Cepharanthine synergistically promotes methylprednisolone pharmacodynamics against human peripheral blood mononuclear cells possibly via regulation of P-glycoprotein/glucocorticoid receptor translocation.

BACKGROUND: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. METHODS: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. RESULTS: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 µM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 µM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 µM of CEP did not influence P-glycoprotein expression. 0.03~0.3 µM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 µM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP.

Fangchinoline inhibits growth and biofilm of Candida albicans by inducing ROS overproduction.

Infections caused by Candida species, especially Candida albicans, threaten the public health and create economic burden. Shortage of antifungals and emergence of drug resistance call for new antifungal therapies while natural products were attractive sources for developing new drugs. In our study, fangchinoline, a bis-benzylisoquinoline alkaloid from Chinese herb Stephania tetrandra S. Moore, exerted antifungal effects on planktonic growth of several Candida species including C. albicans, with MIC no more than 50 µg/mL. In addition, results from microscopic, MTT and XTT reduction assays showed that fangchinoline had inhibitory activities against the multiple virulence factors of C. albicans, such as adhesion, hyphal growth and biofilm formation. Furthermore, this compound could also suppress the metabolic activity of preformed C. albicans biofilms. PI staining, followed by confocal laser scanning microscope (CLSM) analysis showed that fangchinoline can elevate permeability of cell membrane. DCFH-DA staining suggested its anti-Candida mechanism also involved overproduction of intracellular ROS, which was further confirmed by N-acetyl-cysteine rescue tests. Moreover, fangchinoline showed synergy with three antifungal drugs (amphotericin B, fluconazole and caspofungin), further indicating its potential use in treating C. albicans infections. Therefore, these results indicated that fangchinoline could be a potential candidate for developing anti-Candida therapies.

Cepharanthine maintains integrity of the blood-brain barrier (BBB) in stroke via the VEGF/VEGFR2/ZO-1 signaling pathway.

Dysfunction of tight junctions such as zonula occludens protein-1 (ZO-1)-associated aggravation of blood-brain barrier (BBB) permeability plays an important role in the progression of stroke. Cepharanthine (CEP) is an extract from the plant Stephania cepharantha. However, the effects of CEP on stroke and BBB dysfunction have not been previously reported. In this study, we report that CEP improved dysfunction in neurological behavior in a middle cerebral artery occlusion (MCAO) mouse model. Importantly, CEP suppressed blood-brain barrier (BBB) hyperpermeability by increasing the expression of ZO-1. Notably, we found that CEP inhibited the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in the cortex of MCAO mice. Additionally, the results of in vitro experiments demonstrate that treatment with CEP ameliorated cytotoxicity of human bEnd.3 brain microvascular endothelial cells against hypoxia/reperfusion (H/R). Also, CEP attenuated H/R-induced aggravation of endothelial permeability in bEND.3 cells by restoring the expression of ZO-1. Further study proved that the protective effects of CEP are mediated by inhibition of VEGF-A and VEGFR2. Based on the results, we conclude that CEP might possess a therapeutic prospect in stroke through protecting the integrity of the BBB mediated by the VEGF/VEGFR2/ZO-1 axis.

[Study on the Effect and Mechanism of Tetrandrine on Bone Marrow Mesenchymal Stem Cell-Mediated Drug Resistance in Leukemia].

OBJECTIVE: To explore the role of bone marrow mesenchymal stem cells (BMSC)，an essential element of the bone marrow microenvironment, in multidrug resistance(MDR) of K562 cells, as well as the reversal effect of tetrandrine (TET) on BMSC-mediated MDR and its potential mechanism. METHODS: A mixed co-culture system and a transwell co-culture system for BMSC and K562 cells were established, and the cells were divided into different groups and treated with daunorubicin (DNR) alone or combined with TET and DNR. The CCK-8 assay was used to detect the proliferation of K562 cells in each group, and the cell inhibition rate was calculated. Cytometric bead array (CBA) was used to detect the expression levels of IFN, IL-2, IL-6 and IL-10 in the supernatant of different groups. RT-qPCR and Western blot were used to detected the expression of STAT3 at mRNA and protein levels, respectively. RESULTS: Compared with K562+DNR group, the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR group was significantly decreased (P < 0.05), while the levels of IL-6 in the culture supernatant and phosphorylated STAT3 in K562 cells were significantly increased (P < 0.05). Compared with K562+BMSC+DNR group, the inhibition rate of DNR on K562 cell proliferation in K562+BMSC+DNR+TET group was significantly increased (P < 0.05), while the level of IL-6 and phosphorylated STAT3 was significantly decreased (P < 0.05). CONCLUSION: BMSC can promote the drug resistance of leukemia cells, and TET may reverse the BMSC-mediated drug resistance via inhibiting IL-6/STAT3 signaling pathway.

Preparation, Characterization, and Anti-Lung Cancer Activity of Tetrandrine-Loaded Stealth Liposomes.

Background: Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, is a potential candidate for cancer chemotherapy. However, Tet has poor aqueous solubility and a short half-life, which limits its bioavailability and efficacy. Liposomes have been widely utilized to enhance the bioavailability and efficacy of drugs. Methods: In this study, Tet-loaded stealth liposomes (S-LPs@Tet) were prepared by ethanol injection method. Furthermore, physicochemical characterisation, biopharmaceutical behaviour, therapeutic efficacy, and biocompatibility of S-LPs@Tet were assessed. Results: The prepared S-LPs@Tet had an average particle size of 65.57 ± 1.60 nm, a surface charge of -0.61 ± 0.10 mV, and an encapsulation efficiency of 87.20% ± 1.30%. The S-LPs@Tet released Tet in a sustained manner, and the results demonstrated that the formulation remained stable for one month. More importantly, S-LPs significantly enhanced the inhibitory ability of Tet on the proliferation and migration of lung cancer cells, and enabled Tet to escape phagocytosis by immune cells. Furthermore, in vivo studies confirmed the potential for long-circulation and potent tumor-suppressive effects of S-LPs@Tet. Moreover, ex vivo and in vivo safety experiments demonstrated that the carrier material S-LPs exhibited superior biocompatibility. Conclusion: Our research suggested that S-LPs@Tet has potential applications in lung cancer treatment.

Pharmacological profiling of a berbamine derivative for lymphoma treatment.

ABSTRACT: Ca2+/calmodulin-dependent protein kinase II γ (CAMKIIγ) has been identified as a potential target for treating cancer. Based on our previous study of berbamine (BBM) as a CAMKIIγ inhibitor, we have synthesized a new BBM derivative termed PA4. Compared with BBM, PA4 showed improved potency and specificity and was more cytotoxic against lymphoma and leukemia than against other types of cancer. In addition to indirectly targeting c-Myc protein stability, we demonstrated that its cytotoxic effects were also mediated via increased reactive oxygen species production in lymphoma cells. PA4 significantly impeded tumor growth in vivo in a xenograft T-cell lymphoma mouse model. Pharmacokinetics studies demonstrated quick absorption into plasma after oral administration, with a maximum concentration of 1680 ± 479 ng/mL at 5.33 ± 2.31 hours. The calculated oral absolute bioavailability was 34.1%. Toxicity assessment of PA4 showed that the therapeutic window used in our experiments was safe for future development. Given its efficacy, safety, and favorable pharmacokinetic profile, PA4 is a potential lead candidate for treating lymphoma.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/ß-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/ß-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

Tetrandrine (TET) inhibits African swine fever virus entry into cells by blocking the PI3K/Akt pathway.

African Swine Fever Virus (ASFV) infection causes an acute and highly contagious disease in swine, resulting in significant economic losses and societal harm worldwide. Currently, there are no effective vaccines or antiviral drugs available for ASFV. Tetrandrine (TET) is extracted from the traditional Chinese herb Stephania tetrandrae, possesses diverse biological functions such as anti-inflammatory, anti-tumor, and antiviral activities. The study comprehensively evaluated the anti-ASFV effect of TET and validated it through biological assays. The dose-dependent inhibition of TET against ASFV was confirmed and a novel mechanism of TET's anti-ASFV activity was elucidated. TET effectively inhibits ASFV during internalization by blocking macropinocytosis through the inhibition of the PI3K/Akt pathway. The specific inhibitor LY294002, targeting the PI3K/Akt pathway, exhibits similar antiviral activity against ASFV as TET. Furthermore, the inhibitory effect of TET against other viruses such as Lumpy Skin Disease Virus (LSDV) and Porcine Epidemic Diarrhea Virus (PEDV) was also identified. Our findings suggest that TET effectively inhibits ASFV and reveal the potential for broad-spectrum antiviral drugs targeting the PI3K/Akt pathway.

Herbal compound cepharanthine attenuates inflammatory arthritis by blocking macrophage M1 polarization.

OBJECTIVE: Cepharanthine (CEP) is a drug candidate for tumor, viral infection, and some inflammatory diseases, but its effect on rheumatoid arthritis (RA) and the underlying mechanism are incompletely understood. METHODS: CEP was administered intraperitoneally to a collagen-induced arthritis (CIA) model. Joints went radiological and histological examination and serum cytokines were examined with cytometry-based analysis. M1 macrophages were induced from THP-1 cells or mouse bone marrow-derived macrophages with LPS and IFN-γ. Bulk RNA-seq was performed on macrophage undergoing M1-polarizatioin. Western blotting was applied to determine pathways involved in monocyte chemotaxis and polarization. Glycolysis metabolites were measured by chemiluminescence while glycolytic enzymes were examined by quantitative PCR. RESULTS: We found CEP significantly ameliorated synovial inflammation and joint destruction of CIA mice. It downregulated TNF-α levels in serum and in joints. The number of M1 macrophages were reduced in CEP-treated mice. In vitro, CEP inhibited monocyte chemotaxis to MCP-1 by downregulating CCR2 and reducing ERK1/2 signaling. Additionally, CEP suppressed M1 polarization of macrophages induced by LPS and IFN-γ. Genes involved in IFN-γ signaling, IL-6-JAK/STAT3 signaling, glycolysis, and oxidative phosphorylation process were downregulated by CEP. Several enzymes critically involved in glycolytic metabolism were suppressed by CEP, which resulted in reduced citrate in M1-polarizing macrophages. The inhibitory effect of CEP on macrophage polarization might be attributed to the blockage of TLRs-MyD88/IRAK4-IRF5 signaling pathway together with suppression of overactivated glycolytic metabolism in M1-polarizing macrophages. CONCLUSION: CEP attenuated joint inflammation by suppressing monocyte chemotaxis and proinflammatory differentiation. It has the potential to be developed into a complementary or alternative therapy for RA.

Bisbenzylisoquinoline alkaloid fangchinoline derivative HY-2 inhibits breast cancer cells by suppressing BLM DNA helicase.

The high expression of BLM (Bloom syndrome) DNA helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to study the antitumor effect of fangchinoline derivative HY-2 by targeting BLM642-1290 DNA helicase, and then explore its inhibitory mechanism on proliferation of MDA-MB-435 breast cancer cells. We confirmed that the mRNA and protein levels of BLM DNA helicase in breast cancer were higher than those in normal tissues. HY-2 could inhibit the DNA binding, ATPase and DNA unwinding of BLM642-1290 DNA helicase with enzymatic assay. HY-2 could also inhibit the DNA unwinding of DNA helicase in cells. In addition, HY-2 showed an inhibiting the MDA-MB-435, MDA-MB-231, MDA-MB-436 breast cancer cells expansion. The mRNA and protein levels of BLM DNA helicase in MDA-MB-435 cells increased after HY-2 treatment, which might contribute to HY-2 inhibiting the DNA binding, ATPase and DNA unwinding of BLM DNA helicase. The mechanism of HY-2 inhibition on BLM DNA helicase was further confirmed with the effect of HY-2 on the ultraviolet spectrogram of BLM642-1290 DNA helicase and Molecular dynamics simulation of the interacting between HY-2 and BLM640-1291 DNA helicase. Our study provided some valuable clues for the exploration of HY-2 in the living body and developing it as an anticancer drug.

Liensinine and neferine exert neuroprotective effects via the autophagy pathway in transgenic Caenorhabditis elegans.

BACKGROUND: Liensinine and neferine are the main bisbenzylisoquinoline alkaloids obtained from the seeds of Nelumbo nucifera, which commonly used as edible food and traditional medicine in Asia. It was reported that liensinine and neferine could inhibit the activities of acetylcholinesterase and cross the blood-brain barriers, suggesting their therapeutic potential for the management of Alzheimer's disease. METHODS: Here, we employed SH-SY5Y human neuroblastoma cells stably transfected with the human Swedish amyloid precursor protein (APP) mutation APP695 (APP695swe SH-SY5Y) as an in vitro model and transgenic Caenorhabditis elegans as an in vivo model to investigate the neuroprotective effects and underlying mechanism of liensinine and neferine. RESULTS: We found that liensinine and neferine could significantly improve the viability and reduce ROS levels in APP695swe SH-SY5Y cells, inhibit ß-amyloid and tau-induced toxicity, and enhance stress resistance in nematodes. Moreover, liensinine and neferine had obviously neuroprotective effects by assaying chemotaxis, 5-hydroxytryptamine sensitivity and the integrity of injured neurons in nematodes. Preliminary mechanism studies revealed that liensinine and neferine could upregulate the expression of autophagy related genes (lgg-1, unc-51, pha-4, atg-9 and ced-9) and reduce the accumulation of ß-amyloid induced autophagosomes, which suggested autophagy pathway played a key role in neuroprotective effects of these two alkaloids. CONCLUSIONS: Altogether, our findings provided a certain working foundation for the use of liensinine and neferine to treat Alzheimer's disease based on neuroprotective effects.

Enhanced Antitumor Efficacy of Novel Biomimetic Platelet Membrane-Coated Tetrandrine Nanoparticles in Nonsmall Cell Lung Cancer.

Nonsmall cell lung cancer (NSCLC) remains one of the leading causes of cancer-related death worldwide, posing a serious threat to global health. Tetrandrine (Tet) is a small molecule in traditional Chinese medicine with proven primary efficacy against multiple cancers. Although previous studies have demonstrated the potential anticancer effects of Tet on NSCLC, its poor water solubility has limited its further clinical application. Herein, a novel nanoparticle-based drug delivery system, platelet membrane (PLTM)-coated Tet-loaded polycaprolactone-b-poly(ethylene glycol)-b-polycaprolactone nanoparticles (PTeNPs), is proposed to increase the potency of Tet against NSCLC. First, tetrandrine nanoparticles (TeNPs) are created using an emulsion solvent evaporation method, and biomimetic nanoparticles (PTeNPs) are prepared by coating the nanoparticles with PLTMs. When coated with PLTMs, PTeNPs are considerably less phagocytized by macrophages than Tet and TeNPs. In addition, compared with Tet and TeNPs, PTeNPs can significantly inhibit the growth and invasion of NSCLC both in vitro and in vivo. With reliable biosafety, this drug delivery system provides a new method of sustained release and efficient anticancer effects against NSCLC, facilitating the incorporation of Tet in modern nanotechnology.

Induction of apoptotic cell death of cholangiocarcinoma cells by tiliacorinine from Tiliacora triandra: A mechanistic insight.

BACKGROUND: Cholangiocarcinoma (CCA) exhibits poor response to the present chemotherapeutic agents and frequently develops drug resistance. Finding novel anticancer drugs might enhance patient outcomes. Tiliacorinine, a bisbenzylisoquinoline alkaloid from the Thai medicinal plant Tiliacora triandra, effectively induced apoptosis of human CCA cell lines and inhibited tumor growth in mice. Here, we elucidate further the molecular mechanisms underlining the cytotoxicity of tiliacorinine and its implication in overcoming gemcitabine-resistance of CCA cells. METHODS: Cytotoxicity of tiliacorinine against CCA cell lines was assessed using MTT assay. The molecular signaling was determined using Western blot analysis. Molecular docking simulations were applied to predict the binding affinity and orientation of tiliacorinine to the possible binding site(s) of the target proteins. RESULTS: Tiliacorinine induced apoptotic cell death of CCA cells in a dose- and time-dependent manner. Tiliacorinine significantly suppressed the expression of anti-apoptotic proteins, Bcl-xL and XIAP; activated apoptotic machinery proteins, caspase-3, caspase-9, and PARP; and decreased the levels of pAkt and pSTAT3. EGF/EGFR activation model and molecular docking simulations revealed EGFR, Akt, and STAT3 as potent targets of tiliacorinine. Molecular docking simulations indicated a strong binding affinity of tiliacorinine to the ATP-binding pockets of EGFR, PI3K, Akt, JAK2, and SH2 domain of STAT3. Tiliacorinine could synergize with gemcitabine and restore the cytotoxicity of gemcitabine against gemcitabine-resistant CCA cells. CONCLUSION: Tiliacorinine effectively induced apoptosis via binding and blocking the actions of EGFR, Akt, and STAT3. GENERAL SIGNIFICANCE: Tiliacorinine is a novel multi-kinase inhibitor and possibly a potent anti-cancer agent, in cancers with high activation of EGFR.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

A mechanistic updated overview on Cepharanthine as potential anticancer agent.

The antitumor effects of traditional drugs have received increasing attention and active antitumor components extracted from traditional drugs have shown good efficacy with minimal adverse events. Cepharanthine(CEP for short) is an active component derived from the Stephania plants of Menispermaceae, which can regulate multiple signaling pathways alone or in combination with other therapeutic drugs to inhibit tumor cell proliferation, induce apoptosis, regulate autophagy, and inhibit angiogenesis, thereby inhibiting tumor progression. Therefore, we retrieved studies concerning CEP's antitumor effects in recent years and summarized the antitumor mechanism and targets, in order to gain new insights and establish a theoretical basis for further development and application of CEP.

Pharmacological Activity of Cepharanthine.

Cepharanthine, a natural bisbenzylisoquinoline (BBIQ) alkaloid isolated from the plant Stephania Cephalantha Hayata, is the only bisbenzylisoquinoline alkaloid approved for human use and has been used in the clinic for more than 70 years. Cepharanthine has a variety of medicinal properties, including signaling pathway inhibitory activities, immunomodulatory activities, and antiviral activities. Recently, cepharanthine has been confirmed to greatly inhibit SARS-CoV-2 infection. Therefore, we aimed to describe the pharmacological properties and mechanisms of cepharanthine, mainly including antitumor, anti-inflammatory, anti-pathogen activities, inhibition of bone resorption, treatment of alopecia, treatment of snake bite, and other activities. At the same time, we analyzed and summarized the potential antiviral mechanism of cepharanthine and concluded that one of the most important anti-viral mechanisms of cepharanthine may be the stability of plasma membrane fluidity. Additionally, we explained its safety and bioavailability, which provides evidence for cepharanthine as a potential drug for the treatment of a variety of diseases. Finally, we further discuss the potential new clinical applications of cepharanthine and provide direction for its future development.

Optimization of Neferine Purification Based on Response Surface Methodology and Its Anti-Metastasis Mechanism on HepG2 Cells.

Liver cancer continues to be a focus of scientific research due to its low five-year survival rate. One of its main core issues is the high metastasis of cells, for which there is no effective treatment. Neferine was originally isolated from Plumula nelumbinis and demonstrated to have a good antitumor effect. In order to extract high-purity Neferine in a more efficient and environmentally friendly manner, response surface methodology (RSM) was used to optimize the isolation and purification procedures in this study. The extract conditions of a 7:3 ratio for the eluent of dichloromethane: methanol, 1:60 for the mass ratio of the extract amount: silica gel, and 3 mL/min of the elution flow rate were shown to be the optimal conditions. These conditions resulted in the highest yield of 6.13 mg per 66.60 mg of starting material, with productivity of 8.76% and purity of 87.04%. Compared with the previous methods, this method can prepare Neferine in large quantities more quickly. We subsequently evaluated the antitumor activity of the purified Neferine against HepG2 hepatic cancer cells. The purified Neferine was found to inhibit the proliferation of HepG2 cells through the CCK-8 assay, with an IC50 of 33.80 µM in 24 h, 29.47 µM in 48 h, 24.35 µM in 72 h and 2.78 µM in 96 h of treatment. Neferine at a concentration of 3 µM could significantly inhibit the migration and invasion abilities of the HepG2 cells in vitro. We also explored the mechanism of action of Neferine via Western blot. We showed that Neferine could reduce RhoA expression by effectively inhibiting the phosphorylation of MYPT1, thereby effectively exerting anti-metastasis activity against HepG2 cells. Thus, we have optimized the isolation procedures for highly pure Neferine by response surface methodology (RSM) in this study, and purified Neferine is shown to play an essential role in the anti-metastasis process of liver cancer cells. The Neferine purification procedure may make a wide contribution to the follow-up development of other anti-metastasis lead compounds.

Cepharanthine Dry Powder Inhaler for the Treatment of Acute Lung Injury.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) induces a severe cytokine storm that may cause acute lung injury/acute respiratory distress syndrome (ALI/ARDS) with high clinical morbidity and mortality in infected individuals. Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid isolated and extracted from Stephania cepharantha Hayata. It exhibits various pharmacological effects, including antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, and antiviral activities. The low oral bioavailability of CEP can be attributed to its poor water solubility. In this study, we utilized the freeze-drying method to prepare dry powder inhalers (DPI) for the treatment of acute lung injury (ALI) in rats via pulmonary administration. According to the powder properties study, the aerodynamic median diameter (Da) of the DPIs was 3.2 µm, and the in vitro lung deposition rate was 30.26; thus, meeting the Chinese Pharmacopoeia standard for pulmonary inhalation administration. We established an ALI rat model by intratracheal injection of hydrochloric acid (1.2 mL/kg, pH = 1.25). At 1 h after the model's establishment, CEP dry powder inhalers (CEP DPIs) (30 mg/kg) were sprayed into the lungs of rats with ALI via the trachea. Compared with the model group, the treatment group exhibited a reduced pulmonary edema and hemorrhage, and significantly reduced content of inflammatory factors (TNF-α, IL-6 and total protein) in their lungs (p < 0.01), indicating that the main mechanism of CEP underlying the treatment of ALI is anti-inflammation. Overall, the dry powder inhaler can deliver the drug directly to the site of the disease, increasing the intrapulmonary utilization of CEP and improving its efficacy, making it a promising inhalable formulation for the treatment of ALI.

Fangchinoline derivatives inhibits PI3K signaling in vitro and in vivo in non-small cell lung cancer.

Fangchinoline (Fan) are extracted from the traditional Chinese medicine Stephania tetrandra S., which is a bis-benzyl isoquinoline alkaloids with anti-tumor activity. Therefore, 25 novel Fan derivatives have been synthesized and evaluated for their anti-cancer activity. In CCK-8 assay, these fangchinoline derivatives displayed higher proliferation inhibitory activity on six tumor cell lines than the parental compound. Compared to the parent Fan, compound 2h presented the anticancer activity against most cancer cells, especially A549 cells, with an IC50 value of 0.26 µM, which was 36.38-fold, and 10.61-fold more active than Fan and HCPT, respectively. Encouragingly, compound 2h showed low biotoxicity to the human normal epithelial cell BEAS-2b with an IC50 value of 27.05 µM. The results indicated compound 2h remarkably inhibited the cell migration by decreasing MMP-2 and MMP-9 expression and inhibited the proliferation of A549 cells by arresting the G2/M cell cycle. Meanwhile, compound 2h could also induce A549 cell apoptosis by promoting endogenous pathways of mitochondrial regulation. In nude mice presented that the growth of tumor tissues was markedly inhibited by the consumption of compound 2h in a dose-dependent manner, and it was found that compound 2h could inhibit the mTOR/PI3K/AKT pathway in vivo. In docking analysis, high affinity interaction between 2h and PI3K was responsible for drastic kinase inhibition by the compound. To conclude, this derivative compound may be useful as a potent anti-cancer agent for treatment of NSCLC.

Berbamine promotes macrophage autophagy to clear Mycobacterium tuberculosis by regulating the ROS/Ca2+ axis.

Drug-resistant tuberculosis (TB) poses a major threat to global TB control; consequently, there is an urgent need to develop novel anti-TB drugs or strategies. Host-directed therapy (HDT) is emerging as an effective treatment strategy, especially for drug-resistant TB. This study evaluated the effects of berbamine (BBM), a bisbenzylisoquinoline alkaloid, on mycobacterial growth in macrophages. BBM inhibited intracellular Mycobacterium tuberculosis (Mtb) growth by promoting autophagy and silencing ATG5, partially abolishing the inhibitory effect. In addition, BBM increased intracellular reactive oxygen species (ROS), while the antioxidant N-acetyl-L-cysteine (NAC) abolished BBM-induced autophagy and the ability to inhibit Mtb survival. Furthermore, the increased intracellular Ca2+ concentration induced by BBM was regulated by ROS, and BAPTA-AM, an intracellular Ca2+-chelating agent, could block ROS-mediated autophagy and Mtb clearance. Finally, BBM could inhibit the survival of drug-resistant Mtb. Collectively, these findings provide evidence that BBM, a Food and Drug Administration (FDA)-approved drug, could effectively clear drug-sensitive and -resistant Mtb through regulating ROS/Ca2+ axis-mediated autophagy and has potential as an HDT candidate for TB therapy. IMPORTANCE It is urgent to develop novel treatment strategies against drug-resistant TB, and HDT provides a promising approach to fight drug-resistant TB by repurposing old drugs. Our studies demonstrate, for the first time, that BBM, an FDA-approved drug, not only potently inhibits intracellular drug-sensitive Mtb growth but also restricts drug-resistant Mtb by promoting macrophage autophagy. Mechanistically, BBM activates macrophage autophagy by regulating the ROS/Ca2+ axis. In conclusion, BBM could be considered as an HDT candidate and may contribute to improving the outcomes or shortening the treatment course of drug-resistant TB.