RESUMO
BACKGROUND: Elemene, an active anticancer extract derived from Curcuma wenyujin, has well-documented anticarcinogenic properties. Nevertheless, the role of elemene in prostate cancer (PCa) and its underlying molecular mechanism remain elusive. PURPOSE: This study focuses on investigating the anti-PCa effects of elemene and its underlying mechanisms. METHODS: Cell-based assays, including CCK-8, scratch, colony formation, cell cycle, and apoptosis experiments, to comprehensively assess the impact of elemene on PCa cells (LNCaP and PC3) in vitro. Additionally, we used a xenograft model with PC3 cells in nude mice to evaluate elemene in vivo efficacy. Targeted metabolomics analysis via HILIC-MS/MS was performed to investigate elemene potential target pathways, validated through molecular biology experiments, including western blotting and gene manipulation studies. RESULTS: In this study, we discovered that elemene has remarkable anti-PCa activity in both in vitro and in vivo settings, comparable to clinical chemotherapeutic drugs but with fewer side effects. Using our established targeted metabolomics approach, we demonstrated that ß-elemene, elemene's primary component, effectively inhibits glycolysis in PCa cells by downregulating 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Furthermore, we found that ß-elemene accomplishes this downregulation by upregulating p53 and FZR1. Knockdown and overexpression experiments conclusively confirmed the pivotal role of PFKFB3 in mediating ß-elemene's anti-PCa activity. CONCLUSION: This finding presents compelling evidence that elemene exerts its anti-PCa effect by suppressing glycolysis through the downregulation of PFKFB3. This study not only improves our understanding of elemene in PCa treatment but also provides valuable insights for developing more effective and safer therapies for PCa.
Assuntos
Neoplasias da Próstata , Sesquiterpenos , Espectrometria de Massas em Tandem , Masculino , Animais , Camundongos , Humanos , Camundongos Nus , Linhagem Celular Tumoral , Neoplasias da Próstata/tratamento farmacológico , Glicólise , Proliferação de Células , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Curcuma wenyujin Y.H. Chen & C. Ling, also known as Wen-E-Zhu, has been used for cancer treatment since ancient times, with roots dating back to the Song Dynasty. Elemene (EE), a sesquiterpene extract with potent anticancer properties, is extracted from Wen-E-Zhu, with ß-elemene (BE) being its main active compound, along with trace amounts of ß-caryophyllene (BC), γ-elemene and δ-elemene isomers. EE has demonstrated broad-spectrum anti-cancer effects and is commonly used in clinical treatments for various types of malignant cancers, including lung cancer. Studies have shown that EE can arrest the cell cycle, inhibit cancer cell proliferation, and induce apoptosis and autophagy. However, the exact mechanism of its anti-lung cancer activity remains unclear and requires further research and investigation. AIM OF THE STUDY: In this study, the possible mechanism of EE and its main active components, BE and BC, against lung adenocarcinoma was investigated by using A549 and PC9 cell lines. MATERIALS AND METHODS: The subcutaneous tumor model of nude mice was constructed to evaluate the efficacy of EE in vivo, then the in vitro half-inhibitory concentration (IC50) of EE and its main active components, BE and BC, on A549 and PC9 cells at different concentrations were determined by CCK-8. Flow cytometry was used to detect the apoptosis and cycle of A549 and PC9 cells treated with different concentrations of BE and BC for 24 h. Non-targeted metabolomics analysis was performed on A549 cells to explore potential target pathways, which were subsequently verified through kit detection and western blot analysis. RESULTS: Injection of EE in A549 tumor-bearing mice effectively suppressed cancer growth in vivo. The IC50 of EE and its main active components, BE and BC, was around 60 µg/mL. Flow cytometry analysis showed that BE and BC blocked the G2/M and S phases of lung adenocarcinoma cells and induced apoptosis, leading to a significant reduction in mitochondrial membrane potential (MMP). Results from non-targeted metabolomics analysis indicated that the glutathione metabolism pathway in A549 cells was altered after treatment with the active components. Kit detection revealed a decrease in glutathione (GSH) levels and an increase in the levels of oxidized glutathione (GSSG) and reactive oxygen (ROS). Supplementation of GSH reduced the inhibitory activity of the active components on lung cancer and also decreased the ROS content of cells. Analysis of glutathione synthesis-related proteins showed a decrease in the expression of glutaminase, cystine/glutamate reverse transporter (SLC7A11), and glutathione synthase (GS), while the expression of glutamate cysteine ligase modified subunit (GCLM) was increased. In the apoptosis-related pathway, Bax protein and cleaved caspase-9/caspase-9 ratio were up-regulated and Bcl-2 protein was down-regulated. CONCLUSIONS: EE, BE, and BC showed significant inhibitory effects on the growth of lung adenocarcinoma cells, and the mechanism of action was linked to the glutathione system. By down-regulating the expression of proteins related to GSH synthesis, EE and its main active components BE and BC disrupted the cellular redox system and thereby promoted cell apoptosis.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Sesquiterpenos , Animais , Camundongos , Caspase 9/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Camundongos Nus , Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/patologia , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Apoptose , Glutationa/metabolismo , Proliferação de Células , Linhagem Celular TumoralRESUMO
BACKGROUND: Due to the diversity of the ingredients, the complexity of the mechanism of action, the uncertainty of the effective ingredients, coupled with the multiple species and multiple growing areas, the quality control (QC) of Traditional Chinese Medicines (TCMs) is challenging. Discovering and identifying effective compounds from the complex extracts of TCMs and then establishing a scientific QC method is the key to the holistic QC of TCMs. PURPOSE: To develop an anti-lung-cancer-guided spectrum-effect relationship approach for the discovery of QC markers of the rhizome of Curcuma wenyujin (WEZ) and establish a bioactive compounds-based holistic QC method. METHODS: The chemical profiling of the volatile oil (WVO) from 42 batches of WEZ collected from different growing areas was performed by GC-MS. The anti-lung cancer activity of different WVO samples was determined by CCK-8 assay against human lung cancer cells (A549). The apoptosis and cell cycle analysis under different concentrations of WVO were detected by flow cytometry. SIMCA-P software was used to perform multivariate statistical analysis on the chemical composition of different WVO samples and to find the different components. Active compounds were screened using a PLSR model of the spectrum-effect relationship. Bioactive compounds-based fingerprint and quantification of the leading bioactive compounds were developed by GC-MS and GC-FID, respectively. RESULTS: Seventy-eight compounds were detected in WVO and 54 were successfully identified. The multivariate statistical analysis uncovered that WVO components and the anti-A549 activity of WVO at the concentration of 60 nl/ml differ greatly according to the origin of the plant. The WVO at the concentration of 60 nl/ml (IC50) increased A549 cells apoptosis significantly with late and early apoptosis of 15.61% and 7.80%, and the number of cells in the G2/M phase were also increased significantly under this concentration. The spectrum-effect relationship analysis revealed that 44 compounds were positively correlated with their activities, and the result was verified by A549 cell viability assay. Sixteen positively correlated compounds were further selected as QC markers according to their relative amount > 0.5% and anticancer activity. Finally, the 16 QC markers-based GC-MS fingerprint was established to holistically control the quality of WEZ, and a GC-FID method was developed for the quantification of leading bioactive compounds, ß-elemene and ß-caryophyllene. CONCLUSION: Based on an anti-lung-cancer-guided spectrum-effect relationship approach, the bioactive compounds-based holistic QC method was successfully developed for WEZ, which could provide a valuable reference for the QC of TCMs.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biomarcadores/análise , Curcuma/química , Medicamentos de Ervas Chinesas/química , Células A549 , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Biomarcadores/química , Medicamentos de Ervas Chinesas/farmacologia , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Óleos Voláteis/química , Sesquiterpenos Policíclicos/análise , Sesquiterpenos Policíclicos/farmacologia , Controle de Qualidade , Rizoma/química , Sesquiterpenos/análise , Sesquiterpenos/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicines (HMs) often exert integration effects, including synergistic, additive and antagonistic effects, in such ways that they act on multiple targets and multiple pathways on account of their multiple components. Turmeric, made from the rhizome of Curcuma longa L., is a well-known HM prescribed in the polyherbal formulas for cancer treatment in traditional Chinese medicines (TCMs). However, neither the multiple anticancer compounds of turmeric nor the integration effects of these components are fully known. AIM OF THE STUDY: This work aims to develop a systematic approach to reveal the integration effect mechanisms of multiple anticancer compounds in turmeric against prostate cancer PC3 cells. MATERIALS AND METHODS: Combination index and omics technologies were applied to profile the integration effect mechanisms of bioactive compounds in proportions naturally found in turmeric. PC3 cell line (a prostate cancer cell line) fishing and high resolution mass spectrometry were employed to screen and identify the anticancer compounds from turmeric. The combinations which contain different cell-bound compounds in natural proportions were prepared for further evaluation of anti-cancer activity by using cell viability assays, and assessment of cell apoptosis and cell cycle analysis. Combination index analysis was applied to study the integration effects of the anticancer compounds in their natural proportions. Finally, quantitative glycoproteomics/proteomics and Western blot were implemented to reveal the potential synergistic effect mechanisms of the anticancer compounds based on their natural proportions in turmeric. RESULTS: Three curcuminoids (curcumin, CUR; demethoxycurcumin, DMC; bisdemethoxycurcumin, BDMC) in turmeric were discovered and shown to possess significant synergistic anticancer activities. Combination index analysis revealed an additive effect of CUR combined with DMC or BDMC and a slight synergistic effect of DMC combined with BDMC in natural proportions in turmeric, while a combination of all three curcuminoids (CUR, DMC and BDMC) at a ratio of 1:1:1 yielded superior synergistic effects. Interestingly, the presence of BDMC and DMC are essential for synergistic effect. Glycoproteomics and proteomics demonstrated that different curcuminoids regulate various protein pathways, such as ribosome, glycolysis/gluconeogenesis, biosynthesis of amino acids, and combination of CUR + DMC + BDMC showed the most powerful effects on down-regulation of protein expression. CONCLUSIONS: Our analytical approach provides a systematic understanding of the holistic activity and integration effects of the anti-cancer compounds in turmeric and three curcuminoids of turmeric showed a synergistic effect on PC3 cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Curcuma , Diarileptanoides/farmacologia , Glicômica , Glicoproteínas/metabolismo , Extratos Vegetais/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteômica , Antineoplásicos Fitogênicos/isolamento & purificação , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Curcuma/química , Diarileptanoides/isolamento & purificação , Sinergismo Farmacológico , Humanos , Masculino , Células PC-3 , Extratos Vegetais/isolamento & purificação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Mapas de Interação de Proteínas , Transdução de SinaisRESUMO
The stems of Dendrobium officinale, a well-known and expensive food material and herbal medicine in Asia, has recently suffered adulterants and counterfeits by using lower-price confusing Dendrobium species such as D. devonianum or D. transparens in the herbal market. However, robust methods that could authenticate D. officinale from its confusing species effectively are still lacking, especially for the dried samples. This study committed to discover specific peptides biomarkers for the authentication of D. officinale from the other two Dendrobium species using label-free proteomics by nanoLC LTQ Orbitrap mass spectrometry. Multivariate statistical analysis was applied to visualize the difference between the three Dendrobium species. As a result, 29 peptides among a total of 343 measurable peptides were selected to be potential biomarkers for the classification of these Dendrobium species. The validation of the representative peptide biomarkers was carried out by the synthesized peptides and 3 peptide biomarkers were found significant for the authentication of D. officinale. Further analysis showed that peptide ALGLELDLSER may also be a biomarker for the discrimination of the D. officinale originated from different geographical regions.