticancer activity of bioactive compounds from Boesenbergia rotunda (L.) Mansf. (fingerroot). However, the effect and the underlying mechanisms of fingerroot-derived nanovesicles (FDNVs) against colorectal cancer are still unknown. We isolated the nanovesicles from fingerroot and demonstrated their anticancer activity against two colorectal cancer cell lines, HT-29 and HCT116. The IC50 values were 63.9 ± 2.4, 57.8 ± 4.1, 47.8 ± 7.6 µg/ml for HT-29 cells and 57.7 ± 6.6, 47.2 ± 5.2, 34 ± 2.9 µg/ml for HCT116 cells at 24, 48, and 72 h, respectively. Interestingly, FDNVs were not toxic to a normal colon epithelial cell line, CCD 841 CoN. FDNVs exhibited selective uptake by the colorectal cancer cell lines but not the normal colon epithelial cell line. Moreover, dose- and time-dependent FDNV-induced apoptosis was only observed in the colorectal cancer cell lines. In addition, reactive oxygen species levels were substantially increased in colorectal cancer cells, but total glutathione decreased after treatment with FDNVs. Our results show that FDNVs exhibited selective anticancer activity in colorectal cancer cell lines via the disruption of intracellular redox homeostasis and induction of apoptosis, suggesting the utility of FDNVs as a novel intervention for colorectal cancer patients.

A radiosensitizer, gallotannin-rich extract from Bouea macrophylla seeds, inhibits radiation-induced epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: Radioresistance can pose a significant obstacle to the effective treatment of breast cancers. Epithelial-mesenchymal transition (EMT) is a critical step in the acquisition of stem cell traits and radioresistance. Here, we investigated whether Maprang seed extract (MPSE), a gallotannin-rich extract of seed from Bouea macrophylla Griffith, could inhibit the radiation-induced EMT process and enhance the radiosensitivity of breast cancer cells. METHODS: Breast cancer cells were pre-treated with MPSE before irradiation (IR), the radiosensitizing activity of MPSE was assessed using the colony formation assay. Radiation-induced EMT and stemness phenotype were identified using breast cancer stem cells (CSCs) marker (CD24-/low/CD44+) and mammosphere formation assay. Cell motility was determined via the wound healing assay and transwell migration. Radiation-induced cell death was assessed via the apoptosis assay and SA-ß-galactosidase staining for cellular senescence. CSCs- and EMT-related genes were confirmed by real-time PCR (qPCR) and Western blotting. RESULTS: Pre-treated with MPSE before irradiation could reduce the clonogenic activity and enhance radiosensitivity of breast cancer cell lines with sensitization enhancement ratios (SERs) of 2.33 and 1.35 for MCF7 and MDA-MB231cells, respectively. Pretreatment of breast cancer cells followed by IR resulted in an increased level of DNA damage maker (γ-H2A histone family member) and enhanced radiation-induced cell death. Irradiation induced EMT process, which displayed a significant EMT phenotype with a down-regulated epithelial marker E-cadherin and up-regulated mesenchymal marker vimentin in comparison with untreated breast cancer cells. Notably, we observed that pretreatment with MPSE attenuated the radiation-induced EMT process and decrease some stemness-like properties characterized by mammosphere formation and the CSC marker. Furthermore, pretreatment with MPSE attenuated the radiation-induced activation of the pro-survival pathway by decrease the expression of phosphorylation of ERK and AKT and sensitized breast cancer cells to radiation. CONCLUSION: MPSE enhanced the radiosensitivity of breast cancer cells by enhancing IR-induced DNA damage and cell death, and attenuating the IR-induced EMT process and stemness phenotype via targeting survival pathways PI3K/AKT and MAPK in irradiated breast cancer cells. Our findings describe a novel strategy for increasing the efficacy of radiotherapy for breast cancer patients using a safer and low-cost natural product, MPSE.

Bioactive peptide isolated from sesame seeds inhibits cell proliferation and induces apoptosis and autophagy in leukemic cells.

Leukemia is the most common type of hematological malignancies. Several natural products including bioactive peptides have been explored and studied for their anti-leukemic activities. In the present study, anti-leukemic peptide, IGTLILM (IM-7), was isolated and identified from the protein hydrolysate of sesame seeds by reverse phase-solid phase extraction, off-gel fractionation and nano LC-MS/MS. The cytotoxic effects of IM-7 were studied in MOLT-4 and NB4 acute leukemic cell lines using an MTT assay. The induction of apoptosis and autophagy was investigated by flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies, respectively. The mRNA alterations of apoptotic and autophagic-related genes were determined by reverse transcription-quantitative PCR. The present study found that IM-7 inhibited the proliferation of MOLT-4 and NB4 cells in dose-dependent manner, but it showed a minimal effect on healthy mononuclear cells. IM-7 activated apoptosis and autophagy through the upregulation of CASP3, ULK1 and BECN1 and the downregulation of BCL2. In addition, IM-7 enhanced the cytotoxic effect of the anti-leukemic drug, daunorubicin. The findings suggested that IM-7 was potent to suppress the proliferation of MOLT-4 and NB4 leukemic cells and induce apoptosis and autophagy through the regulation of caspase 3-Bcl-2 and ULK1-Beclin1, respectively.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines.

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches.

BACKGROUND: Cryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1-2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1-2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography-tandem mass spectrometry. RESULTS: A PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1-2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age. CONCLUSIONS: The present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

Proteomics for Toxicological Pathways Screening: A Case Comparison of Low-concentration Ionic and Nanoparticulate Silver.

LC-MS/MS-based proteomics coupled with an online bioinformatics platform was under evaluation for applicability to toxicological pathways evaluation at low cytotoxic concentration (LC10) of silver nanoparticles (AgNP) and ionic silver in human carcinoma cells after 48 h of exposure. Significantly, differentially-expressed proteins (One-way ANOVA, p < 0.05) with more than 4-fold compared to the control were subjected to functional pathway analysis by STITCH. SOTA clustering indicated a similarity of the protein expression between AgNP and the control group. We established a resemblance of proteins in the cell cycle pathway affected by both Ag substances. The differences in the toxicological pathways from AgNO3 were involved in the cellular organization and metabolic process of macromolecules, while the nucleic acid metabolic process was altered by AgNP. The present study supported the practicability of LC-MS/MS-based proteomics coupled with STITCH for the identification of toxicological pathways in both silvers. We appraised this platform technology to be promising and powerful for a toxicological screening of other new substances.

Salivary proteomics of canine oral tumors using MALDI-TOF mass spectrometry and LC-tandem mass spectrometry.

Canine oral tumors are relatively common neoplasms in dogs. For disease monitoring and early diagnosis, salivary biomarkers are appropriate because saliva collection is non-invasive and requires no professional skills. In the era of omics, matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) coupled with liquid chromatography-tandem MS (LC-MS/MS) are suitable to identify potential disease-associated peptides and proteins. The present study aimed to use MALDI-TOF MS and LC-MS/MS to search for particular peptide mass fingerprints (PMFs) and conceivable biomarkers in saliva of dogs with early- and late-stage oral melanoma (EOM and LOM, respectively), oral squamous cell carcinoma (OSCC), benign oral tumors (BN), and periodontitis and healthy controls (CP). Pooled saliva samples in each group were used to be representative of population change. Unique PMFs were obtained and specific peptide fragments were sequenced by LC-MS/MS and BLAST-searched with mammalian protein databases. Seven peptide fragments appeared in the tumor groups (EOM, LOM, OSCC and BN) at 1096, 1208, 1322, 1794, 1864, 2354 and 2483 Da, two peptide fragments appeared in the LOM and OSCC groups at 2450 and 3492 Da, and in the CP controls at 2544 and 3026 Da. Also, protein-chemotherapy drug interaction networks were exhibited. Using western blot analysis, the expression of sentrin-specific protease 7 (SENP7), a peptide fragment at 1096 Da, in OSCC was significantly increased, as was the expression of TLR4, a peptide fragment at 3492 Da, in LOM and OSCC, compared with the CP group. The expression of nuclear factor kappa B (NF-κB), a TLR4 partner, was notably increased in OSCC compared with CP, BN and EOM. The expression was also enhanced in LOM compared with EOM. Expressed protein sequences from western blots were verified by LC-MS/MS. Western blots were then performed with individual samples in each group. The results showed the elevated expression of TLR4 in LOM and OSCC, compared with that in CP and BN, the increased expression of NF-κB in LOM and OSCC, compared with CP and in LOM compared with BN, and the enhanced expression of SENP7 in LOM and OSCC, compared with that in CP and BN. In conclusion, discrete clusters of EOM, LOM, OSCC, BN and CP groups and potential protein candidates associated with the diseases were demonstrated by salivary proteomics. Western blot analysis verified SENP7, TLR4 and NF-κB as potential salivary biomarkers of canine oral tumors.

Novel bioactive peptides demonstrating anti-dengue virus activity isolated from the Asian medicinal plant Acacia Catechu.

The therapeutic activities of food-derived bioactive proteins and peptides are attracting increased attention within the research community. Medicinal plants used in traditional medicines are an excellent source of bioactive proteins and peptides, especially those traditionally prepared by water extraction for use as tea or food supplement. In this study, novel bioactive peptides were isolated from enzymatic digests of 33 Thai medicinal plants. The inhibitory activity of each against dengue virus (DENV) infection was investigated. Of 33 plants, peptides from Acacia catechu extract demonstrated the most pronounced anti-DENV activity. Half maximal inhibitory concentration of 0.18 µg/ml effectively inhibited DENV foci formation. Treatment with 1.25 µg/ml crude peptide extract could reduce virus production less than 100-fold with no observable cell toxicity. Peptide sequences were determined by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Two bioactive peptides isolated from Acacia catechu inhibited DENV foci formation >90% at the concentration of 50 µM; therefore, they are recommended for further investigation as antiviral peptides against DENV infection.