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.

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.

The C-terminally shortened analogs of a hexapeptide derived from Lingzhi hydrolysate with enhanced tyrosinase-inhibitory activity.

Ganoderma lucidum or Lingzhi (Chinese) is a medicinal fungus widely used in traditional medicine as a health supplement. This study was conducted to identify an approach to enhance the anti-tyrosinase activity of a peptide from G. lucidum by chemical modification of its C-terminus. The original peptide was obtained from protease-digested Lingzhi proteins, followed by ultrafiltration (molecular weight cut-off 3 kDa) and C18 solid-phase extraction. The hexapeptide (NH2 -VLTCGF-COOH) possessing the anti-tyrosinase activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This hexapeptide was subjected to shortening to enhance the anti-tyrosinase activity. Both the original peptide and the shortened peptides were synthesized by solid-phase peptide synthesis. The purity and mass of the synthetic peptide and the modified peptide were evaluated by high-performance liquid chromatography and LC-MS, respectively. Comparison of the tyrosinase activities showed that the modified peptide demonstrated more than 23.27 ± 1.07% activity, which was better than that of the hexapeptide. The structure-related biological activity was explained by molecular docking, wherein the VLT-tyrosinase complex showed two interaction forces: Asn260 and Gly281 through H-bonding and Glu256 through electrostatic interaction. This information could help toward gaining further understanding of the correlation between the anti-tyrosinase activity and the molecular structure of the modified hexapeptide and support its potential use as a safe cosmetic ingredient with tyrosinase-suppressing ability.

Vernonia amygdalina Leaf Extract Induces Apoptosis in HeLa Cells: A Metabolomics and Proteomics Study.

Medicinal plants produce various bioactive molecules with potential anti-cancer properties with favorable safety profiles. We aimed to investigate the comprehensive composition of Vernonia amygdalina leaf extract and its cytotoxic effects via apoptosis in HeLa cells. The metabolomics approach using LC-MS/MS was conducted to gather the metabolite profile of the extract. Proteomics was performed to understand the comprehensive mechanistic pathways of action. The apoptosis was visualized by cellular staining and the apoptotic proteins were evaluated. V. amygdalina leaf extract exhibited dose-dependent cytotoxic effects on both HeLa and Vero cells after 24 h of exposure in the MTT assay with the IC50 values of 0.767 ± 0.0334 and 4.043 ± 0.469 µg mL-1, respectively, which demonstrated a higher concentration required for Vero cell cytotoxicity. The metabolomic profile of 112 known metabolites specified that the majority of them were alkaloids, phenolic compounds, and steroids. Among these metabolites, deacetylvindoline and licochalcone B were suggested to implicate cytotoxicity. The cytotoxic pathways involved the response to stress and cell death which was similar to doxorubicin. The upstream regulatory proteins, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and X-box binding protein 1 (XBP1), were significantly altered, supporting the regulation of apoptosis and cell death. The levels of apoptotic proteins, c-Jun N-terminal kinases (JNK), p53, and caspase-9 were significantly increased. The novel insights gained from the metabolomic profiling and proteomic pathway analysis of V. amygdalina leaf extract have identified crucial components related to apoptosis induction, highlighting its potential to develop future chemotherapy.

Electrosprayed Nanoparticles Containing Mangiferin-Rich Extract from Mango Leaves for Cosmeceutical Application.

Mango (Mangifera indica L.) is one of the most economically important fruits in Thailand. Mango has been used as a traditional medicine because it possesses many biological activities, such as antioxidant properties, anti-inflammatory properties, microorganism-growth inhibition, etc. Among its natural pharmacologically active compounds, mangiferin is the main active component found in mango leaves. Mangiferin has the potential to treat a variety of diseases due to its multifunctional activities. This study aims to prepare a mangiferin-rich extract (MRE) from mango leaves and develop nanoparticles containing the MRE using an electrospraying technique to apply it in a cosmeceutical formulation. The potential cosmeceutical mechanisms of the MRE were investigated using proteomic analysis. The MRE is involved in actin-filament organization, the positive regulation of cytoskeleton organization, etc. Moreover, the related mechanism to its cosmeceutical activity is metalloenzyme-activity regulation. Nanoparticles were prepared from 0.8% w/v MRE and 2% w/v Eudragit® L100 solution using an electrospraying process. The mean size of the MRE-loaded nanoparticles (MNPs) received was 247.8 nm, with a PDI 0.271. The MRE entrapment by the process was quantified as 84.9%, indicating a high encapsulation efficiency. For the skin-retention study, the mangiferin content in the MNP-containing emulsion-gel membranes was examined and found to be greater than in the membranes of the MRE solution, illustrating that the MNPs produced by the electrospraying technique help transdermal delivery for cosmetic applications.

Potential of electro-sprayed purified mangiferin nanoparticles for anti-aging cosmetic applications.

The fabrication of mangiferin nanoparticles using an electrospraying technique is a new and promising method for developing nanoparticles with higher efficiency and safety. This study aimed to fabricate mangiferin nanoparticles (MNPs) using cellulose acetate (CA) as a polymer at various parameters using electrospraying. Commercial mangiferin (CM) was purified from 88.46 to 95.71% by a recrystallization method to improve its purity and biological activities and remove any residue. The properties of recrystallized mangiferin (RM) were characterized using DSC, FTIR, X-ray diffraction (XRD) and HPLC. Then its biological activity and proteomics were determined. Proteomics analysis of RM showed that up-regulated proteins were involved in more biological processes than CM. MNPs were fabricated by varying the electrospraying parameters including voltage, the distance between the needle-tip-collector and flow rate. Skin permeation, release and irritation were also evaluated. The results revealed that the average particle size of the MNPs ranged between 295.47 ± 5.58 and 448.87 ± 3.00 nm, and had a smooth spherical morphology in SEM images. The MNPs also showed good potential in antioxidant and anti-aging properties. The encapsulation efficiency of MNPs was determined to be 85.31%. From skin permeation studies of CM, RM, and MNPs, the mangiferin content was found in the stratum corneum and dermis skin layers. Moreover, the MNPs solution had 23.68 ± 0.27% and 11.98 ± 0.13% of mangiferin in the stratum corneum and viable epidermis and dermis, respectively. Additionally, the irritation test by HET-CAM was mild and safe. Therefore, MNPs produced by electrospraying are a promising delivery system for cosmetic/cosmeceutical applications.

Utilizing Quantitative Proteomics to Identify Species-Specific Protein Therapeutic Targets for the Treatment of Leishmaniasis.

Leishmaniasis is a tropical disease caused by Leishmania parasites, which are transmitted through the bites of infected sandflies. We focused on the emergence of leishmaniasis in Thailand caused by a species (Leishmania orientalis). Treatment by chemotherapy is not effective against L. orientalis. Hence, we intended to solve this issue using a proteomics approach to investigate protein profiles and in silico analysis for the identification of antigenic proteins from L. orientalis, Leishmania martiniquensis, and Leishmania donovani. Using principal component analysis (PCA), protein profile comparisons indicated that different species of Leishmania are different at the protein level. Proteomics analysis identified 6099 proteins. Among these proteins, 1065 proteins were used for further analysis. There were 16 proteins that were promising candidates for therapeutic aspects as they were abundantly expressed and common to all species. In silico analysis of protein's antigenicity revealed that eight proteins had the potential for the development of antigenic molecules. Protein profile information and these antigenic proteins may play key roles in the pathogeny of leishmaniasis and can be used as novel therapeutic targets against leishmaniasis in the future.

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.

Clinical Prediction Scoring Scheme for 24 h Mortality in Major Traumatic Adult Patients.

A death rate of approximately 32.7 in 100,000 traffic injury victims was reported in Thailand. The prediction of early death would identify and enable prioritization of the most severe patients for resuscitation and consequently reduce the number of deaths. This study aimed to develop a clinical prediction scoring system for 24 h mortality in adult major trauma patients. Retrospective-prognostic clinical prediction was applied in the case of 3173 adult trauma patients who were classified into three groups: death within 8 h, death between 8 and 24 h, and alive at 24 h. The predictors were obtained by univariable and multivariable logistic regression, and the coefficient of parameters was converted to predict early death. The numbers of patients who died within 8 h and between 8 and 24 h were 46 (1.5%) and 123 (3.8%), respectively. The predictors included systolic blood pressure <90 mmHg, heart rate ≥120 bpm, Glasgow coma scale ≤8, traffic injury, and assault injury. The scores of 4 indicated a mortality rate of 12% with a high specificity of 0.89. The suggested TERMINAL-24 scoring system can be used for the prediction of early death in the Emergency Department. However, its discrimination ability and precision should be validated before practical use.

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.

Study of the Lipolysis Effect of Nanoliposome-Encapsulated Ganoderma lucidum Protein Hydrolysates on Adipocyte Cells Using Proteomics Approach.

Excessive lipid accumulation is a serious condition. Therefore, we aimed at developing safe strategies using natural hypolipidemic products. Lingzhi is an edible fungus and potential lipid suppression stimulant. To use Lingzhi as a functional hyperlipidemic ingredient, response surface methodology (RSM) was conducted to optimize the time (X1) and enzyme usage (X2) for the hydrolysate preparation with the highest degree of hydrolysis (DH) and % yield. We encapsulated the hydrolysates using nanoscale liposomes and used proteomics to study how these nano-liposomal hydrolysates could affect lipid accumulation in adipocyte cells. RSM analysis revealed X1 at 8.63 h and X2 at 0.93% provided the highest values of DH and % yields were 33.99% and 5.70%. The hydrolysates were loaded into liposome particles that were monodispersed. The loaded nano-liposomal particles did not significantly affect cell survival rates. The triglyceride (TG) breakdown in adipocytes showed a higher TG increase compared to the control. Lipid staining level upon the liposome treatment was lower than that of the control. Proteomics revealed 3425 proteins affected by the liposome treatment, the main proteins being TSSK5, SMU1, GRM7, and KLC4, associated with various biological functions besides lipolysis. The nano-liposomal Linzghi hydrolysate might serve as novel functional ingredients in the treatment and prevention of obesity.

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.

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease.

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials.