Low-concentration exogenous 3-indoleacetic acid improves fruit-setting rate of Marsdenia tenacissima by inhibiting the expression of embryo abortion-related genes.

Marsdenia tenacissima is a medicinal plant characterized by many flowers, few fruits, and a low fruit-setting rate. Exogenous auxins can improve the fruit-setting rate of plants; however, their impacts on M. tenacissima and regulatory mechanisms remain unclear. In this study, we conducted a field experiment to determine the fruit-setting rate, seed-setting rate, fruit size, and changes in transcriptional expression of related genes by spraying 10 and 50 mg·L-1 of 3-indoleacetic acid (IAA). The control plants were sprayed with distilled water. Our results indicated that the fruit-setting rate was 0.15 when treated with 10 mg·L-1 of IAA, which was 2.76-fold higher than that of the control. Compared with that of the control, the number of differentially expressed genes (DEGs) regulated by 10 mg·L-1 of IAA was 28.6-fold higher than that regulated by 50 mg·L-1 of IAA. These DEGs were closely related to hormone metabolism and fruit development. By transcriptome analysis, spraying 10 mg·L-1 of IAA increased the expressions of STP6, MYB17, and LAX3 and reduced those of CXE18, ILR1-like 3, and SAUR50; this possibly affected the ovule, embryo, and fruit development, thereby elevating the fruit-setting rate of M. tenacissima. Our results indicated that low IAA concentration increased the fruit-setting rate of M. tenacissima, providing theoretical and practical support for promoting the seed yield of M. tenacissima.

The Protective Effect of Marsdenia tenacissima against Cisplatin-Induced Nephrotoxicity Mediated by Inhibiting Oxidative Stress, Inflammation, and Apoptosis.

Cisplatin (Cis) is considered to be one of the most effective drugs for killing cancer cells and remains a first-line chemotherapeutic agent. However, Cis's multiple toxicities (especially nephrotoxicity) have limited its clinical use. Marsdenia tenacissima (Roxb.) Wight et Arn. (MT), a traditional Chinese medicine (TCM) employed extensively in China, not only enhances the antitumor effect in combination with Cis, but is also used for its detoxifying effect, as it reduces the toxic side effects of chemotherapy drugs. The aim of this study was to explore the therapeutic effect of MT on Cis-induced nephrotoxicity, along with its underlying mechanisms. In this study, liquid-mass spectrometry was performed to identify the complex composition of the extracts of MT. In addition, we measured the renal function, antioxidant enzymes, and inflammatory cytokines in mice with Cis-induced nephrotoxicity and conducted renal histology evaluations to assess renal injury. The expressions of the proteins related to antioxidant, anti-inflammatory, and apoptotic markers in renal tissues was detected by Western blotting (WB). MT treatment improved the renal function, decreased the mRNA expression of the inflammatory factors, and increased the antioxidant enzyme activity in mice. A better renal histology was observed after MT treatment. Further, MT inhibited the expression of the phospho-NFκB p65 protein/NFκB p65 protein (p-p65)/p65, phospho-inhibitor of nuclear factor kappa B kinase beta subunit/inhibitor of nuclear factor kappa B kinase beta subunit (p-IKKß/IKKß), Bcl-2-associated X (Bax), and Cleaved Caspase 3/Caspase 3 proteins, while the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Recombinant NADH Dehydrogenase, Quinone 1 (NQO1), and B-cell lymphoma-2 (Bcl-2) was increased. The present study showed that MT ameliorated renal injury, which mainly occurs through the regulation of the Nrf2 pathway, the NF-κB pathway, and the suppression of renal tissue apoptosis. It also suggests that MT can be used as an adjuvant to mitigate the nephrotoxicity of Cis chemotherapy.

Combinatorics-based chemical characterization and bioactivity comparison of different parts of traditional Chinese medicinal plants through LC-Q-TOF-MS/MS, multivariate statistical analysis and bioassay: Marsdenia tenacissima as an example.

Marsdenia tenacissima is a traditional Chinese medicinal plant used for treating cancer, and its main medicinal part is the stem. Considering the resource shortage of M. tenacissima, it is of great significance to improve its utilization efficiency. Steroids and caffeoylquinic acids, the two main components of M. tenacissima, are composed of several basic structures. Based on this rule, a novel strategy of combinatorics-based chemical characterization was proposed to analyze the constituents of roots, stems and leaves of M. tenacissima. Combinatorics was used to generate a compound library for structure alignment, which has the advantages of wide coverage and high specificity. Steroids are composed of four basic parts: core skeleton (C), substituent at position 11 (A), substituent at position 12 (B) and sugar moiety (S). Based on combinatorics, a compound library consisting of 1080 steroids was generated. Diagnostic neutral loss has been used to effectively predict the substituents at position 11 and 12 of steroids, including acetyl, 2-methylpropionyl, tigloyl, 2-methylbutyryl and benzoyl. As a result, 131, 131 and 99 components were detected from the roots, stems and leaves of M. tenacissima, respectively. Principal component analysis (PCA) was used to analyze the differences of roots, stems and leaves, and orthogonal partial least squares-discriminant analysis (OPLS-DA) was further applied to find differential components. Tenacissoside H, a critical indicator component for quality evaluation of the stem, has been proved to be a differential component between roots and stems. Notably, the relative content of tenacissoside H in the roots was significantly higher than that in the stems. The bioactivity comparison showed that roots, stems and leaves of M. tenacissima had similar scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. However, their α-glucosidase inhibitory activity was ranked as leaves > stems > roots. Therefore, besides stems, the other parts of M. tenacissima have potential medicinal value. This study not only helps to develop the resource of M. tenacissima, but also provides a paradigm for the research of other similar medicinal plants.

Marsdenia tenacissima enhances immune response of tumor infiltrating T lymphocytes to colorectal cancer.

Introduction: Tumor-infiltrating T lymphocytes in the tumor microenvironment are critical factors influencing the prognosis and chemotherapy outcomes. As a Chinese herbal medicine, Marsdenia tenacissima extract (MTE) has been widely used to treat cancer in China. Its immunoregulatory effects on tumor-associated macrophages is well known, but whether it regulates tumor-infiltrating T-cell functions remains unclear. Method: We collected 17 tumor samples from MTE-administered colorectal cancer patients, 13 of which showed upregulation of CD3+/CD8+ tumor-infiltrating T cells. Further in vitro and in vivo experiments were performed to investigate the regulatory effects of MTE on tumor-infiltrating T cells and immune escape of tumors. Results: Under single and co-culture conditions, MTE inhibited TGF-ß1 and PD-L1 expression in the colorectal cancer (CRC) cell lines HCT116 and LoVo. In Jurkat cells, MTE inhibited FOXP3 and IL-10 expression, increased IL-2 expression, but had no effect on PD-1 expression. These findings were confirmed in vitro using subcutaneous and colitis-associated CRC mouse models. MTE also increased the density of CD3+/CD8+ tumor-infiltrating T cells and exhibited considerable tumor-suppressive effects in these two tumor mouse models. Conclusions: Our findings suggested that MTE inhibits the immune escape of cancer cells, a precipitating factor increasing the immune response of T lymphocytes.

C21 steroids from the roots of Marsdenia tenacissima.

A phytochemical investigation of the dichloromethane soluble fraction of the ethanolic extract obtained from the roots of Marsdenia tenacissima led to the discovery of the sixteen undescribed pregnane C21 steroids (1-16) and isolation of eleven known C21 steroidal analogues (17-27). Their chemical structures were elucidated by one- and two-dimensional nuclear magnetic resonance spectroscopy and, high resolution-electrospray ionization mass spectrometry and their absolute configurations were determined using electronic circular dichroism or single-crystal X-ray diffraction. The in vitro anti-proliferative effects of 1-16 were evaluated against HepG2 (human hepatocellular cancer), A549 (lung cancer), and MCF-7 (human breast cancer) cell lines. Even though some of them showed moderate cytotoxic activities, marsectohexol derivative 12 exhibited significant cytotoxicity against A549 cells with an IC50 value of 5.2 µM.

Pregnane steroid glycosides with multidrug resistance reversal activity from the stems of Marsdenia tenacissima.

Eighteen previously unreported pregnane glycosides, namely marsdenosides S1-S18, along with 15 known analogues, have been isolated from the stems of Marsdenia tenacissima. The structures of the undescribed compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation, X-ray crystallography and acid hydrolysis. All the isolates were evaluated for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and nine ones displayed moderate MDR reversal activity with reversal folds in the range of 2.45-9.01. The most active 12-O-acetyl-20-O-benzoyl-(14,17,18-orthoacetate)-dihydrosarcostin-3-O-ß-d-thevetopyranosyl-(1 â†’ 4)-O-ß-d-oleandropyranosyl-(1 â†’ 4)-O-ß-d-cymaropyranoside increased the sensitivity of MCF-7/ADR cell to adriamycin comparably to the reference drug verapamil (RF = 8.93).

[Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification].

The present study aimed to explore the main active components and underlying mechanisms of Marsdenia tenacissima in the treatment of ovarian cancer(OC) through network pharmacology, molecular docking, and in vitro cell experiments. The active components of M. tenacissima were obtained from the literature search, and their potential targets were obtained from SwissTargetPrediction. The OC-related targets were retrieved from Therapeutic Target Database(TTD), Online Mendelian Inheritance in Man(OMIM), GeneCards, and PharmGKB. The common targets of the drug and the disease were screened out by Venn diagram. Cytoscape was used to construct an "active component-target-disease" network, and the core components were screened out according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened out according to the node degree. GO and KEGG enrichment analyses of potential therapeutic targets were carried out with DAVID database. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock. Finally, the anti-OC activity of M. tenacissima extract was verified based on SKOV3 cells in vitro. The PI3K/AKT signaling pathway was selected for in vitro experimental verification according to the results of GO function and KEGG pathway analyses. Network pharmacology results showed that 39 active components, such as kaempferol, 11α-O-benzoyl-12ß-O-acetyltenacigenin B, and drevogenin Q, were screened out, involving 25 core targets such as AKT1, VEGFA, and EGFR, and the PI3K-AKT signaling pathway was the main pathway of target protein enrichment. The results of molecular docking also showed that the top ten core components showed good binding affinity to the top ten core targets. The results of in vitro experiments showed that M. tenacissima extract could significantly inhibit the proliferation of OC cells, induce apoptosis of OC cells through the mitochondrial pathway, and down-regulate the expression of proteins related to the PI3K/AKT signaling pathway. This study shows that M. tenacissima has the characteristics of multi-component, multi-target, and multi-pathway synergistic effect in the treatment of OC, which provides a theoretical basis for in-depth research on the material basis, mechanism, and clinical application.

Marsdenia tenacissima extract induces endoplasmic reticulum stress-associated immunogenic cell death in non-small cell lung cancer cells through targeting AXL.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia Tenacissima (Roxb.) Wight et Arn. is a traditional Chinese medicine. Its standardized extract (MTE), with the trade name Xiao-Ai-Ping injection, is widely used for cancer treatment. The pharmacological effects of MTE-inducing cancer cell death have been primarily explored. However, whether MTE triggers tumor endoplasmic reticulum stress (ERS)-associated immunogenic cell death (ICD) is unknown. AIM OF THE STUDY: To determine the potential role of endoplasmic reticulum stress in the anti-cancer effects of MTE, and uncover the possible mechanisms of endoplasmic reticulum stress-associated immunogenic cell death induced by MTE. MATERIAL AND METHODS: The anti-tumor effects of MTE on non-small cell lung cancer (NSCLC) were examined through CCK-8 and wound healing assay. Network pharmacology analysis and RNA-sequencing (RNA seq) were performed to confirm the biological changes of NSCLCs after MTE treatment. Western blot, qRT-PCR, reactive oxygen species (ROS) assay, and mitochondrial membrane potential (MMP) assay were used to explore the occurrence of endoplasmic reticulum stress. Immunogenic cell death-related markers were tested by ELISA and ATP release assay. Salubrinal was used to inhibit the endoplasmic reticulum stress response. SiRNA and bemcentinib (R428) were used to impede the function of AXL. AXL phosphorylation was regained by recombinant human Gas6 protein (rhGas6). The effects of MTE on endoplasmic reticulum stress and immunogenic cell death response were also proved in vivo. The AXL inhibiting compound in MTE was explored by molecular docking and confirmed by Western blot. RESULTS: MTE inhibited cell viability and migration of PC-9 and H1975 cells. Enrichment analysis identified that differential genes after MTE treatment were significantly enriched in endoplasmic reticulum stress-related biological processes. MTE decreased mitochondrial membrane potential (MMP) and increased ROS production. Meanwhile, endoplasmic reticulum stress-related proteins (ATF6, GRP-78, ATF4, XBP1s, and CHOP) and immunogenic cell death-related markers (ATP, HMGB1) were upregulated, and the AXL phosphorylation level was suppressed after MTE treatment. However, when salubrinal (an endoplasmic reticulum stress inhibitor) and MTE were co-treated cells, the inhibitory effects of MTE on PC-9 and H1975 cells were impaired. Importantly, inhibition of AXL expression or activity also promotes the expression of endoplasmic reticulum stress and immunogenic cell death-related markers. Mechanistically, MTE induced endoplasmic reticulum stress and immunogenic cell death by suppressing AXL activity, and these effects were attenuated when AXL activity recovered. Moreover, MTE significantly increased the expression of endoplasmic reticulum stress-related markers in LLC tumor-bearing mouse tumor tissues and plasma levels of ATP and HMGB1. Molecular docking illustrated that kaempferol has the strongest binding energy with AXL and suppresses AXL phosphorylation. CONCLUSION: MTE induces endoplasmic reticulum stress-associated immunogenic cell death in NSCLC cells. The anti-tumor effects of MTE are dependent upon endoplasmic reticulum stress. MTE triggers endoplasmic reticulum stress-associated immunogenic cell death by inhibiting AXL activity. Kaempferol is an active component that inhibits AXL activity in MTE. The present research revealed the role of AXL in regulating endoplasmic reticulum stress and enriched the anti-tumor mechanisms of MTE. Moreover, kaempferol may be considered a novel AXL inhibitor.

Pregnane steroids and steroid glycosides with multidrug resistance reversal activity from Marsdenia tenacissima.

Twenty compounds comprising four pregnane steroids (2-4 & 20) and 16 pregnane glycosides (1 & 5-19) have been obtained from the ethanol extract of the roots of a Dai ethnological herb, Marsdenia tenacissima. Their structures were characterized on the basis of comprehensive spectroscopic analyses with 17 ones (1-17) being reported for the first time, including the rare cases (2 & 3) of free C21 steroids with 17α-acetyl substitution, compounds 4-7 bearing an unusual 14α-OH, and the first examples with simultaneous 14α-OH/17α-acetyl substitution (7) and glycosylation at C-12 position (10 & 11). An empirical rule for the identification of C-17 configuration, in C21 steroids incorporating the marsdenin constitution structure, was also proposed. All the isolates, along with an array of previously reported analogues in our compound library, were screened for their chemo-reversal ability against P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in MCF-7/ADR cell line, and six compounds exhibited moderate MDR reversal activity with reversal folds ranging from 1.92 to 4.44.

Nine New Pregnane Glycosides from the Cultivated Medicinal Plant Marsdenia tenacissima.

The ethnobotanical plant Marsdenia tenacissima has been used for hundreds of years for Dai people in Yunnan Province, China. Previously, chemical investigations on this plant have revealed that pregnane glycosides were the main biological constituents. Nine new pregnane glycosides, marsdeosides A-I (1-9), were isolated from cultivated dried stems of the medicinal plant Marsdenia tenacissima in this study. The structures were analyzed by extensive spectroscopic analysis, including 1D, 2D NMR, HRESIMS, and IR spectroscopic analysis. The absolute configurations of the sugar moieties were identified by comparing the Rf values and specific optical rotations with those of the commercially available standard samples and the data reported in the literature. Marsdeosides A (1) featured an unusual 8,14-seco-pregnane skeleton. Compounds 1, 8, and 9 showed activity against nitric oxide production in lipopolysaccharide-activated macrophage RAW264.7, with IC50 values of 37.5, 38.8, and 42.8 µM (L-NMMA was used as a positive control, IC50 39.3 µM), respectively. This study puts the knowledge of the chemical profile of the botanical plant M. tenacissima one step forward and, thereby, promotes the sustainable utilization of the resources of traditional folk medicinal plants.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3ß/STAT3 signaling axis.

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3ßSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3ß, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3ß/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Marsdenia tenacissima extract prevents the malignant progression of glioma through upregulating lncRNA MEG3 and SFRP1-dependent inhibition of Wnt/ß-catenin pathway.

BACKGROUND/AIM: Recent studies have highlighted the tumor-suppressive effect of Marsdenia tenacissima extract (MTE) on human cancers. This research unveils the potential impact of MTE on glioma and ascertains the relevant molecular mechanisms. METHODS: Glioma cells were treated with MTE, with normal human astrocytes (NHAs) as controls. A battery of function experiments, including the CCK-8 viability test, colony formation assay, scratch migration assay, and Transwell invasion assay, was executed to address the responses of glioma cells to MTE treatment and gain or loss of function of lncMEG3, miR-542-3p, and SFRP1. FISH, RIP, and dual-luciferase reporter assays were adopted for assessing gene interactions. U251-GFP-Luc cells were delivered into nude mice through intracranial injection to develop an orthotopic glioma model for in vivo validation. RESULTS: 200 mg/mL MTE could suppress the proliferating, migrating, and invading properties of glioma cells but not affect those of NHAs. MTE treatment enhanced the expression of lncMEG3, which competes with SFRP1 for binding miR-542-3p. SFRP1 could inactivate the Wnt/ß-catenin pathway. Animal experimentation substantiated the antitumor activity and mechanism of MTE in nude mice. CONCLUSIONS: MTE suppresses glioma via the lncMEG3/miR-542-3p/SFRP1/Wnt/ß-catenin axis. These findings contribute to a theoretical basis for the use of MTE for glioma patients.

Polyoxypregnane Glycosides from Root of Marsdenia tenacissima and Inhibited Nitric Oxide Levels in LPS Stimulated RAW 264.7 Cells.

Six new polyoxypregnane glycosides, marstenacisside F1−F3 (1−3), G1−G2 (4−5) and H1 (6), as well as 3-O-ß-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-ß-D-allopyranosyl-(1→4)-ß-D-oleandropyranosyl-11α,12ß-di-O-benzoyl-tenacigenin B (7), were isolated from the roots of Marsdenia tenacissima. Their structures were established by an extensive interpretation of their 1D and 2D NMR and HRESIMS data. Compounds 1−7 were tenacigenin B derivatives with an oligosaccharide chain at C-3. This was the first time that compound 7 had been isolated from the title plant and its 1H and 13C NMR data were reported. Compounds 4 and 5 were the first examples of C21 steroid glycoside bearing unique ß-glucopyranosyl-(1→4)-ß-glucopyranose sugar moiety. All the isolated compounds were evaluated for anti-inflammatory activity by inhibiting nitric oxide (NO) production in the lipopolysaccharide-induced RAW 264.7 cells. The results showed that marstenacisside F1 and F2 exhibited significant NO inhibitory activity with an inhibition rate of 48.19 ± 4.14% and 70.33 ± 5.39%, respectively, at 40 µM, approximately equal to the positive control (L-NMMA, 68.03 ± 0.72%).

Marsdenia tenacissima genome reveals calcium adaptation and tenacissoside biosynthesis.

Marsdenia tenacissima is a medicinal plant widely distributed in the calcium-rich karst regions of southwest China. However, the lack of a reference genome has hampered the implementation of molecular techniques in its breeding, pharmacology and domestication. We generated the chromosome-level genome assembly in Apocynaceae using combined SMRT sequencing and Hi-C. The genome length was 381.76 Mb, with 98.9% of it found on 11 chromosomes. The genome contained 222.63 Mb of repetitive sequences and 21 899 predicted gene models, with a contig N50 of 6.57 Mb. Phylogenetic analysis revealed that M. tenacissima diverged from Calotropis gigantea at least 13.43 million years ago. Comparative genomics showed that M. tenacissima underwent ancient shared whole-genome duplication. This event, together with tandem duplication, contributed to 70.71% of gene-family expansion. Both pseudogene analysis and selective pressure calculations suggested calcium-related adaptive evolution in the M. tenacissima genome. Calcium-induced differentially expressed genes (DEGs) were mainly enriched in cell-wall-related processes. Domains (e.g. Fasciclin and Amb_all) and cis-elements (e.g. MYB and MYC) frequently occurred in the coding and promoter regions of cell-wall DEGs, respectively, and the expression levels of these genes correlated significantly with those of calcium-signal-related transcription factors. Moreover, calcium addition increased tenacissoside I, G and H contents. The availability of this high-quality genome provides valuable genomic information for genetic breeding and molecular design, and lends insights into the calcium adaptation of M. tenacissima in karst areas.

Steroid glycosides from the roots of Marsdenia tenacissima.

Eleven undescribed glycosylated C21 steroids and nine known homologous glycosides with diverse acyl substituents, as well as their common steroid aglycone, have been obtained from the roots of Marsdenia tenacissima. Their structures were elucidated mainly by comprehensive spectroscopic analyses and comparison with previously reported analogues, with the absolute configuration assignment being supported by chemical degradation, X-ray crystallography and ECD exciton chirality method. Among them, two pairs of regioisomers were found to exist as inseparable equilibrium mixtures due to an interesting intramolecular transesterification, and nicotinoyl substitution was first reported for metabolites from the title plant. Screening of these compounds in a panel of bioassays revealed that two glycosides displayed mild inhibition against butyrylcholinesterase.

Marsdenia tenacissima injection induces the apoptosis of prostate cancer by regulating the AKT/GSK3β/STAT3 signaling axis / 中国天然药物

Marsdenia tenacissima injection, a standard Marsdenia tenacissima extract (MTE), has been approved as an adjuvant therapeutic agent for various cancers. Our previous study showed that MTE inhibited the proliferation and metastasis of prostate cancer (PCa) cells. However, the underlying mechanisms and active ingredients of MTE against PCa were not completely understood. This study revealed that MTE induced significant decreases in cell viability and clonal growth in PCa cells. In addition, MTE induced the apoptosis of DU145 cells by reducing the mitochondrial membrane potential and increasing the expression of Cleaved Caspase 3/7, Cyt c, and Bax. In vivo, DU145 xenografted NOD-SCID mice treated with MTE showed significantly decreased tumor size. TUNEL staining and Western blot confirmed the pro-apoptotic effects of MTE. Network pharmacology analysis collected 196 ingredients of MTE linked to 655 potential targets, and 709 PCa-associated targets were retrieved, from which 149 overlapped targets were screened out. Pathway enrichment analysis showed that the HIF-1, PI3K-AKT, and ErbB signaling pathways were closely related to tumor apoptosis. Western blot results confirmed that MTE increased the expression of p-AKTSer473 and p-GSK3βSer9, and decreased the expression of p-STAT3Tyr705in vitro and in vivo. A total of 13 compounds in MTE were identified by HPLC-CAD-QTOF-MS/MS and UPLC-QTOF-MS/MS. Molecular docking analysis indicated that six compounds may interact with AKT, GSK3β, and STAT3. In conclusion, MTE induces the endogenous mitochondrial apoptosis of PCa by regulating the AKT/GSK3β/STAT3 signaling axis, resulting in inhibition of PCa growth in vitro and in vivo.

Mechanism of Marsdenia tenacissima against ovarian cancer based on network pharmacology and experimental verification / 中国中药杂志

The present study aimed to explore the main active components and underlying mechanisms of Marsdenia tenacissima in the treatment of ovarian cancer(OC) through network pharmacology, molecular docking, and in vitro cell experiments. The active components of M. tenacissima were obtained from the literature search, and their potential targets were obtained from SwissTargetPrediction. The OC-related targets were retrieved from Therapeutic Target Database(TTD), Online Mendelian Inheritance in Man(OMIM), GeneCards, and PharmGKB. The common targets of the drug and the disease were screened out by Venn diagram. Cytoscape was used to construct an "active component-target-disease" network, and the core components were screened out according to the node degree. The protein-protein interaction(PPI) network of the common targets was constructed by STRING and Cytoscape, and the core targets were screened out according to the node degree. GO and KEGG enrichment analyses of potential therapeutic targets were carried out with DAVID database. Molecular docking was used to determine the binding activity of some active components to key targets by AutoDock. Finally, the anti-OC activity of M. tenacissima extract was verified based on SKOV3 cells in vitro. The PI3K/AKT signaling pathway was selected for in vitro experimental verification according to the results of GO function and KEGG pathway analyses. Network pharmacology results showed that 39 active components, such as kaempferol, 11α-O-benzoyl-12β-O-acetyltenacigenin B, and drevogenin Q, were screened out, involving 25 core targets such as AKT1, VEGFA, and EGFR, and the PI3K-AKT signaling pathway was the main pathway of target protein enrichment. The results of molecular docking also showed that the top ten core components showed good binding affinity to the top ten core targets. The results of in vitro experiments showed that M. tenacissima extract could significantly inhibit the proliferation of OC cells, induce apoptosis of OC cells through the mitochondrial pathway, and down-regulate the expression of proteins related to the PI3K/AKT signaling pathway. This study shows that M. tenacissima has the characteristics of multi-component, multi-target, and multi-pathway synergistic effect in the treatment of OC, which provides a theoretical basis for in-depth research on the material basis, mechanism, and clinical application.

Laticifer ontogenesis and the chemical constituents of Marsdenia zehntneri (Apocynaceae) latex in a semiarid environment.

MAIN CONCLUSION: Anastomosed laticifers with intrusive growth produce latex containing methyl comate and betulin with economic and ecological value in arid environments. Climatic factors influence laticifer development in the apical meristem and vascular cambium. Latex is a complex emulsion with high medicinal as well as ecological value related to plant survival. Marsdenia zehntneri is a shrubby plant that grows on limestone outcrops in the semiarid regions of Brazil. We sought to characterize the ontogenesis of the laticifers of this species and to relate that process to climatic seasonality and phenology through anatomical, ultrastructural, and micro-morphometric evaluations of the apical meristem and vascular cambium. The histochemistry of the secretory structure was investigated and the chemical composition of the latex was analyzed. Phenological assessments were performed by monitoring phenological events for 1 year. The laticifers network of M. zehntneri permeates the entire primary and secondary body of the plant, providing a wide distribution system of defensive compounds. Its laticifers, of a distinct mixed type (anastomosed, with intrusive growth), are numerous and voluminous in the apical meristem but scarce and minute in the secondary phloem. Latex secretion involves the participation of oleoplasts, polysomes, and dictyosomes. Methyl 2,3-dihydroxy-ursan-23-oate, methyl 3-hydroxy-ursan-23-oate, and betulin are encountered in high proportions in the latex and have ecological and medicinal functions. The development of primary laticifers is related to the resumption of apical meristem activity with increasing day length at the end of the austral winter. The development of secondary laticifers is related to high summer temperatures and rainfall that favor vascular cambium activity. The wide distribution of laticifers, their seasonal pattern of secretion, and their latex composition contribute to the adaptation of M. zehntneri to its natural environment.

Marsdenia tenacissima extract disturbs the interaction between tumor-associated macrophages and non-small cell lung cancer cells by targeting HDGF.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight et Arn. is a traditional Chinese herbal medicine, and its water-soluble ingredient Marsdenia tenacissima extract (MTE), was widely used for cancer treatment. The multi-pharmacological efficacies and mechanisms of MTE in directly inhibiting tumor cells have been extensively studied. However, the anti-tumor effects of MTE in the tumor-associated macrophages (TAMs) microenvironment remain unclear. AIM OF THE STUDY: To uncover the role of hepatoma-derived growth factor (HDGF) in the interaction between TAMs and non-small cell lung cancer (NSCLC) cells. To evaluate the anti-tumor effects of MTE on the vicious crosstalk between TAMs and NSCLC by targeting HDGF. MATERIALS AND METHODS: HDGF-overexpression PC-9 and H292 NSCLC cell lines were constructed and verified. RNA-sequencing (RNA-seq) was performed in HDGF-overexpression PC-9 cells to probe the differential expression of genes. THP-1-derived macrophages were characterized using specific markers after stimulation with phorbol-12-myristate 13-acetate (PMA) and rhIL-4 or rhHDGF. The role of HDGF both in NSCLC cells and TAMs was determined using approaches like Western blot, qRT-PCR, ELISA, and flow cytometry. The interaction between tumor cells and TAMs were assessed by indirect co-culture H1975, PC-9 cells with M2 type macrophages. The effects of MTE on anti-tumor and macrophage polarization were evaluated in vitro and in vivo. RESULTS: RNA-seq results identified IL-4 as a critical response to HDGF in NSCLC. HDGF induced macrophages polarizing toward M2 type, and promoted NSCLC cells proliferation, migration and invasion in vitro. On the one hand, HDGF dose-dependently promoted IL-4 expression in NSCLC cells. On the other hand, HDGF induced M2 macrophage polarization through the IL-4/JAK1/STAT3 signaling pathway. MTE treatment significantly decreased the expression and secretion of HDGF in NSCLC cells. Meanwhile, MTE treatment led to M2 macrophage repolarization, as evidenced by decreased expression of M2 markers and increased levels of M1 markers. Importantly, MTE treatment significantly suppressed tumor development in C57BL/6 mice bearing Lewis lung cancer (LLC) cells in vivo, accompanied by decreased plasma HDGF levels, reduced M2 macrophages infiltration and increased M1 macrophages proportion in mice tumor tissues. CONCLUSIONS: HDGF upregulated IL-4 expression in NSCLC cells, and promoted M2 polarization by the IL-4/JAK1/STAT3 signaling pathway in macrophages. MTE disturbed the interaction between NSCLC and TAMs in vitro, and inhibited tumor growth in vivo, at least in part, by suppressing HDGF. Therefore, our present study revealed a novel anti-tumor mechanism of MTE through inhibiting HDGF expression and enhancing macrophage polarization from M2 to M1 phenotype.

An ester derivative of tenacigenin B from Marsdenia tenacissima (Roxb.) Wight et Arn reversed paclitaxel-induced MDR in vitro and in vivo by inhibiting both P-gp and MRP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima is a medicinal plant, used as a raw material for cancer treatment in China. In our previous studies, 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B (MT2), the main steroid aglycone isolated from M. tenacissima, was found to significantly enhance the antitumor activity of paclitaxel (PTX) in vivo. However, it is unclear whether MT2 reverses multidrug resistance (MDR) in tumors. AIM OF THE STUDY: To determine the role and mechanism of MT2 in reversing tumor MDR. MATERIALS AND METHODS: MDR cell line HeLa/Tax was established from the human cervical carcinoma cell line HeLa by long-term exposure to subtoxic concentrations of PTX and was used to evaluate the ability of MT2 to restore chemosensitivity of cells both in vitro and in a nude mouse model. The expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) was determined using western blotting and immunohistochemistry. The substrate transport function was assessed using an MDR function assay kit. The binding modes of MT2 and P-gp were determined using the conformation-sensitive anti-P-gp antibodies. The permeability and transport properties of MT2 were analyzed in Caco-2 cell monolayers. RESULTS: Compared to parental cells, HeLa/Tax cells overexpress P-gp and MRP2 and are approximately 100-360 fold more resistant to the anticancer drugs PTX, docetaxel, and vinblastine. MT2 at 5 or 10 µmol/L significantly increased the sensitivity of HeLa/Tax to these three anticancer drugs (18-56-fold decrease in IC50 value) and suppressed the expression of P-gp and MRP2. Knockdown of P-gp with small interfering RNA partially reversed MT2-induced sensitivity to PTX in HeLa/Tax cells. Moreover, MT2 directly inhibited P-gp-mediated substrate transport while interacting with membrane P-gp in non-substrate ways. MT2 was highly permeable and could not be transported in the Caco-2 cell monolayers. In nude mice bearing HeLa/Tax xenografts, the combination treatment with MT2 and PTX exerted a synergistic inhibitory effect on the growth of tumors and the expression of P-gp and MRP2 without increasing toxicity. CONCLUSION: MT2 is a potential agent for reversing MDR. It impedes membrane drug efflux pumps by suppressing P-gp and MRP2 expression, and directly inhibiting the transport function of P-gp.