Macropinocytosis inhibits alkaliptosis in pancreatic cancer cells through fatty acid uptake.

Alkaliptosis, a form of regulated cell death, is characterized by lysosomal dysfunction and intracellular pH alkalinization. The pharmacological induction of alkaliptosis using the small molecule compound JTC801 has emerged as a promising anticancer strategy in various types of cancers, particularly pancreatic ductal adenocarcinoma (PDAC). In this study, we investigate a novel mechanism by which macropinocytosis, an endocytic process involving the uptake of extracellular material, promotes resistance to alkaliptosis in human PDAC cells. Through lipid metabolomics analysis and functional studies, we demonstrate that the inhibition of alkaliptosis by fatty acids, such as oleic acid, is not dependent on endogenous synthetic pathways but rather on exogenous uptake facilitated by macropinocytosis. Consequently, targeting macropinocytosis through pharmacological approaches (e.g., using EIPA or EHoP-016) or genetic interventions (e.g., RAC1 knockdown) effectively enhances JTC801-induced alkaliptosis in human PDAC cells. These findings provide compelling evidence that the modulation of macropinocytosis can increase the sensitivity of cancer cells to alkaliptosis inducers.

Predicting the prognosis of glioma patients with TERT promoter mutations and guiding the specific immune profile of immune checkpoint blockade therapy.

The telomerase reverse transcriptase promoter (TERTp) is frequently mutated in gliomas. This study sought to identify immune biomarkers of gliomas with TERTp mutations. Data from TCGA were used to identify and validate survival-associated gene signatures, and immune and stromal scores were calculated using the ESTIMATE algorithm. High stromal or immune scores in patients with TERTp-mutant gliomas correlated with shorter overall survival compared to cases with low stromal or immune scores. Among TERTp-mutant gliomas with both high immune and high stromal scores, 213 commonly shared DEGs were identified. Among 71 interacting DEGs representing candidate hub genes in a PPI network, HOXC6, WT1, CD70, and OTP showed significant ability in establishing subgroups of high- and low-risk patients. A risk model based on these 4 genes showed strong prognostic potential for gliomas with mutated TERTp, but was inapplicable for TERTp-wild-type gliomas. TERTp-mutant gliomas with high-risk scores displayed a greater percentage of naïve B cells, plasma cells, naïve CD4 T cells, and activated mast cells than low-risk score gliomas. TIDE analysis indicated that immune checkpoint blockade (ICB) therapy may benefit glioma patients with TERTp mutations. The present risk model can help predict prognosis of glioma patients with TERTp mutations and aid ICB treatment options.

Targeting paraptosis in cancer: opportunities and challenges.

Cell death can be classified into two primary categories: accidental cell death and regulated cell death (RCD). Within RCD, there are distinct apoptotic and non-apoptotic cell death pathways. Among the various forms of non-apoptotic RCD, paraptosis stands out as a unique mechanism characterized by distinct morphological changes within cells. These alterations encompass cytoplasmic vacuolization, organelle swelling, notably in the endoplasmic reticulum and mitochondria, and the absence of typical apoptotic features, such as cell shrinkage and DNA fragmentation. Biochemically, paraptosis distinguishes itself by its independence from caspases, which are conventionally associated with apoptotic death. This intriguing cell death pathway can be initiated by various cellular stressors, including oxidative stress, protein misfolding, and specific chemical compounds. Dysregulated paraptosis plays a pivotal role in several critical cancer-related processes, such as autophagic degradation, drug resistance, and angiogenesis. This review provides a comprehensive overview of recent advancements in our understanding of the mechanisms and regulation of paraptosis. Additionally, it delves into the potential of paraptosis-related compounds for targeted cancer treatment, with the aim of enhancing treatment efficacy while minimizing harm to healthy cells.

Regulation of Cellular Signaling with an Aptamer Inhibitor to Impede Cancer Metastasis.

Tumor invasion and metastasis are the main causes of tumor progression and are the leading causes of death among cancer patients. In the present study, we propose a strategy to regulate cellular signaling with a tumor metastasis-relevant cytoskeleton-associated protein 4 (CKAP4) specific aptamer for the achievement of tumor metastasis inhibition. The designed aptamer could specifically bind to CKAP4 in the cell membranes and cytoplasm to block the internalization and recycling of α5ß1 integrin, resulting in the disruption of the fibronectin-dependent cell adhesion and the weakening of the cell traction force. Moreover, the aptamer is able to impede the interaction between CKAP4 and Dickkopf1 (DKK1) to further block the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which subsequently reduces AKT phosphorylation and inhibits the reorganization of the actin cytoskeleton in cell migration. The synergetic function of the designed aptamer in inhibiting cancer cell adhesion and blocking the PI3K signaling pathway enables efficient tumor cell metastasis suppression. The aptamer with specific targeting ability in regulating cellular signaling paves the way for cancer treatment and further provides a guiding ideology for inhibiting tumor metastasis.

Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes.

While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400 µs separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 µm2/s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ∼60-70% of that in vitro, up to ∼250 µm2/s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments.

T52 attenuates oncogenic STAT3 signaling and suppresses osteosarcoma.

BACKGROUND: T52 is a steroidal saponin extracted from the traditional Chinese herb Rohdea fargesii (Baill.), and it is reported to possess strong anti-proliferative capabilities in human pharyngeal carcinoma cell lines. However, whether T52 has anti-osteosarcoma properties, and its potential mechanism is remains unknown. PURPOSE: To examine the outcome and underlying mechanism of T52 in osteosarcomas (OS). METHODS/STUDY DESIGNS: The physiological roles of T52 in OS cells were examined using CCK-8, colony formation (CF), EdU staining, cell cycle/apoptosis and cell migration/invasion assays. The relevant T52 targets against OS were assessed via bioinformatics prediction, and the binding sites were analyzed by molecular docking. Western blot analysis was carried out to examine the levels of factors associated with apoptosis, cell cycle, and STAT3 signaling pathway activation. RESULTS: T52 markedly diminished the proliferation, migration, and invasion of OS cells, and promoted G2/M arrest and apoptosis in a dose-dependent fashion (DDF) in vitro. Mechanistically, molecular docking predicted that T52 stably associated with STAT3 Src homology 2 (SH2) domain residues. Western blot revealed that T52 suppressed the STAT3 signaling pathway, as well as the expression of the downstream targets, such as, Bcl-2, Cyclin D1, and c-Myc. In addition, the anti-OS property of T52 were partially reversed by STAT3 reactivation, which confirmed that STAT3 signaling is critical for regulating the anti-OS property of T52. CONCLUSION: We firstly demonstrated that T52 possessed strong anti-osteosarcoma property in vitro, which was brought on by the inhibition of the STAT3 signaling pathway. Our findings provided pharmacological support for treating OS with T52.

Total steroidal saponins from black nightshade (Solanum nigrum L.) overcome tumor multidrug resistance by inducing autophagy-mediated cell death in vivo and in vitro.

Multiple drug resistance (MDR) often occurs after prolonged chemotherapy, leading to refractory tumors and cancer recurrence. In this study, we demonstrated that the total steroidal saponins from Solanum nigrum L. (SN) had broad-spectrum cytotoxic activity against various human leukemia cancer cell lines, especially in adriamycin (ADR)-sensitive and resistant K562 cell lines. Moreover, SN could effectively inhibit the expression of ABC transporter in K562/ADR cells in vivo and in vitro. In vivo, by establishing K562/ADR xenograft tumor model, we demonstrated that SN might overcome drug resistance and inhibit the proliferation of tumors by regulating autophagy. In vitro, the increased LC3 puncta, the expression of LC3-II and Beclin-1, and the decreased expression of p62/SQSTM1 in SN-treated K562/ADR and K562 cells demonstrated autophagy induced by SN. Moreover, using the autophagy inhibitors or transfecting the ATG5 shRNA, we confirmed that autophagy induced by SN was a key factor in overcoming MDR thereby promoting cell death in K562/ADR cells. More importantly, SN-induced autophagy through the mTOR signaling pathway to overcome drug resistance and ultimately induced autophagy-mediated cell death in K562/ADR cells. Taken together, our findings suggest that SN has the potential to treat multidrug-resistant leukemia.

Single-molecule displacement mapping indicates unhindered intracellular diffusion of small (<~1 kDa) solutes.

While fundamentally important, the intracellular diffusion of small (<~1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400-µs separation, we extend single-molecule displacement/diffusivity mapping (SM d M), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 µm 2 /s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ~60-70% of that in vitro , up to ~250 µm 2 /s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water, but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments.

Single-molecule displacement mapping unveils sign-asymmetric protein charge effects on intraorganellar diffusion.

Using single-molecule displacement/diffusivity mapping (SM d M), an emerging super-resolution microscopy method, here we quantify, at nanoscale resolution, the diffusion of a typical fluorescent protein (FP) in the endoplasmic reticulum (ER) and mitochondrion of living mammalian cells. We thus show that the diffusion coefficients D in both organelles are ~40% of that in the cytoplasm, with the latter exhibiting higher spatial inhomogeneities. Moreover, we unveil that diffusions in the ER lumen and the mitochondrial matrix are markedly impeded when the FP is given positive, but not negative, net charges. Calculation shows most intraorganellar proteins as negatively charged, thus a mechanism to impede the diffusion of positively charged proteins. However, we further identify the ER protein PPIB as an exception with a positive net charge, and experimentally show that the removal of this positive charge elevates its intra-ER diffusivity. We thus unveil a sign-asymmetric protein charge effect on the nanoscale intraorganellar diffusion.

Single-Molecule Displacement Mapping Unveils Sign-Asymmetric Protein Charge Effects on Intraorganellar Diffusion.

Using single-molecule displacement/diffusivity mapping (SMdM), an emerging super-resolution microscopy method, here we quantify, at nanoscale resolution, the diffusion of a typical fluorescent protein (FP) in the endoplasmic reticulum (ER) and mitochondrion of living mammalian cells. We thus show that the diffusion coefficients D in both organelles are ∼40% of that in the cytoplasm, with the latter exhibiting higher spatial inhomogeneities. Moreover, we unveil that diffusions in the ER lumen and the mitochondrial matrix are markedly impeded when the FP is given positive, but not negative, net charges. Calculation shows most intraorganellar proteins as negatively charged, hence a mechanism to impede the diffusion of positively charged proteins. However, we further identify the ER protein PPIB as an exception with a positive net charge and experimentally show that the removal of this positive charge elevates its intra-ER diffusivity. We thus unveil a sign-asymmetric protein charge effect on the nanoscale intraorganellar diffusion.

Synergistic Combination of the Total Steroidal Saponins from the Berries of Black Nightshade and Adriamycin to Overcome Leukemia Multidrug Resistance.

The berries of black nightshade (Solanum nigrum L.) are consumed as a favorite fruit in some regions and have been reported to possess a range of biological activities. Previous studies have found that the steroidal saponins from the berries of S. nigrum (SN) showed potential antileukemic activity, although the underlying mechanism remains to be revealed. This study investigated the effects and mechanisms of SN in combination with adriamycin to reverse leukemia multidrug resistance in vivo and in vitro. The results indicated that the combination of SN and adriamycin displayed enhanced suppression ability both in vitro and in vivo by the modulation of drug efflux proteins. Further study revealed that SN and adriamycin co-treatment induced cell apoptosis in K562/ADR cells through caspase pathways and autophagy through the PI3K/Akt/mTOR and MAPK signaling pathway. This study provides a new prospect of the berries of black nightshade in multidrug resistance therapy of cancer.

Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers.

The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based on the CRISPR-dPspCas13b system with fluorescent RNA aptamers in sgRNA (CasFAS) in live cells. Using modified sgRNA attached to fluorescent RNA aptamers that showed reduced background fluorescence, this approach provides a simple, sensitive way to image and track endogenous RNA with high accuracy and efficiency. In addition, color switching can be easily achieved by changing the fluorogenic dye analogues in living cells through user-friendly washing and restaining operations. CasFAS is compatible with orthogonal fluorescent aptamers, such as Broccoli and Pepper, enabling multiple colors RNA labeling or intracellular RNA-RNA interaction imaging. Finally, the visualization of severe fever with thrombocytopenia syndrome virus (SFTSV) was achieved by CasFAS, which may facilitate further studies on this virus.

Protein diffusion in Escherichia coli cytoplasm scales with the mass of the complexes and is location dependent.

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli. We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

Aged green tea reduces high-fat diet-induced fat accumulation and inflammation via activating the AMP-activated protein kinase signaling pathway.

Background: Obesity is a global public health concern and increases the risk of metabolic syndrome and other diseases. The anti-obesity effects of various plant-derived bioactive compounds, such as tea extracts, are well-established. The mechanisms underlying the anti-obesity activity of Jinxuan green tea (JXGT) from different storage years are still unclear. Objective: The aim of this study was to evaluate the effects of JXGTs from three different years on the high fat diet (HFD)-fed mouse model. Design: The mice were divided into six groups, the control group received normal diet and the obese model group received HFD. We analyzed the effects of JXGTs from 2005, 2008, and 2016 on HFD-fed obese mice over a period of 7 weeks. Results: The JXGTs reduced the body weight of the obese mice, and also alleviated fat accumulation and hepatic steatosis. Mechanistically, JXGTs increased the phosphorylation of AMP-activated protein kinase (p-AMPK)/AMP-activated protein kinase (AMPK) ratio, up-regulated carnitine acyl transferase 1A (CPT-1A), and down-regulated fatty acid synthase (FAS), Glycogen synthase kinase-3beta (GSK-3ß), Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α), Interleukin 6 (IL-6), and Tumour necrosis factor alpha (TNFα). Thus, JXGTs can alleviate HFD-induced obesity by inhibiting lipid biosynthesis and inflammation, thereby promoting fatty acid oxidation via the AMPK pathway. Discussion: The anti-obesity effect of three aged JXGTs were similar. However, JXGT2016 exhibited a more potent activation of AMPK, and JXGT2005 and JXGT2008 exhibited a more potent inhibiting glycogen synthase and inflammation effect. Furthermore, the polyphenol (-)-epicatechin (EC) showed the strongest positive correlation with the anti-obesity effect of JXGT. Conclusions: These findings demonstrate that JXGT treatment has a potential protection on HFD-induced obesity mice via activating the AMPK/CPT-1A and down-regulating FAS/GSK-3ß/PGC-1α and IL-6/TNFα. Our study results also revealed that different storage time would not affect the anti-obesity and anti-inflammation effect of JXGT.

S-20, a steroidal saponin from the berries of black nightshade, exerts anti-multidrug resistance activity in K562/ADR cells through autophagic cell death and ERK activation.

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Adriamycin (ADR) has been widely used to treat cancer, however, as a substrate of the adenosine triphosphate binding cassette (ABC) transporter, it is easy to develop drug resistance during the treatment. Here, we demonstrated that steroidal saponin S-20 isolated from the berries of black nightshade has comparable cytotoxicity in ADR-sensitive and resistant K562 cell lines. Autophagy is generally considered to be a protective mechanism to mediate MDR during treatment. However, we found that S-20-induced cell death in K562/ADR is associated with autophagy. We further explored the underlying mechanisms and found that S-20 induces caspase-dependent apoptosis in ADR-sensitive and resistant K562 cell lines. Most importantly, S-20-induced autophagy activates the ERK pathway and then inhibits the expression of drug resistance protein, which is the main reason to overcome K562/ADR resistance, rather than apoptosis. Taken together, our findings emphasize that S-20 exerts anti-multidrug resistance activity in K562/ADR cells through autophagic cell death and ERK activation, which may be considered as an effective strategy.

Single Molecules Are Your Quanta: A Bottom-Up Approach toward Multidimensional Super-resolution Microscopy.

The rise of single-molecule localization microscopy (SMLM) and related super-resolution methods over the past 15 years has revolutionized how we study biological and materials systems. In this Perspective, we reflect on the underlying philosophy of how diffraction-unlimited pictures containing rich spatial and functional information may gradually emerge through the local accumulation of single-molecule measurements. Starting with the basic concepts, we analyze the uniqueness of and opportunities in building up the final picture one molecule at a time. After brief introductions to the more established multicolor and three-dimensional measurements, we highlight emerging efforts to extend SMLM to new dimensions and functionalities as fluorescence polarization, emission spectra, and molecular motions, and discuss rising opportunities and future directions. With single molecules as our quanta, the bottom-up accumulation approach provides a powerful conduit for multidimensional microscopy at the nanoscale.

Total saponins from Tupistra chinensis baker inhibits growth of human gastric cancer cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Tupistra chinensis Baker (syn. Rohdea chinensis), an antitumor folk herb mainly distributed in China, its rhizome has been historically used to treat gastric cancer. Studies showed that the steroidal saponins were the main bioactive components in the rhizome of T. chinensis. Our previous studies have confirmed that the steroidal saponins have a variety of anti-tumor activities. However, the underlying anti-tumor mechanism of the total steroidal saponins of T. chinensis (TCS) remains to be revealed. AIM OF THE STUDY: In the present study, we studied the potential anti-proliferative activity and anti-tumor mechanism of TCS on gastric cancer in vitro and in vivo. METHODS: In vitro, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to detect the proliferation ability of TCS on SGC-7901 cells and AGS cells. Flow cytometry were performed to analyze cell apoptosis, cell cycle, mitochondrial membrane potential and reactive oxygen species expression level. Western blotting was performed to validate the expression of proteins in related pathways. In vivo, a xenograft model was established by injecting SGC-7901 cells into nude mice. RESULTS: In vitro, TCS inhibited the proliferation of gastric cancer cells. TCS effectively induced apoptosis by PI3K/Akt/mTOR signaling pathway in SGC-7901 cells, and promoted apoptosis via p53-mediated pathway in AGS cells. TCS also exhibited inhibitory activity in blocking the migration of gastric cancer cells. In vivo, TCS significantly inhibited the growth of xenograft tumor. CONCLUSION: These results indicated that TCS exhibited significant anti-gastric cancer effects in vitro and in vivo.

Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing.

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

A spirostanol saponin isolated from Tupistra chinensis Baker simultaneously induces apoptosis and autophagy by regulating the JNK pathway in human gastric cancer cells.

T-17, a bioactive spirostanol saponin extracted from Tupistra chinensis Baker, was previously reported with anti-inflammatory and cytotoxic activities. However, the mechanism underlying of its anti-proliferation activity remains to be elucidated. In this study, we investigated the anti-gastric cancer cell growth activity of T-17 in terms of cell viability, colony formation, cell cycle, induction of apoptosis/autophagy, and JNK pathway. T-17 showed dose-dependent cytotoxicity in SGC-7901 and AGS cell lines, it induced caspase-mediated apoptosis as well as G0/G1 phase arrest and modulation of cyclinE2 and p21 expression. In addition, T-17 promoted the cancer cell autophagy as evidenced with increased expression of Beclin-1 and decreased p62 in western blot and formation of GFP-LC3 puncta. Furthermore, T-17-induced autophagy decreased gastric cancer cell apoptosis as assessed by pharmacological autophagy inhibitors and ATG5 siRNA usage. Importantly, the activation of JNK pathway was simultaneously involved in T-17-induced apoptosis and autophagy. Taken together, the results suggest that T-17 is a promising cytotoxic agent for therapeutic treatment of human gastric adenocarcinoma, which provides a good foundation for further research and development of Tupistra chinensis Baker.

Conductance and configuration of molecular gold-water-gold junctions under electric fields.

Water molecules can mediate charge transfer in biological and chemical reactions by forming electronic coupling pathways. Understanding the mechanism requires a molecular-level electrical characterization of water. Here, we describe the measurement of single water molecular conductance at room temperature, characterize the structure of water molecules using infrared spectroscopy, and perform theoretical studies to assist in the interpretation of the experimental data. The study reveals two distinct states of water, corresponding to a parallel and perpendicular orientation of the molecules. Water molecules switch from parallel to perpendicular orientations on applying an electric field, producing switching from high to low conductance states, thus enabling the determination of single water molecular dipole moments. The work further shows that water-water interactions affect the atomic scale configuration and conductance of water molecules. These findings demonstrate the importance of the discrete nature of water molecules in electron transfer and set limits on water-mediated electron transfer rates.