Arenobufagin enhances T-cell anti-tumor immunity in colorectal cancer by modulating HSP90ß accessibility.

BACKGROUND: Colorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin (Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. PURPOSE: This study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90ß-STAT3-PD-L1 axis activity. METHODS: We investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits CRC. Utilizing target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90ß (HSP90ß) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. RESULTS: We observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90ß's accessibility significantly decreased upon Are binding. We demonstrated a decrease in the activity of the HSP90ß-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. CONCLUSION: Contrary to previous research focusing on the direct cytotoxicity of Are towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Are and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Are.

α-hederin regulates glucose metabolism in intestinal epithelial cells by increasing SNX10 expression.

BACKGROUND: Sorting nexin 10 (SNX10) has recently been identified as a critical regulator of colorectal carcinogenesis, whose deletion promoted cell proliferation and survival in human CRC cells, and promoted colorectal tumor growth and upregulated amino-acid metabolism in mice. However, what happens when silencing SNX10 in normal human intestinal epithelial cells (IECs) remains unknown, and no drugs targeting SNX10 have been reported. Here, we first investigated the biological function and underlying mechanisms of SNX10 in normal human IECs, and found that α-hederin, a pentacyclic triterpenoid saponin, has a regulatory effect on SNX10 expression. PURPOSE: This study aimed to explore the function of SNX10 in IECs to provide a new target for the prevention and treatment of malignant transformation and the intervention mechanism of α-hederin for further development of potential novel agents targeting SNX10. METHODS: The transfection approach was used to construct SNX10 stable knockdown cells. Cell proliferation was detected by CCK8, clone formation, EdU, flow cytometry, and wound healing assays. Enzyme activity assays for glucose metabolism, qRT-PCR, western blotting, and immunofluorescence staining were performed to investigate the protein expression of signaling pathways. RESULTS: Silencing SNX10 promoted cell proliferation and cycle transition in IECs and increased the activity of key enzymes involved in glucose metabolism. Moreover, DEPDC5 expression was significantly decreased following SNX10 knockdown, followed by activation of the mTORC1 pathway. α-hederin reversed the accelerated cell proliferation, cycle progression, and glucose metabolic activity, as well as the activated mTORC1 pathway caused by SNX10 knockdown, by notably increasing SNX10 expression in a dose-dependent manner. CONCLUSION: We first reported that knockdown of SNX10 in normal human IECs promoted cell proliferation and activated glucose metabolism by activating the mTORC1 pathway. Meanwhile, we first found that α-hederin down-regulated glucose metabolism activity and slowed cell proliferation by increasing SNX10 expression in IECs.

Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway.

PURPOSE: Scutellaria barbata D. Don (SB), mainly containing flavonoids, has been frequently used for cancer treatment. However, little research has investigated the antitumor activity of flavonoids from SB (FSB). The current study aimed to assess the antitumor effect of TFSB and elucidate the probable underlying mechanism in vivo and in vitro. STUDY DESIGN: FSB was prepared, and its chemical composition was characterized by HPLC-MS. Colorectal HCT116 cells were treated with various concentration of FSB. The viability, proliferation, apoptosis, migration, and autophagy of HCT116 cells were studied, as were further confirmed in tumor xenografts. METHODS: Cell viability and proliferation were respectively examined by MTT and EdU staining. ROS was determined with DCFH-DA, and cell apoptosis was detected using flow cytometry. Transwell and wound-healing assays were performed to evaluate cell migration. Immunofluorescence was employed to evaluate sestrin2 and ATF4 level. The protein expressions of p-AMPK, p-ULK1, p-mTOR, 4E-BP1, LC3-I/II, cleaved-caspase-3, Bax, and bcl-2 were investigated by western blot. ATF4 was overexpressed in experiments to explore the role of ATF4/sestrin2 pathway in FSB-mediated efficacy. RESULTS: FSB clearly reduced the cell viability, promoted ROS generation, and induced apoptosis in HCT116 cells by down-regulated Bcl-2, and increased cleaved-caspase-3 and Bax. Furthermore, FSB significantly inhibited migration of colorectal cells in a dose-dependent manner. Further mechanistic study indicated that FSB upregulated p-mTOR protein level, and reduced p-AMPK, p-ULK1, p-mTOR, p-4E-BP1 and LC3-I/II expression, which were major autophagy-related genes. In addition, FSB could cause downregulation of endogenous mTOR inhibitor sestrin2 and ATF4 expression. Transient overexpression of ATF4 resulted in mTOR and sestrin2 inhibition, and significantly compromised the effects of FSB on apoptosis and autophagy in HCT116 cells. CONCLUSION: Our results reveal, for the first time, that FSB exerts antitumor activity through autophagy inhibition and apoptosis induction via ATF4/sestrin2 pathway in colorectal cancer cells. Scutellaria barbata D. Don may have great potential in the application for the prevention and treatment of human colorectal cancer.

Characterization of the chemical constituents and in vivo metabolic profile of Scutellaria barbata D. Don by ultra high performance liquid chromatography with high-resolution mass spectrometry.

Scutellaria barbata D. Don (S. barbata) is one of the most frequently used anticancer herb medicine in China. Mechanistic understanding of the biological activities of S. barbata is hindered by limited knowledge regarding its components and metabolic profile. In this study, ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (quadrupole time-of-flight mass spectrometry) was used to identify the chemical constituents in S. barbata and their metabolic profiles in rats. By applying cleavage rules and comparison with reference substances, 89 components were identified in S. barbata, which included 45 flavonoids, 28 diterpenoids, 10 phenolics, and 6 others. A total of 110 compounds, including 32 prototype compounds and 78 metabolites, were identified or tentatively characterized in vivo. Methylation, sulfonation, and glucuronidation were the main metabolic pathways, which could be attributed to the fact that several of the compounds in S. barbata have phenolic hydroxyl groups. This is the first systematic study on the chemical constituents and in vivo metabolic profile of S. barbata. The analytical method features a quick and comprehensive dissection of the chemical composition and metabolic profile of S. barbata and provides a basis for exploring its various biological activities.

Identification of the absorbed components and metabolites of Xiao-Ai-Jie-Du decoction and their distribution in rats using ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Xiao-Ai-Jie-Du decoction (XAJDD), a traditional Chinese medicine formula, has long been used for the treatment of hepatocarcinoma, gastric cancer and colorectal cancer. It is composed of six herbal medicines, including Scutellariae Barbatae Herba, Pseudostellariae Radix, Ophiopogonis Radix, Cremastrae Pseudobulbus, Curcumae Rhizoma and Akebiae Fructus. Despite the in-depth study on its pharmacological effects on cancer prevention and treatment, the comprehensive analysis of the chemical components and the absorbed bioactive constituents are not well studied. Thus, an ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was established to detect and identify the chemical constituents in XAJDD. The absorbed components and metabolites after oral administration of XAJDD in rats were also studied. In total, 102 components were identified or tentatively characterized in XAJDD, including 30 flavonoids, 19 triterpenoids, 12 organic acids, 9 steroidal saponins, 9 cyclic peptides, 7 phenanthrenes, 5 amino acids, 3 alkaloids and 8 other compounds. After analysing the metabolites in rat plasma and urine after oral administration of XAJDD, a total of 70 compounds were identified, including 15 primary components and 55 metabolites, and metabolic pathways, including hydrogenation, hydroxylation, methylation, sulfonation, and glucuronidation were evaluated. Among these, methylation and glucuronidation were the main metabolic pathways. In conclusion, the developed UHPLC-Q-TOF-MS method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of XAJDD in vitro and characterizing the primary components and their metabolites in vivo; moreover, the results will provide essential data for further studying the relationship between the chemical components and pharmacological activity of XAJDD.

α-Hederin inhibits interleukin 6-induced epithelial-to-mesenchymal transition associated with disruption of JAK2/STAT3 signaling in colon cancer cells.

Colon cancer is the third most frequently diagnosed malignancy and has high morbidity worldwide. Epithelial-mesenchymal transition (EMT) has been increasingly implicated in colon cancer progression and metastasis. The present study was aimed to evaluate the potential antitumor activity of α-hederin, a monodesmosidic triterpenoid saponin isolated from Hedera helix, in human SW620 colon cancer cells stimulated with interleukin 6 (IL-6) for mimicking the tumor inflammatory microenvironment in vivo. Cell viability assay showed that IL-6 at 6.25 ng/ml significantly enhanced viability of SW620 cells, and thus this concentration was used to stimulate SW620 cells throughout this study. We observed that α-hederin concentration-dependently inhibited cell viability, migration and invasion in IL-6-treated SW620 cells. Moreover, α-hederin significantly restored IL-6-induced decrease in E-cadherin expression and abolished IL-6-induced increase in N-cadherin, vimentin, fibronectin, twist and snail at both mRNA and protein levels in SW620 cells. These data suggested that α-hederin suppressed IL-6-indcued EMT in colon cancer cells. Further molecular examinations showed that α-hederin inhibited phosphorylation of Janus Kinase 2 (JAK2) and Signal Transducer and Activator of Transcription 3(STAT3), and halted the nuclear translocation of phosphorylated STAT3 in IL-6-treated SW620 cells. In addition, JAK2/STAT3 signaling inhibitor AG490 not only produced similar inhibitory effects on EMT markers as α-hederin, but also synergistically enhanced α-hederin's inhibitory effects on EMT markers in IL-6-treated SW620 cells. Altogether, we demonstrated that α-hederin suppressed IL-6-induced EMT associated with disruption of JAK2/STAT3 signaling in colon cancer cells. Our data strongly suggested α-hederin as a promising candidate for intervention of colon cancer and metastasis.

LC-MS/MS analysis and evaluation of the anti-inflammatory activity of components from BushenHuoxue decoction.

CONTEXT: BushenHuoxue decoction (BSHXD) is a Chinese medicine prescription, which is composed of nine Chinese medical materials, used to treat osteoarthritis (OA). OBJECTIVE: This study develops sensitive and convenient LC-MS/MS methods to analyze chemical components from BSHXD, and assess the anti-inflammatory activities thereof. MATERIALS AND METHODS: The chemical composition from BSHXD water extract was qualitative analyzed by high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS). Twelve reference compounds were analyzed by UPLC-ESI-MS/MS. Anti-inflammatory activities of target components were assessed by ELISA at 20 and 100 µg/mL. RESULTS: It is the first time that 88 compounds were qualitatively identified from BSHXD, of which 12 with potential in treating OA according to the literature were quantified. Within BSHXD the contents of quercetin, isopsoralen, icarisideII, osthole, and isoimperatorin increased remarkably compared with those in single herb which make up BSHXD, the contents were 0.1999, 0.4634, 0.0928, 0.5364, and 0.1487 mg/g. ELISA data displayed that BSHXD and the five compounds mentioned inhibited the expressions of TNF-α, IL-6 and NO released from LPS-stimulated RAW264.7 cell, with maximum inhibition rates of 104.05% (osthole, 100 µg/mL), 100.03% (osthole, 100 µg/mL), and 93.46% (isopsoralen, 20 µg/mL), respectively. DISCUSSION AND CONCLUSION: Content changes of 12 compounds in BSHXD and single herbs which comprise the prescription were measured and analyzed. Contents of five compounds increased may be explained by solubilization between drugs and chemical reaction. ELISA results reported that the increased contents of the five compounds could inhibit expression of the inflammatory factors.

Longikaurin E induces apoptosis of pancreatic cancer cells via modulation of the p38 and PI3K/AKT pathways by ROS.

Pancreatic cancer is a devastating disease with a poor prognosis. It ranks as the fourth or fifth most common cancer in men and women and has the lowest 5-year survival rate. Therefore, there is an urgent need to develop novel therapeutic agents for pancreatic cancer. Longikaurin E (LE), which is derived from the traditional herbal medicine Rabdosia longituba, had been reported to have anti-proliferative and pro-apoptotic properties in several types of cancers. In this study, we investigated the cytotoxic properties of LE against pancreatic cancer cells and explored the mechanism behind the observed apoptosis. Pancreatic cancer cell lines cultured in the presence of LE exhibited dose- and time-dependent growth suppression by clone formation, methylthiazoltetrazolium assay, lactate dehydrogenase cytotoxicity assay, and fluorescence-activated cell sorting analysis, respectively. In addition, these culture conditions also induced the generation of cellular reactive oxygen species (ROS). In order to determine the mechanisms underlying LE-induced cytotoxicity, we used reverse transcription polymerase chain reaction and Western blot analysis in the pancreatic cancer cell line PANC1. The results showed that the expression of Bax was noticeably upregulated and the expression levels of Bcl-2, Bcl-XL, survivin, and c-Myc were significantly downregulated. We also observed increased p38 phosphorylation and decreased phosphorylation of the PI3K/AKT pathway. Interestingly, we also found that LE activated caspase-3. However, N-acetyl-L-cysteine, a kind of antioxidant, reversed all of these cellular activities. In conclusion, this study suggested that LE induced apoptosis of pancreatic cancer cells via ROS generation to modulate the p38 and PI3K/AKT pathways and could be a promising anti-pancreatic agent.