Novel Arf1 Inhibitors Drive Cancer Stem Cell Aging and Potentiate Anti-Tumor Immunity.

The small G protein Arf1 has been identified as playing a selective role in supporting cancer stem cells (CSCs), making it an attractive target for cancer therapy. However, the current Arf1 inhibitors have limited translational potential due to their high toxicity and low specificity. In this study, two new potent small-molecule inhibitors of Arf1, identified as DU101 and DU102, for cancer therapy are introduced. Preclinical tumor models demonstrate that these inhibitors triggered a cascade of aging in CSCs and enhance anti-tumor immunity in mouse cancer and PDX models. Through single-cell sequencing, the remodeling of the tumor immune microenvironment induced by these new Arf1 inhibitors is analyzed and an increase in tumor-associated CD8+ CD4+ double-positive T (DPT) cells is identified. These DPT cells exhibit superior features of active CD8 single-positive T cells and a higher percentage of TCF1+PD-1+, characteristic of stem-like T cells. The frequency of tumor-infiltrating stem-like DPT cells correlates with better disease-free survival (DFS) in cancer patients, indicating that these inhibitors may offer a novel cancer immunotherapy strategy by converting the cold tumor immune microenvironment into a hot one, thus expanding the potential for immunotherapy in cancer patients.

Ovatodiolide inhibited hepatocellular carcinoma stemness through SP1/MTDH/STAT3 signaling pathway.

Hepatocellular carcinoma (HCC) is characterized with high recurrence and mortality, and the clinical treatments for HCC are very limited. Hepatocellular carcinoma stem cells are the root of HCC progress, recurrence, and multidrug resistance. Ovatodiolide (OVA) is a bioactive diterpenoid served as an inflammatory and immunotherapeutic responses modulator. In this research, we found OVA inhibited HCC stemness through inhibiting MTDH gene transcription. Moreover, we firstly discovered transcription factor SP1 bound to the promoter region of MTDH to transcriptionally regulate MTDH level. Mechanically, we demonstrated OVA decreased SP1 protein stability to transcriptionally inhibit MTDH gene, and inhibited the nuclear translocation of p65, and then diminished IL-6 level to suppress JAK/STAT3 signaling pathway, eventually decreases CD133 level and the stemness of HCC. Furthermore, we demonstrated ACT004, OVA derivative with high metabolic stability towards cytochrome P450 enzymes, showed no genotoxicity and no accumulative or delayed toxicities after long-term administration in rats. And the in vivo efficacy experiments indicated ACT004 inhibited tumor growth of hepatocellular carcinoma. In conclusion, we revealed the mechanism of OVA in regulating HCC stemness, detected the toxicity of OVA derivative and evaluated the in vivo efficacy which lays a foundation for further discovery of anti-HCC stem cell agents and provide a new strategy for the application of OVA in clinical treatment.

Hypocretenolides: collective total syntheses and activities toward metastatic colon cancer.

A concise and collective synthetic route to hypocretenolides was developed for the first time. This route features one-pot addition-alkylation and intramolecular 1,3-dipolar cycloaddition to efficiently assemble the 5/7/6 ring system. Our syntheses enabled multigram preparation of hypocretenolide which facilitated further biological evaluation. Preliminary CCK-8 cytotoxic results of hypocretenolide indicated its IC50 values within 1 µM against 4 colon cancer cell lines. Wound healing and transwell assays suggested the promising inhibitory activities of hypocretenolide toward the migratory capabilities of colon cancer cells in vitro. The animal results confirmed that hypocretenolide can inhibit metastasis of colon cancer cells.

Synthesis of melampomagnolide B derivatives as potential anti-Triple Negative Breast Cancer agents.

Triple-negative breast cancer (TNBC) is the most malignant and aggressive subtype of breast cancer. Currently, the treatment options to TNBC are limited and the discovery of new drugs and novel therapeutic strategies for treatment of TNBC is urgently needed. In this study, a series of melampomagnolide B (MMB) derivatives were designed, synthesized, and evaluated for their anti-TNBC activities. Compound 7 and 13a showed highly potent activity against different TNBC cells with IC50 values ranging from 0.37 µM to 1.52 µM, which demonstrated 3.6- to 54-fold improvement comparing to the parent compound MMB. The phenotypic effect revealed that compound 7 and 13a could inhibit metastasis, induce apoptosis and arrest cell cycle distribution of TNBC cells. Furthermore, the mechanism research indicated compounds 7 and 13a bound IKKß and inhibited the IKKß-mediated phosphorylation of IκB and p65, then inhibited the nuclear translocation of p65 and eventually regulated the genes related to metastasis, apoptosis and cell cycle under NF-κB control. Moreover, compound 7 inhibited the tumor growth in vivo, and the weights of spleens and livers were also reduced compared with control group which indicated that compound 7 could inhibit metastasis of TNBC in vivo. These findings indicate that compound 7 may be used as a promising lead compound for ultimate discovery of anti-TNBC drug.

Dual hypoxia-responsive supramolecular complex for cancer target therapy.

The prognosis with pancreatic cancer is among the poorest of any human cancer. One of the important factors is the tumor hypoxia. Targeting tumor hypoxia is considered a desirable therapeutic option. However, it has not been translated into clinical success in the treatment of pancreatic cancer. With enhanced cytotoxicities against hypoxic pancreatic cancer cells, BE-43547A2 (BE) may serve as a promising template for hypoxia target strategy. Here, based on rational modification, a BE prodrug (NMP-BE) is encapsulated into sulfonated azocalix[5]arene (SAC5A) to generate a supramolecular dual hypoxia-responsive complex NMP-BE@SAC5A. Benefited from the selective load release within cancer cells, NMP-BE@SAC5A markedly suppresses tumor growth at low dose in pancreatic cancer cells xenograft murine model without developing systemic toxicity. This research presents a strategy for the modification of covalent compounds to achieve efficient delivery within tumors, a horizon for the realization of safe and reinforced hypoxia target therapy using a simple approach.

ACT001 inhibited CD133 transcription by targeting and inducing Olig2 ubiquitination degradation.

Lung cancer is the most lethal malignancies with high aggressive and poor prognosis. Until now, the five-year survival rate has not been improved which brings serious challenge to human health. Lung cancer stem cells (LCSCs) serve as the root of cancer occurrence, progression, recurrence, and drug resistance. Therefore, effective anti-cancer agents and molecular mechanisms which could specifically eliminate LCSCs are urgently needed for drug design. In this article, we discovered Olig2 was overexpressed in clinical lung cancer tissues and acted as a transcription factor to regulate cancer stemness by regulating CD133 gene transcription. The results suggested Olig2 could be a promising target in anti-LCSCs therapy and new drugs targeted Olig2 may exhibit excellent clinical results. Furthermore, we verified ACT001, a guaianolide sesquiterpene lactone in phase II clinical trial with excellent glioma remission, inhibited cancer stemness by directly binding to Olig2 protein, inducing Olig2 ubiquitination degradation and inhibiting CD133 gene transcription. All these results suggested that Olig2 could be an excellent druggable target in anti-LCSCs therapy and lay a foundation for the further application of ACT001 in the treatment of lung cancer in clinical.

Cytotoxic Terpenes from the Flowers of Inula japonica against Human Hepatocarcinoma Cells.

Two new 1,10-seco-eudesmanolides (1 and 2) were isolated from the flowers of Inula japonica together with two eudesmanolide analogs (3 and 4) and two monoterpene derivatives (5 and 6). Their structures were established on the basis of detailed spectroscopic analyses and electronic circular dichroism data. All isolates were evaluated for their antiproliferative activities against human hepatocarcinoma HepG2 and SMMC-7721 cells. Japonipene B (3) exhibited the most potent effect with the IC50 values of 14.60±1.62 and 22.06±1.34 µM against HepG2 and SMMC-7721 cells, respectively. Furthermore, japonipene B (3) showed significant efficacies of arresting the cell cycle at the S/G2-M stages, inducing mitochondria-mediated apoptosis, and inhibiting cell migration in HepG2 cells.

Influence of Stent Length on Periprocedural Outcomes After Primary Percutaneous Coronary Intervention in Patients with ST Segment Elevation Myocardial Infarction.

Purpose: A longer stent is associated with adverse events after percutaneous coronary intervention (PCI). However, little information is available on the relationship between stent length and periprocedural prognosis in patients with ST segment elevation myocardial infarction (STEMI). We aimed to assess the target vessel stent length influence on angiographic outcomes and in-hospital major adverse cardiovascular event (MACE) during primary PCI in patients with STEMI. Patients and Methods: This single-center retrospective observational study included 246 patients with STEMI admitted to the Zhejiang Provincial People's Hospital between January 2019 and December 2021, who underwent primary PCI and successful stent implantation. The exclusion criteria included left main lesion, multiple diseased vessel-stenting, bleeding disorders, contrast allergy, and incomplete data. Patients were divided into two groups based on the median stents length: group A (≤29 mm, n=125) and group B (>29mm, n=121). Periprocedural outcomes were slow flow/no-reflow (SF-NR) and in-hospital MACE, which included acute heart failure, malignant arrhythmia, cardiovascular death, non-fatal stroke, non-fatal myocardial infarction, and urgent revascularization. Multivariate logistic analyses were used to explore the correlation between stent length and SF-NR. Results: A total of 246 patients (82.9% males) with a mean age of 59.9±12.6 years were included in the analysis. The incidence of SF-NR was significantly higher in group B than in group A (36.4% vs 23.2%, p=0.024). However, the in-hospital MACE incidence rate was similar between the two groups (7.2% vs 7.4%, p=0.943). Multivariate logistic regression analysis showed that stent length and diameter, and peak troponin I level were independent risk factors for SF-NR. Conclusion: Excessive stent length is an independent risk factor for SF-NR, without any significant influence on the risk of MACE during hospitalization.

Probe Synthesis Reveals Eukaryotic Translation Elongation Factor†1 Alpha†1 as the Anti-Pancreatic Cancer Target of BE-43547A2.

The natural product, BE-43547A2 , decreases pancreatic cancer cell stemness. However, its anticancer molecular mechanisms have not been fully established. Based on structure-activity relationships of BE-43547A2 , we synthesized a probe and investigated its potential targets using an in situ click reaction. We found that BE-43547A2 exerts its anticancer effects by covalently binding the cysteine234 (C234) residue of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1). This binding mode was confirmed by a series of experiments including a xenograft mouse model. We also determined that eEF1A1 plays an important role in regulating pancreatic cancer cell stemness. Analyses of 99 clinical pancreatic cancer samples revealed that eEF1A1 expressions are closely correlated with clinicopathological grade and patient survival. In conclusion, eEF1A1 is involved in pancreatic cancer progression and is therefore, a promising novel covalent target for pancreatic cancer treatment.

Levistilide A Promotes Expansion of Human Umbilical Cord Blood Hematopoietic Stem Cells by Enhancing Antioxidant Activity.

Several approaches to expand human hematopoietic stem cells (hHSCs) clinically along with retainable capability of multipotential differentiation have been reported, but only a few have advanced to evaluation in clinical trials, which limits the application of HSC-based therapy. Here we show a phthalide derivative, Levistilide A (LA), can serve as a promising molecule to expand functional human umbilical cord blood (UCB) HSCs ex vivo. An in-house screen identified LA out of nine natural products as an outstanding candidate for hHSCs expansion. Additionally, our data indicated that LA treatment not only increased the numbers of phenotype-defined HSCs, but also enhanced their colony formation ability. Xenotransplantation assays showed that LA treatment could maintain unaffected engraftment of hHSCs with multilineage differentiation capacity. Further experiments revealed that LA enhanced the antioxidant activity of hHSCs by reducing intracellular and mitochondrial reactive oxygen species (ROS) levels. The identification of LA provides a new strategy in solving the clinical issue of limited numbers of UCB HSCs.

A Two-Phase Approach to Fusicoccane Synthesis To Uncover a Compound That Reduces Tumourigenesis in Pancreatic Cancer Cells.

Alterbrassicicene D (1) and 3(11)-epoxyhypoestenone (2) were synthesised via a two-phase approach featuring concise construction of the 5-8-5 tricyclic intermediate and a tandem base-mediated epoxide opening-transannular oxa-Michael addition cascade to forge the complex skeleton of 2. The route is scalable and we generated 15 g of the tricyclic intermediate in 8 steps from (R)-limonene and 720 mg of the penultimate bioactive intermediate in a protecting-group-free manner. Our synthesis enabled the structural determination of 2 and provided materials for preliminary anticancer evaluation. The penultimate intermediate showed therapeutic potential in terms of its ability to dramatically reduce the tumourigenic potential of PANC-1 pancreatic cancer cells according to a limiting dilution tumour-initiating assay. Our synthetic approach will facilitate unified access to naturally occurring fusicoccanes and their derivatives for anticancer evaluation.

Enhanced self-renewal of human long-term hematopoietic stem cells by a sulfamoyl benzoate derivative targeting p18INK4C.

The use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18INK4C (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.

Total Synthesis and Structure Revision of Boholamide A.

The 15-membered cyclic depsipeptide boholamide A and an epimer were prepared by total synthesis for the first time, thus leading to a revision of C6 stereochemistry in the originally proposed structure of natural boholamide A. This convergent route features achievement of a macro-lactamization step in a gram scale. The revised boholamide A was sythesized with 16 linear steps in 5.46% overall yield. This work facilitates the investigations of boholamide A as a potential hypoxia-selective anticancer agent.

Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells.

BACKGROUND: TNBC is the most aggressive breast cancer with higher recurrence and mortality rate than other types of breast cancer. There is an urgent need for identification of therapeutic agents with unique mode of action for overcoming current challenges in TNBC treatment. METHODS: Different inhibitors were used to study the cell death manner of DMOCPTL. RNA silencing was used to evaluate the functions of GPX4 in ferroptosis and apoptosis of TNBC cells and functions of EGR1 in apoptosis. Immunohistochemical assay of tissue microarray were used for investigating correlation of GPX4 and EGR1 with TNBC. Computer-aided docking and small molecule probe were used for study the binding of DMOCPTL with GPX4. RESULTS: DMOCPTL, a derivative of natural product parthenolide, exhibited about 15-fold improvement comparing to that of the parent compound PTL for TNBC cells. The cell death manner assay showed that the anti-TNBC effect of DMOCPTL mainly by inducing ferroptosis and apoptosis through ubiquitination of GPX4. The probe of DMOCPTL assay indicated that DMOCPTL induced GPX4 ubiquitination by directly binding to GPX4 protein. To the best of our knowledge, this is the first report of inducing ferroptosis through ubiquitination of GPX4. Moreover, the mechanism of GPX4 regulation of apoptosis is still obscure. Here, we firstly reveal that GPX4 regulated mitochondria-mediated apoptosis through regulation of EGR1 in TNBC cells. Compound 13, the prodrug of DMOCPTL, effectively inhibited the growth of breast tumor and prolonged the lifespan of mice in vivo, and no obvious toxicity was observed. CONCLUSIONS: These findings firstly revealed novel manner to induce ferroptosis through ubiquitination of GPX4 and provided mechanism for GPX4 inducing mitochondria-mediated apoptosis through up-regulation of EGR1 in TNBC cells. Moreover, compound 13 deserves further studies as a lead compound with novel mode of action for ultimate discovery of effective anti-TNBC drug.

Lmod3 promotes myoblast differentiation and proliferation via the AKT and ERK pathways.

Mutations in the Lmod3 gene have been identified as a genetic cause of nemaline myopathy. However, the mechanism underlying this disease and the function of Lmod3 remain largely unknown. In this study, we found that Lmod3 knockdown in C2C12 cells impaired myoblast differentiation, whereas enforced Lmod3 expression enhanced such differentiation. We also discovered that myoblast proliferation was promoted by Lmod3 overexpression but impeded by its knockdown. Additionally, knockdown of Lmod3 led to apoptosis in myoblasts. Concurrently, forced Lmod3 expression in C2C12 cells contributed to activation of the AKT and ERK pathways during myoblast differentiation and proliferation, respectively. Conversely, knockdown of Lmod3 in C2C12 cells produced the opposite results. Furthermore, administration of IGF-1, a booster of both AKT and ERK pathways, partially rescued the inhibitory effect of Lmod3 knockdown on both differentiation and proliferation of C2C12 cells. These results suggest that Lmod3 promotes differentiation and proliferation of myoblasts through the AKT and ERK pathways, respectively.

TNFSF15 facilitates human umbilical cord blood haematopoietic stem cell expansion by activating Notch signal pathway.

The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB-derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)-derived HSC. TNFSF15-treated UCB-HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi-SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle-treated group. Additionally, signal proteins of the Notch pathway are highly up-regulated in TNFSF15-treated UCB-HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB-HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB-HSC application or study.

Sesquiterpenoids isolated from the flower of Inula japonica as potential antitumor leads for intervention of paclitaxel-resistant non-small-cell lung cancer.

Three new sesquiterpene lactone dimers (1-3) were isolated from the flowers of Inula japonica together with twenty-two known sesquiterpene derivatives (4-25). Their structures were established on the basis of detailed spectroscopic analyses. All isolates were evaluated for their antiproliferative activities against paclitaxel-resistant human non-small-cell lung cancer cell line A549/PTX. The preliminary structure-activity relationship was discussed. Compound 24 exhibited the most potent effect with the IC50 value of 0.34 ± 0.10 µM, even more active than the clinically used drug paclitaxel (PTX, IC50 = 1.40 ± 0.52 µM). Compound 24 showed significant efficacy of arresting the cell cycle at the G2-M stage, inducing apoptosis through mitochondria-mediated pathway, and inhibiting cell migration and invasion. Furthermore, compound 24 could reverse multidrug resistance through suppressing the expression of ABC family proteins.