Determining the In Vitro Ligand-Target Interaction by Cellular Thermal Shift Assay and Isothermal Dose-Response Fingerprint Assay.

The cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRF CETSA) have been introduced as powerful tools for investigating target engagement by measuring ligand-triggered thermodynamic stabilization of cellular target proteins. Yet, these techniques have rarely been used to evaluate the thermal stability of RNA-binding proteins (RBPs) when exposed to ligands. Here, we present an adjusted approach using CETSA and ITDRFCETSA to determine the interaction between enasidenib and RBM45. Our assay is sensitive and time-efficient and can potentially be adapted for studying the interactions of RBM45 protein with other potential candidates. Key features • This protocol builds upon the method developed by Molina et al. and extends its application to new protein classes, such as RBPs.

Rupatadine inhibits colorectal cancer cell proliferation through the PIP5K1A/Akt/CDK2 pathway.

BACKGROUND: Phosphatidylinositol-4-phosphate 5-kinase type 1 alpha (PIP5K1A) acts upstream of the Akt regulatory pathway and is abnormally expressed in many types of malignancies. However, the role and mechanism of PIP5K1A in colorectal cancer (CRC) have not yet been reported. In this study, we aimed to determine the association between PIP5K1A and progression of CRC and assess the efficacy and mechanism by which rupatadine targets PIP5K1A. METHODS: Firstly, expression and function of PIP5K1A in CRC were investigated by human colon cancer tissue chip analysis and cell proliferation assay. Next, rupatadine was screened by computational screening and cytotoxicity assay and interactions between PIP5K1A and rupatadine assessed by kinase activity detection assay and bio-layer interferometry analysis. Next, rupatadine's anti-tumor effect was evaluated by in vivo and in vitro pharmacodynamic assays. Finally, rupatadine's anti-tumor mechanism was explored by quantitative real-time reverse-transcription polymerase chain reaction, western blot, and immunofluorescence. RESULTS: We found that PIP5K1A exerts tumor-promoting effects as a proto-oncogene in CRC and aberrant PIP5K1A expression correlates with CRC malignancy. We also found that rupatadine down-regulates cyclin-dependent kinase 2 and cyclin D1 protein expression by inhibiting the PIP5K1A/Akt/GSK-3ß pathway, induces cell cycle arrest, and inhibits CRC cell proliferation in vitro and in vivo. CONCLUSIONS: PIP5K1A is a potential drug target for treating CRC. Rupatadine, which targets PIP5K1A, could serve as a new option for treating CRC, its therapeutic mechanism being related to regulation of the Akt/GSK-3ß signaling pathway.

Lasiokaurin Regulates PLK1 to Induce Breast Cancer Cell G2/M Phase Block and Apoptosis.

Aim of the study: To investigate the anti-tumor effects of Lasiokaurin on breast cancer and explore its underlying molecular mechanism. Materials and methods: In this study, MTT assay, plate colony formation assays, soft agar assay, and EdU assay were employed to evaluate the anti-proliferation effects of LAS. Apoptosis and cell cycle distribution were detected by flow cytometry. The molecular mechanism was predicted by performing RNA sequencing and verified by using immunoblotting assays. Breast cancer organiods derived from patient-derived xenografts model and MDA-MB-231 xenograft mouse model were established to assess the effect of LAS. Results: Our study showed that LAS treatment significantly suppressed cell viability of 5 breast cancer cell lines, with the IC50 value of approximately 1-5 µM. LAS also inhibitied the clonogenic ability and DNA synthesis of breast cancer cells, Moreover, LAS induced apoptosis and G2/M cell cycle arrest in SK-BR-3 and MDA-MB-231 cells. Notably, transcriptomic analysis predicted the mechanistic involvement of PLK1 in LAS-suppressed breast cancer progression. Our experiment data further verified that LAS reduced PLK1 mRNA and protein expression in breast cancer, accompanied by downregulating CDC25C and AKT phosphorylation. Ultimately, we confirmed that LAS inhibit breast cancer growth via inhibiting PLK1 pathway in vivo. Conclusions: Collectively, our findings revealed that LAS inhibits breast cancer progression via regulating PLK1 pathway, which provids scientific evidence for the use of traditional Chinese medicine in cancer therapy.

Arnicolide C Suppresses Tumor Progression by Targeting 14-3-3θ in Breast Cancer.

Background: Arnicolide C, which is isolated from Centipeda minima, has excellent antitumor effects. However, the potential impacts and related mechanisms of action of arnicolide C in breast cancer remain unknown. Methods: The viability of breast cancer cells was measured using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and colony formation assays. For analysis of apoptosis and the cell cycle, flow cytometry was used. A molecular docking approach was used to explore the possible targets of arnicolide C. Western blot analysis was used to detect changes in the expression of 14-3-3θ and proteins in related pathways after arnicolide C treatment in breast cancer cells. The anti-breast cancer effect of arnicolide C in vivo was evaluated by establishing cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Results: Arnicolide C inhibited proliferation, increased apoptosis, and induced G1 arrest. In particular, molecular docking analysis indicated that arnicolide C binds to 14-3-3θ. Arnicolide C reduced 14-3-3θ expression and inhibited its downstream signaling pathways linked to cell proliferation. Similar results were obtained in the CDX and PDX models. Conclusion: Arnicolide C can have an anti-breast cancer effect both in vitro and in vivo and can induce cell cycle arrest and increase apoptosis in vitro. The molecular mechanism may be related to the effect of arnicolide C on the expression level of 14-3-3θ. However, the specific mechanism through which arnicolide C affects 14-3-3θ protein expression still needs to be determined.

SLC7A11 promotes EMT and metastasis in invasive pituitary neuroendocrine tumors by activating the PI3K/AKT signaling pathway.

Invasive pituitary neuroendocrine tumors (PitNETs) are the most prevalent types of intracranial and neuroendocrine tumors. Their aggressive growth and difficulty in complete resection result in a high recurrence rate. Cystine transporter solute carrier family 7 member 11 (SLC7A11) is overexpressed in various cancers, which contributes to tumor growth, progression, and metastasis by promoting cystine uptake and glutathione biosynthesis. We identified SLC7A11 as an invasive biomarker based on three Gene Expression Omnibus cohorts. This study aimed to investigate the role of SLC7A11 in invasive PitNETs. Cell proliferation was assessed using CCK-8 and colony formation assays, while cell apoptosis was estimated with flow cytometry. Wound healing assays and transwell assays were utilized to evaluate migration and invasion ability. Our findings demonstrated that SLC7A11 was markedly upregulated in invasive PitNETs, and was associated with the invasiveness of PitNETs. Knockdown of SLC7A11 could largely suppress tumor cell proliferation, migration, and invasion, while inducing apoptosis. Furthermore, SLC7A11 depletion was implicated in regulating epithelial-mesenchymal transition and inactivating the PI3K/AKT signaling pathway. These insights suggest SLC7A11 as a potential therapeutic target for invasive PitNETs.

The cell cycle regulator p16 promotes tumor infiltrated CD8+ T cell exhaustion and apoptosis.

The therapeutic efficacy of adoptive T cell therapy is largely restricted by reduced viability and dysfunction of CD8+ T cells. Continuous antigen stimulation disrupts the expansion, effector function, and metabolic fitness of CD8+ T cells, leading to their differentiation into an exhausted state within the tumor microenvironment (TME). While the function of the cell cycle negative regulator p16 in senescent cells is well understood, its role in T cell exhaustion remains unclear. In this study, we demonstrated that TCR stimulation of CD8+ T cells rapidly upregulates p16 expression, with its levels positively correlating with TCR affinity. Chronic TCR stimulation further increased p16 expression, leading to CD8+ T cell apoptosis and exhaustion differentiation, without inducing DNA damage or cell senescence. Mechanistic investigations revealed that p16 downregulates mTOR, glycolysis, and oxidative phosphorylation (OXPHOS) associated gene expression, resulting in impaired mitochondrial fitness, reduced T cell viability, and diminished effector function. Furthermore, the deletion of p16 significantly enhances the persistence of CD8+ T cells within tumors and suppresses the terminal exhaustion of tumor-infiltrating T cells. Overall, our findings elucidate how increased p16 expression reshapes T cell intracellular metabolism, drives T cell apoptosis and exhaustion differentiation, and ultimately impairs T cell anti-tumor function.

Marsdenia tenacissima extract alters crucial metabolites in cancer, determined by 1 H NMR based metabolomics approach

ABSTRACT Altered metabolites level in the biosystems, is the potential cause of cancer, the primary reason of alteration of metabolism is change in nutrient consumption and waste excretion, as a result genetic mutation leads to cancer initiation and progression. Aberration of specific metabolites such as fumarate, succinate, 2-hydroxyglutarate may alter cell signaling. We collected liver and kidney samples and prepared for 1H NMR analysis, then executed NMR spectroscopy. We used a set of domestic R scripts to perform an unsupervised principal component analysis (PCA) and a supervised orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA). It signifies class discrimination for getting a clear separation, whereas PCA scores plot signifies the model group kept further away from the control group than drug group on the horizontal axis. In another PCA scores plots, most parts of the control group was overlapping with the drug group but was distant from the model group. Marsdenia tenacissima extract (MTE) (Chines name: Xiao-Ai-Ping, XAP) modulates level of crucial metabolites such as fumarate, lactate, succinate, determined by 1H NMR spectroscopy and their altered level contributes major role in cancer

Targeting GLS1 to cancer therapy through glutamine metabolism

Glutamine metabolism is one of the hallmarks of cancers which is described as an essential role in serving as a major energy and building blocks supply to cell proliferation in cancer cells. Many malignant tumor cells always display glutamine addiction. The “kidney-type” glutaminase (GLS1) is a metabolism enzyme which plays a significant part in glutaminolysis. Interestingly, GLS1 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, GLS1 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that its inhibitors could provide a benefit strategy for cancer therapy. Herein, we summarize the prognostic value of GLS1 in multiple cancer type and its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for GLS1 inhibitors. On the basis of case studies, our perspectives for targeting GLS1 and development of GLS1 antagonist are discussed in the final part (AU)