Sesquilignans PD from Zanthoxylum nitidum var. tomentosum exerts antitumor effects via the ROS/MAPK pathway in liver cancer cells.

Sesquilignans PD is a natural phenylpropanoid compound that was isolated from Zanthoxylum nitidum var. tomentosum. In this study, we assessed the antitumor effect of PD on SK-Hep-1 and HepG2 cells and the underlying molecular mechanisms. The results revealed that PD markedly inhibited the proliferation and migration of both liver cancer cells. Moreover, PD induced apoptosis, autophagy, and reactive oxygen species (ROS) production in liver cancer cells. Notably, PD increased the protein levels of p-p38 MAPK and p-ERK1/2 in liver cancer cells. This is the first report on the anticancer effect of PD, which is mediated via increased ROS production and MAPK signaling activation.

Alkaloids from Corydalis saxicola and their antiproliferative activity against cancer cells.

Eight undescribed alkaloids named corydalisine D-K (1-7), including one isoquinoline benzopyranone alkaloid (1), one benzocyclopentanone alkaloid (2), four benzofuranone alkaloids (3, 4, and 5a/5b) and two protoberberine alkaloids (6 and 7), along with fourteen known ones, were isolated from the Corydalis saxicola. Their structures, including absolute configurations, were unambiguously identified using spectroscopic techniques, single-crystal X-ray diffraction and electron circular dichroism calculation. Compounds 2, 14 and 21 exhibit antiproliferative activity against five cancer cell lines. The aporphine alkaloid demethylsonodione (compound 14), which exhibited the best activity (IC50 = 3.68 ± 0.25 µM), was subjected to further investigation to determine its mechanism of action against the T24 cell line. The molecular mechanism was related to the arrest of cell cycle S-phase, inhibition of CDK2 expression, accumulation of reactive oxygen species (ROS), induction of cell apoptosis, inhibition of cell migration, and activation of p38 MAPK signaling pathway. The results indicated that 14 could be used as a potential candidate agent for further development of anti-bladder transitional cell carcinoma.

Discovery of novel 2-oximino-2-indolylacetamide derivatives as potent anticancer agents capable of inducing cell autophagy and ferroptosis.

A series of 2-oximino-2-indolylacetamide derivatives were designed, synthesized and evaluated for their antitumour effects. Among them, 4d exhibited the most potent antiproliferative effect in vitro on the tested human cancer cells. Additionally, 4d significantly induced cell apoptosis, caused mitochondrial dysfunction, promoted Bax, cleaved-PARP and p53 expression and inhibited Bcl-2 expression in 5-8F cells. Moreover, 4d remarkably promoted autophagosome formation, leading to cell apoptosis. Further investigation indicated that 4d could trigger cell death through cell ferroptosis, including increased ROS generation and lipid peroxidation and decreased glutathione peroxidase 4 (GPx4) expression and glutathione (GSH) levels. More importantly, 4d induced 5-8F cell death by activating ROS/MAPK and inhibiting the AKT/mTOR and STAT3 signalling pathways. Interestingly, 4d significantly suppressed tumour growth in a 5-8F cell xenograft model without obvious toxicity to mice. Overall, these results demonstrate that 4d may be a potential compound for cancer therapy.

Undescribed isoquinolines from Zanthoxylum nitidum and their antiproliferative effects against human cancer cell lines.

Eleven previously undescribed alkaloids, including three pairs of enantiomers nitidumalkaloids A-C, a pair of scalemic mixtures nitidumalkaloid D and three optically pure or achiral alkaloids, nitidumalkaloids E-G, along with 20 known alkaloids, were isolated from an ethanolic extract of the whole Zanthoxylum nitidum (Roxb.) DC plant. The chemical structures of the alkaloids were elucidated using a combination of comprehensive nuclear magnetic resonance (NMR) and high-resolution electro-spray ionization mass spectrometry (HR-ESI-MS) analyses. The configuration of the stereogenic centers of all undescribed compounds was precisely established based on single-crystal X-ray diffraction and electronic circular dichroism (ECD) calculations. Racemic mixtures of nitidumalkaloids A-D were purified, and their enantiomers were analyzed via chiral-phase high-performance liquid chromatography with electrochemical detection measurements (HPLC-ECD). Twelve compounds exhibited significant antiproliferative activities against a panel of cancer cell lines. Further studies were designed to investigate the underlying molecular mechanism of (1'S, 6R)-nitidumalkaloid B, which was the most active antiproliferative agent against human cancer A549 cells. G2/M cell cycle arrest, induction of apoptosis, and suppression of the Wnt/ß-catenin signaling pathway were in part associated with the antiproliferative activity of (1'S, 6R)-nitidumalkaloid B. Moreover, (1'S, 6R)-nitidumalkaloid B inhibited cell migration by downregulating the epithelial-mesenchymal transition process in A549 cells. These data suggest that the antiproliferation activity of (1'S, 6R)-nitidumalkaloid B was correlated with the stereoselectivity of the stereoisomers, and (1'S, 6R)-nitidumalkaloid B was prioritized as a potential leading compound for the management of aggressive human non-small-cell lung cancer (NSCLC) from natural products.

Structurally diverse isobutylamides from Zanthoxylum nitidum.

Five pairs of new racemic alkamides (1a/1b and 4a/4b-7a/7b) and two new achiral derivatives (2-3), as well as five known ones (8-12), were purified from the 95% EtOH extract of Zanthoxylum nitidum. Their structures were elucidated based on spectroscopic analyses (NMR and HR-ESI-MS), electronic circular dichroism (ECD) and NMR calculations. The enantiomeric separation was successfully achieved by chiral-phase HPLC-ECD measurements. Among all the isolates, compounds 2, 3, and 10 showed inhibitory effects against five human cancer cell lines, with IC50 values in range of 18.51-48.03 µM.

New alkaloids and their in vitro antitumor activity of Corydalis balansae.

The chemical investigation on Corydalis balansae resulted in the isolation of three previous undescribed compounds (1, 10, and 11) and 17 known compounds. Compound 1 and 2 were obtained as two lignanamide dimers, and compound 11 had a spiro [benzofuranone-benzazepine] skeleton, which was found in Corydalis for the first time. The structures of new compound were determined by the detailed analysis of 1D/2D NMR, UV, and IR data. Absolute configurations of compounds 10 and 11 were defined by their crystal X-ray diffraction data and calculations of electronic circular dichroism (ECD). The CCK-8 method was used to assay the inhibition effect of all the compounds on the growth of Hela, MGC-803, A549, and HepG2 cancer cells. Compound 2, 13, and 14 showed moderate inhibitory activity against the tested cell lines. Compound 2 exhibited potential antitumor activity against MGC-803 cells with an IC50 value of 20.8 µM, while the positive control etoposide was 17.3 µM. Furthermore, results from the cellular-mechanism investigation indicated that compound 2 could induce S-phase cell-cycle arrest and MGC-803 cells apoptosis, which was triggered by the up-regulation of PARP1, caspase-3 and -9, Bax, and down-regulation of Bcl-2. The 2-induced strong apoptosis indicated that compound 2 had good potential as an antitumor lead compound.

Four New Sesquiterpenoids from Zanthoxylum nitidum.

Zanthoxylum nitidum (Roxb.) DC., is one of Guangxi's characteristic national medicines, and is the classic Laoban medicine of Yao people "Ru Shan Hu" and Zhuang medicine "Liang Bei Zhen". It has been used as an anti-inflammatory, analgesic and haemostatic medicine for thousands of years. In this study, four new sesquiterpenoids (1-4), along with six previously described coumarins (5-10), were isolated from 95 % EtOH extract of Zanthoxylum nitidum. Comprehensive spectroscopic analyses (NMR and HR-ESI-MS) were used to elucidate the structures of these isolates. The absolute configurations of nitidumine A-D (1-4) were established by electronic circular dichroism (ECD). Their cytotoxicity of all the isolates against five cancer cell lines (T24, HeLa, MGC-803, A549, and HepG2) was evaluated by MTT experiment and found not to be cytotoxicity.

Pentagalloylglucose suppresses the growth and migration of human nasopharyngeal cancer cells via the GSK3ß/ß-catenin pathway in vitro and in vivo.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a type of malignant squamous cell tumour originating from the nasopharynx epithelium. Pentagalloylglucose (PGG) is a natural polyphenolic compound that exerts anticancer effects in many types of tumours. However, the role and underlying mechanism of PGG in NPC cells have not been fully defined. PURPOSE: This study aimed to investigate the anticancer activity of PGG as well as the potential mechanism in NPC cells. METHODS: The effects of PGG on the proliferation, apoptosis and cell cycle distribution of CNE1 and CNE2 cells were assessed by MTT and flow cytometry assays. Cell migration was evaluated using wound healing and transwell assays. The expression of microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Western blotting was used to explore the levels of related proteins and signalling pathway components. Furthermore, the effects of PGG on NPC cell growth were analysed in a xenograft mouse model in vivo using cisplatin as a positive control. RESULTS: PGG dose-dependently inhibited the proliferation of CNE1 and CNE2 cells. PGG regulated the cell cycle by altering p53, cyclin D1, CDK2, and cyclin E1 protein levels. PGG induced apoptosis and autophagy in NPC cells and elevated the Bax/Bcl-2 ratio and the protein levels of LC3B. Moreover, PGG decreased NPC cell migration by increasing E-cadherin and decreasing N-cadherin, vimentin and CD44 protein levels. Mechanistically, PGG treatment downregulated p-mTOR and ß-catenin expression but upregulated p-p38 MAPK and p-GSK3ß expression. In addition, PGG significantly inhibited NPC cell tumour growth and lung metastasis in vivo. CONCLUSION: PGG may suppress cell proliferation, induce apoptosis and autophagy, and decrease the metastatic capacity of NPC cells through the p38 MAPK/mTOR and Wnt/ß-catenin pathways. The present study provides evidence for PGG as a potential therapy for NPC.

The neurotrophic and antineuroinflammatory effects of phenylpropanoids from Zanthoxylum nitidum var. tomentosum (Rutaceae).

Three novel lignans (1, 5 and 6) and two novel quinic acids (16 and 17) along with 15 known phenylpropanoids were obtained from the ethanol extract of Zanthoxylum nitidum var. tomentosum (Rutaceae). Their structures were confirmed by comprehensive spectroscopic data (NMR and HRESIMS), and the absolute configurations of all novel compounds were elucidated based on electronic circular dichroism (ECD) spectroscopic data. The production of nitric oxide (NO) in BV-2 microglial cells induced through lipopolysaccharide (LPS) was used to evaluate in vitro anti-neuroinflammatory activity of compounds 1-20. Compound 2, 3, 7 and 16 showed excellent inhibition of LPS-induced NO production. The structure-activity relationships of the isolates were investigated. In addition, the mechanism of action of 2 was elucidated by RT-PCR and Western blotting analysis, which indicated that it reduced neuroinflammatory mainly through NLRP3/caspase1 signaling pathways in LPS-induced BV2 microglial cells.

Synthesis and discovery of asiatic acid based 1,2,3-triazole derivatives as antitumor agents blocking NF-κB activation and cell migration.

A series of asiatic acid (AA) based 1,2,3-triazole derivatives were designed, synthesized and subjected to a cell-based NF-κB inhibition screening assay. Among the tested compounds, compound 6k displayed impressive NF-κB inhibitory activity with an IC50 value in the low micromolar range. A molecular docking study was performed to reveal key interactions between 6k and NF-κB in which the 1,2,3-triazole moiety and the hydroxyl groups of the AA skeleton were important for improving the inhibitory activity. Subsequently, surface plasmon resonance analysis validated the high affinity between compound 6k and NF-κB protein with an equilibrium dissociation constant (KD) value of 0.36 µM. Further studies showed that compound 6k observably inhibited the NF-κB DNA binding, nuclear translocation and IκBα phosphorylation. Moreover, in vitro antitumor activity screening showed that compound 6k (IC50 = 2.67 ± 0.06 µM) exhibited the best anticancer activity against A549 cells, at least partly, by inhibition of the activity of NF-κB. Additionally, the treatment of A549 cells with compound 6k resulted in apoptosis induction potency and in vitro cell migration inhibition. Thus, we conclude that AA based 1,2,3-triazole derivatives may be potential NF-κB inhibitors with the ability to induce apoptosis and suppress cell migration.

Wnt3a signaling promotes proliferation, myogenic differentiation, and migration of rat bone marrow mesenchymal stem cells.

AIM: To investigate the effects of the wingless-related MMTV integration site 3A (Wnt3a) signaling on the proliferation, migration, and the myogenic and adipogenic differentiation of rat bone marrow mesenchymal stem cells (rMSC). METHODS: Primary MSC were isolated and cultured from Sprague-Dawley rats and characterized by flow cytometry. Mouse L cells were transfected with Wnt3a cDNA, and conditioned media containing active Wnt3a proteins were prepared. Cell proliferation was evaluated by cell count and 5-bromodeoxyuridine incorporation assay. The migration of rMSC was performed by using a transwell migration and wound healing assay. The myogenic and adipogenic differentiation in rMSC were examined by light microscopy, immunofluorescence, and RT-PCR at different time points after myogenic or adipogenic introduction. RESULTS: Wnt3a signaling induced beta-catenin nuclear translocation and activated the Wnt pathway in rMSC. In the presence of Wnt3a, rMSC proliferated more rapidly than the control cells, keeping their differentiation potential. Moreover, Wnt3a signaling induced 2.62% and 3.76% of rMSC-expressed desmin and myosin heavy chain after being cultured in myogenic medium. The myogenic differentiation genes, including Pax7, MyoD, Myf5, Myf4, and myogenin, were activated after Wnt3a treatment. On the other hand, Wnt3a inhibited the adipogenic differentiation in rMSC through the downregulated expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma). Furthermore, Wnt3a promoted the migration capacity of rMSC. CONCLUSION: The results indicate that Wnt3a signaling can induce myogenic differentiation in rMSC. Wnt3a signaling is also involved in the regulation of the proliferation and migration of rMSC. These results could provide a rational foundation for cell-based tissue repair in humans.

[Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice].

Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.

[Sibling brother and sister both with Duchenne muscular dystrophy].

OBJECTIVE: To investigate the clinical and lab features of sibling brother and sister both with Duchenne muscular dystrophy (DMD). METHODS: We conducted comprehensive clinical and lab investigations including the test of serum enzymes, electromyography (EMG), electrocardiography, color Doppler echocardiography, HE staining of skeletal muscles, immunohistochemical study of dystrophin and utrophin, multiple ligation probe amplification (MLPA) on exon 1-79 of dystrophin gene, and short tandem repeat-poly- merase chain reaction of CA repeats located in dystrophin gene. RESULTS: These two patients were confirmed to suffer from DMD. They were characterized by typical features of DMD including typical clinical manifestations, increased serum enzymes, EMG presenting myogenic impairment, HE staining presentation belonging to DMD, negative dystrophin in brother, and inconstantly positive on the sarcolemma of sister. Furthermore, no deletion or duplication was found in the 1-79 exons of dystrophin gene. The suffering brother and sister carried the same maternal X chromosome. CONCLUSIONS: Carriers of DMD gene show typical clinical and laboratory manifestations of DMD. Comprehensive examinations should be performed for such carriers.

[Transfection and in vitro expression of human microdystrophin gene in rat mesenchymal stem cells].

OBJECTIVE: To construct the eukaryotic expression vector of human microdystrophin gene and observe its expression in rat mesenchymal stem cells (rMSCs) in vitro. METHODS: The plasmid PBSK-MICRO containing human microdystrophin cDNA was digested by restriction endonuclease, and the resultant microdystrophin fragment was inserted into the NotI site of pcDNA3.1(+) to prepare the eukaryotic expression vector-pcDNA3.1(+)/ microdystrophin, which was identified by endonuclease digestion and sequencing. The recombinant plasmid was transfected into rMSCs via lipofectamine, and after G418 selection, the expression of microdystrophin was detected by RT-PCR and indirect immunofluorescence assay. RESULTS: Microdystrophin gene fragment was correctly inserted into the plasmid pcDNA3.1(+), as conformed by sequencing and digestion with Not I and Hind III. The total mRNA of the transfected rMSCs was extracted and microdystrophin mRNA expression was found in the cells by RT-PCR. Indirect immunofluorescence assay for the protein expression of microdystrophin showed bright red fluorescence in the transfected rMSCs. CONCLUSION: Eukaryotic expression plasmid pcDNA3.1(+)/microdystrophin has been constructed successfully and microdystrophin can be expressed in transfected rMSCs in vitro, which may facilitate further research of Duchenne muscular dystrophy treatment by genetically modified allogeneic stem cell transplantation.

[Dystrophin expression and pathology of diaphragm muscles of mdx mice after xenogenic bone marrow stem cell transplantation].

OBJECTIVE: To investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD). METHODS: The bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls. RESULTS: The proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice. CONCLUSION: BMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.