Derived from fangchinoline, LYY-35 exhibits an inhibiting effect on human NSCLC cancer A549 cells.

Although fangchinoline has been widely used as an adjunct therapy for a variety of inflammatory and cancerous diseases, its mechanism of action on tumor cells remains unclear. Fangchinoline derivative LYY-35 reduced the number of A549 cells, deformed cell morphology and increased cell debris. Cell viability was significantly reduced, while the same concentration of LYY-35 had little effect on BEAS-2B viability of normal lung epithelial cells. In addition, LYY-35 can also reduce the migration, proliferation and invasion ability of A549 cells. Levels of ß-catenin, ZO-1 and ZEB-1 proteins, biomarkers of cell adhesion and epithelial mesenchymal transformation, were significantly reduced. The levels of superoxide dismutase and lactate dehydrogenase decreased gradually, while the levels of glutathione, malondialdehyde and intracellular and extracellular ROS increased significantly. At the same time, LYY-35 induced increased apoptosis, increased expression of Bax, cleaved caspase3, cleaved PARP1, and decreased expression of Bcl-xl, which blocked the cell cycle to G0/G1 phase. The expressions of cell cycle checkpoint proteins Cyclin B1, Cyclin E1, CDK6, PCNA and PICH were significantly decreased. With the increase of LYY-35 concentration, the trailing phenomenon was more obvious in single cell gel electrophoresis. DNA damage repair proteins: BLM, BRCA-1 and PARP-1 expression decreased gradually.LYY-35 can inhibit the proliferation of non-small cell lung cancer A549 cells, block cell cycle, promote apoptosis, increase ROS production, cause DNA damage and interfere with DNA replication. LYY-35 is promising for the treatment of non-small cell lung cancer in the future.

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor.

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

Network pharmacology and in vitro experimental verification unveil glycyrrhizin from glycyrrhiza glabra alleviates acute pancreatitis via modulation of MAPK and STAT3 signaling pathways.

Acute pancreatitis (AP) is a severe gastrointestinal inflammatory disease with increasing mortality and morbidity. Glycyrrhiza glabra, commonly known as Liquorice, is a widely used plant containing bioactive compounds like Glycyrrhizin, which possesses diverse medicinal properties such as anti-inflammatory, antioxidant, antiviral, and anticancer activities. The objective of this study is to investigate the active components, relevant targets, and underlying mechanisms of the traditional Chinese medicine Glycyrrhiza glabra in the treatment of AP. Utilizing various computational biology methods, we explored the potential targets and molecular mechanisms through Glycyrrhizin supplementation. Computational results indicated that Glycyrrhizin shows promising pharmacological potential, particularly with mitogen-activated protein kinase 3 (MAPK3) protein (degree: 70), forming stable complexes with Glycyrrhizin through ionic and hydrogen bonding interactions, with a binding free energy (ΔGbind) of -33.01 ± 0.08 kcal/mol. Through in vitro experiments, we validated that Glycyrrhizin improves primary pancreatic acinar cell injury by inhibiting the MAPK/STAT3/AKT signaling pathway. Overall, MAPK3 emerges as a reliable target for Glycyrrhizin's therapeutic effects in AP treatment. This study provides novel insights into the active components and potential targets and molecular mechanisms of natural products.

Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness.

Tonic signaling of chimeric antigen receptor (CAR), i.e., the spontaneous CAR activation in the absence of tumor antigen stimulation, is considered to be a pivotal event controlling CAR-T efficacy. However, the molecular mechanism underlying the spontaneous CAR signals remains elusive. Here, we unveil that positively charged patches (PCPs) on the surface of the CAR antigen-binding domain mediate CAR clustering and result in CAR tonic signaling. For CARs with high tonic signaling (e.g., GD2.CAR and CSPG4.CAR), reducing PCPs on CARs or boosting ionic strength in the culture medium during ex vivo CAR-T cell expansion minimizes spontaneous CAR activation and alleviates CAR-T cell exhaustion. In contrast, introducing PCPs into the CAR with weak tonic signaling, such as CD19.CAR, results in improved in vivo persistence and superior antitumor function. These results demonstrate that CAR tonic signaling is induced and maintained by PCP-mediated CAR clustering. Notably, the mutations we generated to alter the PCPs maintain the antigen-binding affinity and specificity of the CAR. Therefore, our findings suggest that the rational tuning of PCPs to optimize tonic signaling and in vivo fitness of CAR-T cells is a promising design strategy for the next-generation CAR.

Thioredoxin-interacting protein-activated intracellular potassium deprivation mediates the anti-tumour effect of a novel histone acetylation inhibitor HL23, a fangchinoline derivative, in human hepatocellular carcinoma.

INTRODUCTION: Hyperactivated histone deacetylases (HDACs) act as epigenetic repressors on gene transcription and are frequently observed in human hepatocellular carcinoma (HCC). Although multiple pharmacological HDAC inhibitors (HDACis) have been developed, none is available in human HCC. OBJECTIVES: To investigate the pharmacological effects of a fangchinoline derivative HL23, as a novel HDACi and its molecular mechanisms through TXNIP-mediated potassium deprivation in HCC. METHODS: Both in vitro assays and orthotopic HCC mouse models were used to investigate the effects of HL23 in this study. The inhibitory activity of HL23 on HDACs was evaluated by in silico studies and cellular assays. Chromatin immunoprecipitation (ChIP) was conducted to confirm the regulation of HL23 on acetylation mark at TXNIP promoter. Genome-wide transcriptome analysis together with bioinformatic analysis were conducted to identify the regulatory mechanisms of HL23. The clinical significance of TXNIP and HDACs was evaluated by analysing publicly available database. RESULTS: HL23 exerted compatible HDACs inhibition potency as Vorinostat (SAHA) while had superior anti-HCC effects than SAHA and sorafenib. Both in vitro and in vivo studies showed HL23 significantly suppressed HCC progression and metastasis. HL23 significantly upregulated TXNIP expression via regulating acetylation mark (H3K9ac) at TXNIP promoter. TXNIP was responsible for anti-HCC activity of HL23 through mediating potassium channel activity. HDAC1 was predicted to be the target of HL23 and HDAC1lowTXNIPhigh could jointly predict promising survival outcome of patients with HCC. Combination treatment with HL23 and sorafenib could significantly enhance sorafenib efficacy. CONCLUSION: Our study identified HL23 as a novel HDACi through enhancing acetylation at TXNIP promoter to trigger TXNIP-dependent potassium deprivation and enhance sorafenib efficacy in HCC treatment.

Discovery of Mitochondrial Complex I Inhibitors as Anticancer and Radiosensitizer Drugs Based on Compensatory Stimulation of Lactate Release.

Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.

Targeting cancer cells in acidosis with conjugates between the carnitine palmitoyltransferase 1 inhibitor etomoxir and pH (low) insertion peptides.

Targeting enzymes involved in tumor metabolism is a promising way to tackle cancer progression. The inhibition of carnitine palmitoyltransferase 1 (CPT1) by etomoxir (Eto) efficiently slows down the growth of various cancers. Unfortunately, the clinical use of this drug was abandoned because of hepatotoxic effects. We report the development of pH-sensitive peptide (pHLIP)-drug conjugate to deliver Eto selectively to cancer cells exposed to acidic microenvironmental conditions. A newly designed sequence for the pHLIP peptide, named pHLIPd, was compared with a previously published reference pHLIP peptide, named pHLIPr. We showed that the conjugate between pHLIPd and Eto has a better pH-dependent insertion and structuration than the pHLIPr-based conjugate inside POPC vesicles. We observed antiproliferative effects when applied on acid-adapted cancer cells, reaching a larger inhibitory activity than Eto alone. In conclusion, this study brings the first evidence that pHLIP-based conjugates with a CPT1 inhibitor has the potential to specifically target the tumor acidic compartment and exert anticancer effects while sparing healthy tissues.

A novel class of C14-sulfonate-tetrandrine derivatives as potential chemotherapeutic agents for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), the most common malignancy of the liver, exhibits high recurrence and metastasis. Structural modifications of natural products are crucial resources of antitumor drugs. This study aimed to synthesize C-14 derivatives of tetrandrine and evaluate their effects on HCC. Forty C-14 sulfonate tetrandrine derivatives were synthesized and their in vitro antiproliferative was evaluated against four hepatoma (HepG-2, SMMC-7721, QGY-7701, and SK-Hep-1) cell lines. For all tested cells, most of the modified compounds were more active than the lead compound, tetrandrine. In particular, 14-O-(5-chlorothiophene-2-sulfonyl)-tetrandrine (33) exhibited the strongest antiproliferative effect, with half-maximal inhibitory concentration values of 1.65, 2.89, 1.77, and 2.41 µM for the four hepatoma cell lines, respectively. Moreover, 33 was found to induce apoptosis via a mitochondria-mediated intrinsic pathway via flow cytometry and western blotting analysis. In addition, colony formation, wound healing, and transwell assays demonstrated that 33 significantly inhibited HepG-2 and SMMC-7721 cell proliferation, migration, and invasion, indicating that it might potentially be a candidate for an anti-HCC therapy in the future.

An Effective Treatment of Perimenopausal Syndrome by Combining Two Traditional Prescriptions of Chinese Botanical Drugs.

Ethnopharmacological relevance: Two types of traditional Chinese formulas of botanical drugs are prescribed for treating perimenopausal syndrome (PMS), a disorder in middle-aged women during their transition to menopause. One is for treating PMS as kidney deficiency (KD) due to senescence and declining reproductive functions, and the other is for treating it as liver qi stagnation (LQS) in association with stress and anxiety. Despite the time-tested prescriptions, an objective attestation to the effectiveness of the traditional Chinese treatment of PMS is still to be established and the associated molecular mechanism is still to be investigated. Materials and methods: A model for PMS was generated from perimenopausal rats with chronic restraint stress (CRS). The effectiveness of traditional Chinese formulas of botanical drugs and a combination of two of the formulas was evaluated based on 1H NMR plasma metabolomic, as well as behavioral and physiological, indicators. To investigate whether the formulas contained ligands that could compensate for the declining level of estrogen, the primary cause of PMS, the ligand-based NMR technique of saturation transfer difference (STD) was employed to detect possible interacting molecules to estrogen receptors in the decoction. Results: Each prescription of the classical Chinese formula moderately attenuated the metabolomic state of the disease model. The best treatment strategy however was to combine two traditional Chinese formulas, each for a different etiology, to adjust the metabolomic state of the disease model to that of rats at a much younger age. In addition, this attenuation of the metabolomics of the disease model was by neither upregulating the estrogen level nor supplementing an estrogenic compound. Conclusion: Treatment of PMS with a traditional Chinese formula of botanical drugs targeting one of the two causes separately could ameliorate the disorder moderately. However, the best outcome was to treat the two causes simultaneously with a decoction that combined ingredients from two traditional prescriptions. The data also implicated a new paradigm for phytotherapy of PMS as the prescribed decoctions contained no interacting compound to modulate the activity of estrogen receptors, in contrast to the treatment strategy of hormone replacement therapy.

The anti-dysenteric drug fraxetin enhances anti-tumor efficacy of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.

Fraxetin, a natural product isolated and purified from the bark of Fraxinus bungeana A.DC., has anti-inflammatory, analgesic, and anti-dysenteric activities. This study aimed to investigate the anti-tumor effects of fraxetin in pancreatic ductal adenocarcinoma (PDA). The effects of fraxetin on the malignant biological behavior of PDA were evaluated. Besides, the effects of fraxetin on the sensitivity of PCCs to gemcitabine, angiogenesis, the epithelial-mesenchymal transition (EMT), glucose metabolism, reactive oxygen species (ROS), and STAT3 activity were analyzed. By reversing the EMT, fraxetin suppressed proliferation, invasion, and migration, and induced mitochondrial-dependent apoptosis in PCCs. Also, treatment with fraxetin inhibited PDA growth and metastasis in nude mouse models. Furthermore, fraxetin made PCCs more sensitive to the chemotherapy drug gemcitabine. Mechanically, fraxetin treatment suppressed oncogenic KRAS-triggered STAT3 activation in PCCs and PDA tissues. Fraxetin shows significant interactions with STAT3 Src Homology 2 (SH2) domain residues, thereby preventing its homo-dimer formation, which then blocks the activation of downstream signal pathways. The anti-tumor activity of fraxetin in PDA was functionally rescued by a STAT3 activator colivelin. As a result, fraxetin hindered hypoxia-induced angiogenesis by decreasing HIF-1α and VEGFA expression, controlled glucose metabolism by reducing GLUT1 expression, inhibited the EMT by blocking the Slug-E-cadherin axis, and drove ROS-mediated apoptosis by regulating the STAT3-Ref1 axis. In conclusion, fraxetin enhances the anti-tumor activity of gemcitabine and suppresses pancreatic cancer development by antagonizing STAT3 activation.

Discovery of tetrandrine derivatives as tumor migration, invasion and angiogenesis inhibitors.

Metastatic progression of cancer is a complex and clinically daunting process, with migration, invasion and angiogenesis being the key features. Tetrandrine (TET) is a typical dibenzylisoquinoline alkaloid with promising anti-tumor activity. In our previous work, a number of TET derivatives were designed and synthesized with obvious anti-proliferation activities against cancer cells, however, the anti-metastatic effects of these compounds were not evaluated. In the current investigation, five TET derivatives (8, 18, 32, 71, and 72) with pronounced anti-proliferative activities (IC50 values of 1.00, 1.91, 3.43, 3.78, and 1.93 µM, respectively) against human umbilical vein endothelial cells (HUVECs) were screened out. Scratch assays showed that these compounds significantly suppressed the migration of HUVECs and induced their apoptosis. Among them, derivatives 8 and 72 obviously inhibited the proliferation, colony formation and invasion of HCT-15 cells. Tube formation assays revealed that 4 µM of 8 or 72 remarkably inhibited the tube forming capacity of HUVECs. Moreover, 8 and 72 surpressed the formation of filopodia in HUVECs and severely impaired their motility. Both compounds effectively inhibited the angiogenesis in the zebrafish model with low toxicities in vivo. These results indicated that TET derivatives 8 and 72 are promising anti-metastatic inhibitors.

Design, synthesis and bioactivity investigation of tetrandrine derivatives as potential anti-cancer agents.

Twenty-four 14-sulfonamide-tetrandrine derivatives as potential anti-cancer agents were synthesized. The synthetic derivatives were investigated for their cytotoxic activity against human cancer cell lines MDA-MB-231, PC3, WM9, HEL and K562. Initially, the IC50 values (50% inhibitory concentrations) of all of the compounds were determined. These derivatives exhibited potent, but distinct, inhibitory effects on the above-mentioned cell lines. Among them, compound 23, which was modified with a 2-naphthalenesulfonyl group at the 14-amino position, showed impressive inhibition of all five cancer cell lines, and especially of MDA-MB-231 cells with an IC50 value of 1.18 ± 0.14 µM. Further mechanism exploration showed that 23 induced potent apoptotic cell death on MDA-MB-231 cancer cells in a concentration-dependent manner. The results revealed that 23 might be a potential anti-cancer drug candidate.

Design and synthesis of novel C14-urea-tetrandrine derivatives with potent anti-cancer activity.

Tetrandrine is a dibenzyltetrahydroisoquinoline alkaloid, isolated from traditional Chinese medicinal plant Stephania tetrandra, with anti-tumor activity. Our previous study identified several derivatives of tetrandrine showing better activities than parental compound against human hepatocellular carcinoma cells. To increase diversity and cytotoxic activities of the original compound, a series of novel 14-urea-tetrandrine derivatives were synthesized through structural modification of tetrandrine. These derivaties demonstrated a moderate to strong anti-proliferative activities against human cell lines HEL and K562 (Leukemia), prostate (PC3), breast (MDA-MB-231) and melanoma (WM9). Compound 4g showed strongest cytotoxic effect against PC3 cells with IC50 value of 0.64 µM, which was 12-fold, 31-fold and 26-fold lower than the parental tetrandrine, 5-fluorouracil and cisplatin, respectively. Preliminary structure-activity relationship study indicated that urea subsititution was the key pharmacophore for the enhancement of their antitumor activities. Induction of apoprosis by 4g was associated with the activation of pro-apoptotic protein BAX and inhibition of antiapoptosis proteins survivin as well as Bcl-2. Moreover, activation of caspases led to increase cleavage of PARP, which further accelerates apoptotic cell death. These results reveal that the compound 4g may be used as a potential anticancer drug candidate.

Design, Synthesis and Anticancer Evaluation of Fangchinoline Derivatives.

Twenty fangchinoline derivatives were synthesized from the natural product fangchinoline, and their anticancer activities on human breast cancer MDA-MB-231 cell line, human prostate cancer PC3 cell line, human melanoma WM9 cell line and human leukaemia HEL and K562 cell lines were evaluated. The biological result showed that those derivatives exhibited potent activities on inhibiting cancer cell growth, and the structure-activity relationships were investigated. Among them, compound 4g, which was protected by benzoyl group in 7-phenolic position and nitrified in 14-position, showed impressive inhibition on all 5 cancer cell lines, especially WM9 cell line, with an IC50 value of 1.07 µM. Further mechanistic studies demonstrated that compound 4g may induce cancer cell death by apoptotic means. These research results suggested that compound 4g could be a lead for the further development toward an anticancer agent against human melanoma WM9 in the future.

Novel Flavones from the Root of Phytolacca acinosa Roxb.

Two new flavones, 6,7-methylenedioxy-4-hydroxypeltogynan-7'-one (1), cochliophilin B (2), as well as two known ones, cochliophilin A (3) and 6-methoxy-7-hydroxy flavone (4), were isolated from the ethanol extract of the root of Phytolacca acinosa Roxb. Compound 1 is a flavanol framework with one δ-lactone unit, which is rather rare in nature. The structures of the new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D- and 2D-NMR) analyses, the absolute configuration of 1 was established by comparing experimental and calculated electronic circular dichroism spectra. The structures of known compounds were fixed by comparison with literatures data. Compounds 2 and 4 showed modest inhibitory activities against BEL-7402 cell line, with IC50 values of 28.22 and 39.16 µmol/L, respectively.

Design and synthesis of novel tetrandrine derivatives as potential anti-tumor agents against human hepatocellular carcinoma.

Tetrandrine, a lead anti-tumor compound with a bis-benzyltetrahydroisoquinoline skeleton isolated from medicinal plant Stephania tetrandra. In order to obtain active anti-tumor agents and evaluate their structure-activity relationships, a series of novel tetrandrine derivatives were designed and synthesized in this study. Their anti-tumor activities against human hepatocellular carcinoma cell lines (HMCC97L and PLC/PRF/5) were also evaluated. The bioassay results showed that the derivatives exhibited moderate to strong inhibition against the two cell lines. Among them, compound 31 showed prominent cytotoxicity with IC50 = 1.06 µM (15.8 folds than that of tetrandrine, and 30.3 folds than that of Sorafenib). Further studies on the mechanisms demonstrated that the in vitro anti-tumor activity of compound 31 was predominantly due to the inducement of apoptosis of HCC cells. Compound 31 was capable of initiating endoplasmic reticulum stress-associated apoptotic cell death, and the activation of JNK as well as caspase pathways were probably involved. Our results suggest that compound 31, a new 14-position substituted amide tetrandrine derivative, might be a potential candidate for developing novel anti-HCC drugs in the coming future.

New EGFR inhibitor, 453, prevents renal fibrosis in angiotensin II-stimulated mice.

Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis through the over expression of angiotensin (Ang) II, ultimately leading to chronic kidney disease (CKD). As the main peptide in the RAS, Ang II is a key regulator of nephrotic inflammation, fibrogenic destruction and hypertensive nephropathy. Controlled by growth factors such as TGF-ß, Ang II is thought to be affected by other such growth factors including epidermal growth factor (EGF) due to its ability to stimulate growth, regulate angiogenesis, and desensitize cells from apoptotic stimuli. Here we show that epidermal growth factor receptor (EGFR) plays a key role in Ang II induced renal fibrosis and its inhibition for the use as an effective treatment of CKD. 453, an AG1478 analog, was used to block the EGF-EGFR interaction in vivo in 4-week old mice treated with Ang II and 453. Along with the inhibition of EGFR and its downstream signaling pathways (AKT and ERK), 453 also prevented the activation of fibrotic (collagen, CFGF, TGF-ß), inflammatory (COX2, IL-6, IL-1ß, TNF-α), apoptosis and oxidative stress pathways. These findings suggest the use of 453 as a novel EGFR-inhibitor for therapeutic use in CKD kidney dysfunction.