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This study aimed to investigate the neuroprotective effects of 1-methoxylespeflorin G11 (MLG), a pterocarpan, against glutamate-induced neurotoxicity in neuronal HT22 hippocampal cells. The protective effects of MLG were evaluated using MTT assay and microscopic analysis. The extent of apoptosis was studied using flow cytometric analysis performed on the damaged cells probed with annexin V/propidium iodide. Moreover, mitochondrial reactive oxygen species (ROS) were assessed using flow cytometry through MitoSOXTM Red staining. To determine mitochondrial membrane potential, staining with tetramethylrhodamine and JC-1 was performed followed by flow cytometry. The results demonstrated that MLG attenuates glutamate-induced apoptosis in HT22 cells by inhibiting intracellular ROS generation and mitochondrial dysfunction. Additionally, MLG prevented glutamate-induced apoptotic pathway in HT22 cells through upregulation of Bcl-2 and downregulation of cleaved PARP-1, AIF, and phosphorylated MAPK cascades. In addition, MLG treatment induced HO-1 expression in HT22 cells. These results suggested that MLG exhibits neuroprotective effects against glutamate-induced neurotoxicity in neuronal HT22 cells by inhibiting oxidative stress and apoptosis.

Rumex japonicus Houtt (RJH) is a valuable plant used in traditional medicine to treat several diseases, such as scabies and jaundice. In this study, Jurkat cell growth inhibitory extracts of R. japonicus roots were subjected to bioassay-guided fractionation, resulting in the isolation of three naphthalene derivatives (3-5) along with one anthraquinone (6) and two phenolic compounds (1 and 2). Among these compounds, 2-methoxystypandrone (5) exhibited potent anti-proliferative effects on Jurkat cells. Analysis by flow cytometry confirmed that 2-methoxystypandrone (5) could significantly reduce mitochondrial membrane potential and promote increased levels of mitochondrial reactive oxygen species (ROS), suggesting a strong mitochondrial depolarization effect. Real-time quantitative polymerase chain reaction (qPCR) analysis was also performed, and the results revealed that the accumulation of ROS was caused by reduced mRNA expression levels of heme oxygenase (HO-1), catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). In addition, 2-methoxystypandrone (5) triggered strong apoptosis that was mediated by the arrest of the G0/G1 phase of the cell cycle. Furthermore, 2-methoxystypandrone (5) downregulated p-IκB-α, p-NF-κB p65, Bcl2, and Bcl-xl and upregulated BAX proteins. Taken together, these findings revealed that 2-methoxystypandrone (5) isolated from RJH could potentially serve as an early lead compound for leukemia treatment involving intracellular signaling by increasing mitochondrial ROS and exerting anti-proliferative effects.

Breast cancer is now the most common type of cancer worldwide, surpassing lung cancer. This issue is further worsened by the lack of effective therapies for the disease. Recent reports indicate that the inhibition of ubiquitin-like modifier-activating enzyme 5 (UBA5) can impede tumor development. However, there have been few reports regarding UBA5-inhibiting compounds. This work studied usenamine A, a natural product from the lichen Usnea longissimi that exhibits UBA5-inhibitory effects. Bioinformatics analysis was performed using public databases, and the anti-proliferative ability of usenamine A in breast cancer cells was examined through MTS and colony formation assays. Flow cytometry and western blot analysis were also conducted to examine and analyze cell cycle arrest and apoptosis. In addition, LC3B-RFP and UBA5 expression plasmids were used for the analysis of usenamine A-induced autophagy. According to the bioinformatics analysis results, UBA5 was upregulated in breast cancer. According to in vitro studies, usenamine A displayed prominent anti-proliferative activity and resulted in G2/M phase arrest in MDA-MB-231 cells. Moreover, usenamine A induced autophagy and endoplasmic reticulum stress in MDA-MB-231 cells. In conclusion, the findings support the potential of usenamine A as an agent that can attenuate the development and progression of breast cancer.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

Chromatographic purification of a methanol extract of the roots of Lespedeza bicolor led to the isolation of four new pterocarpans (1-4), two new coumestans (6 and 7), two new arylbenzofurans (8 and 9), and the known pterocarpan 1-methoxyerythrabyssin II (5). Their structures were identified using NMR spectroscopy, UV spectroscopy, and mass spectrometry. Cytotoxicity assays showed that compounds 1-9 exerted antiproliferative effects on blood cancer cells. Of these compounds, 1 and 6 induced mitochondrial depolarization and induced apoptosis in Jurkat cells. These compounds promoted cell death by inducing cell-cycle arrest at the G1 stage, reducing levels of BCL2, and increasing cleavage of PARP-1. These findings indicate that 1 and 6 are possible lead compounds for the treatment of human leukemia cells via intracellular signaling.

Esophageal cancer (EC) is one of the malignant cancer with pool survival due to the limited therapeutic and drug-resistance. Narciclasine, a natural compound from Lycoris sanguinea possesses antitumor and anti-inflammatory properties. However, the mechanisms underlying the growth-inhibitory effect of narciclasine against EC have not yet been elucidated. Experimental evidences indicated that narciclasine treatment significantly affected the distribution of FAK and its phosphorylation, resulting in proliferation inhibition and migration inhibition of EC. Our study also showed that narciclasine treatment triggered DNA damage and inhibited DNA replication, leading to cell cycle arrest and apoptosis. Further mechanistic studies indicated that narciclasine inhibited EC cell proliferation and migration through FAK/JNK and p38 pathway. Altogether, these findings suggest that narciclasine could be a potential novel chemotherapeutic agent for esophageal cancer cell proliferation and migration.

Our aim was to verify the potential ability of succinylacetone (SA) to inhibit mitochondrial function, thereby suppressing cancer cell proliferation. SA treatment caused apoptosis in HCT116 and HT29 cells, but not in SW480 cells, with mitochondria playing a key role. We checked for dysfunctional mitochondria after SA treatment. Mitochondria of HT29 cells were swollen, indicating damage, whereas in HCT116 cells, several mitochondria had a diminished size. Damaged mitochondria decreased ATP production and induced reactive oxygen species (ROS) in the cells. To understand SA-induced reduction in ATP production, we investigated the electron transfer chains (ETC) and pyruvate dehydrogenase kinase (PDK) activity, which prevents the transfer of acetyl-CoA to the TCA (tricarboxylic acid) cycle by inhibiting PDH (pyruvate dehydrogenase) activity. In each cell line, the inhibitory mechanism of ATP by SA was different. The activity of complex III consisting of the mitochondrial ETCs in HT29 cells was decreased. In contrast, PDH activity in HCT116 cells was reduced. Nicotinamide nucleotide transhydrogenase (NNT)-removing reactive oxygen species (ROS) was upregulated in HT29 cells, but not in HCT116 cells, indicating that in HT29 cells, a defense mechanism was activated against ROS. Collectively, our study showed a differential mechanism occurs in response to SA in colon cancer cells.

The aim of this study was to investigate the potential anti-cancer effects of probiotic cell-free supernatant (CFS) treatment using Lactobacillus fermentum for colorectal cancer (CRC) in 3D culture systems. Cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assays, whereas apoptosis was monitored through RT-qPCR analysis of Bax, Bak, Noxa, and Bid mRNA expressions in addition to flow cytometry analysis of Lactobacillus cell-free supernatant (LCFS) treatment. Our results showed that the anti-cancer effect of LCFS on cell viability was pronouncedly enhanced in 3D-cultured HCT-116 cells, which was linked to the increased level of cleaved caspase 3. Additionally, upregulation of apoptotic marker gene mRNA transcription was dramatically increased in 3D cultured cells compared to 2D systems. In conclusion, this study suggests that LCFS enhances the activation of intrinsic apoptosis in HCT-116 cells and the potential anti-cancer effects of Lactobacilli mixtures in 3D culture systems. All in all, our study highlights the benefits of 3D culture models over 2D culture modeling in studying the anti-cancer effects of probiotics.

Betula platyphylla bark has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diarylheptanoids are the major constituents of the B. platyphylla bark and possess various pharmacological effects. Our previous study confirmed the selective antiproliferative effect of platyphylloside (BPP) isolated from B. platyphylla on colon cancer and leukemic cells using 60 different cancer cell lines from thr National Cancer Institution (NCI). In line with previous reports, this study focuses on the apoptotic pathway of BPP, a phenolic glycoside composed of two aromatic rings joined by a seven-carbon chain. Cytotoxicity assays in solid tumor and blood cancer cell models demonstrated that BPP possesses potent antiproliferative activity. The level of apoptosis increased with BPP treatment, causing cell cycle arrest at the G1 phase along with the downregulation of IκBα phosphorylation and BCL-2, as well as upregulation of cleaved caspase 3 and BAX proteins. In addition, BPP displayed potent mitochondrial depolarization effects in Jurkat cells. The combined findings revealed that the cytotoxic effects of BPP were mediated by intracellular signaling, possibly through a mechanism involving the upregulation of mitochondrial reactive oxygen species (ROS). Thus, BPP could be a potential multitarget therapeutic agent in leukemia and colon cancer.

This study explores the potential anticancer effects of lesbicoumestan from Lespedeza bicolor against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-&kappa, B pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-&kappa, B pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter&rsquo, s active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.

Mallotus japonicus has been evaluated for the treatment of dermatitis, inflammatory conditions, and cancer. Diterpenes, one of the major constituents of M. japonicus, possess various pharmacological effects. In this study, 2 known diterpenes, anomaluone (6) and 16-epiabbeokutone (7), along with other known compounds, 2-hydroxy ferulic acid (1), bergenin (2), gallocatechin (3), catechin (4), erythro,erythro-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl) ethoxy]-3,5-dimethoxyphenyl]-1,2,3- propanetriol (5), and gallincin (8), were isolated from M. japonicus. Cytotoxicity assays in blood cancer cell models demonstrated that M. japonicus compounds possess potent antiproliferative activity. In addition, treatment with compound 6 increased the number of apoptotic cells, led to cell-cycle arrest at the subG0/G1 phase, and decreased the number of cells in the S and G2/M phases. Compound 6 also displayed potent mitochondrial depolarization effects in Jurkat cells. These findings revealed that the cytotoxic effects of 6 were mediated by intracellular signaling, possibly through a mechanism involving upregulation of mitochondrial reactive oxygen species. Thus, compound 6 could be a potential multi-target therapeutic agent for leukemia.

Hepatic stellate cells (HSCs) are involved in the pathogenesis of liver fibrosis. Resveratrol, 3,5,4&prime, trihydroxystilbene, is a dietary polyphenol found in natural food products. Here, we evaluated the anti-proliferative effects of a synthetic resveratrol derivative, 3,5-diethoxy-3&prime, hydroxyresveratrol (DEHR), on HSCs. Flow cytometry and Western blot analyses showed that DEHR induces apoptosis through the upregulation of cleaved caspase-3 and poly (ADP-ribose) polymerase expression and reduction in the level of an anti-apoptotic protein B-cell lymphoma 2 (Bcl2). As caveolin-1 (CAV1), a competitive inhibitor of heme oxygenase 1 (HO-1), is related to apoptotic proteins in hepatic cells, we focused on the role of CAV1 in DEHR-induced apoptosis in HSCs through Western blot analyses. Our results showed that the inhibitory effect of DEHR on cell viability was stronger in HO-1 siRNA-transfected cells but weakened in CAV1 siRNA-transfected cells. Collagen concentration was significantly reduced, whereas CAV1 expression increased after treatment of a bile duct ligation injury-induced liver fibrosis model with DEHR for four weeks. We confirmed that DEHR treatment significantly reduced fibrous hyperplasia around the central veins, using hematoxylin and eosin and Sirius red staining. DEHR ameliorates liver fibrosis in vitro and in vivo, possibly through a mechanism involving CAV1.

Deuterium oxide (D2O) has been reported to be active toward various in vitro cell lines in combination with phytochemicals. Our objective was to describe, for the first time, the effect of D2O on the proliferation of hepatic stellate cells (HSCs). After D2O treatment, the p53-cyclin-dependent kinase (CDK) pathway was stimulated, leading to inhibition of the proliferation of HSCs and an increase in the [ATP]/[ADP] ratio. We also evaluated the role of aquaporin (AQP) 11 in activated HSCs. We found that D2O treatment decreased AQP11 expression levels. Of note, AQP11 levels elevated by a genetic approach counteracted the D2O-mediated inhibition of proliferation. In addition, the expression levels of AQP11 negatively correlated with those of p53. On the other hand, cells transfected with an AQP11-targeted small interfering RNA (siRNA) showed enhanced inhibition of proliferation. These findings suggest that the inhibition of cell proliferation by D2O in activated HSCs could be AQP11 dependent. Our previous studies have documented that bisdemethoxycurcumin (BDMC) induces apoptosis by regulating heme oxygenase (HO)-1 protein expression in activated HSCs. In the current study, we tested whether cotreatment with BDMC and D2O can modulate the AQP11-dependent inhibition of cell proliferation effectively. We observed that D2O cotreatment with BDMC significantly decreased cell proliferation compared to treatment with D2O alone, and this effect was accompanied by downregulation of HO-1 and an increase in p53 levels.