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).

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.

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.

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.

Marsdenia tenacissima (Roxb.) Moon injection exerts a potential anti-tumor effect in prostate cancer through inhibiting ErbB2-GSK3ß-HIF1α signaling axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima injection (MTE), a traditional Chinese medical injection extracted from the rattan of Marsdenia tenacissima (Roxb.) Moon, has been approved for clinical use in China as an adjuvant therapeutic agent in multiple cancers, including esophageal cancer, gastric cancer, lung cancer, and liver cancer. However, the activity and mechanism of MTE on prostate cancer (PCa) remain to be defined. AIM OF THE STUDY: To investigate the activity and the underlying mechanism of MTE in the treatment of PCa. MATERIALS AND METHODS: The component characterization of MTE was analyzed by HPLC-CAD-QTOF-MS/MS technology. Cell Counting Kit-8 (CCK-8) assay was used to assess PCa cell proliferation. Colony formation assay was applied to detect the clonogenic ability of the cells. MetaboAnalyst5.0 database was employed to analyze the altered metabolites of PC3 cells treated with MTE obtained by UPLC-QTOF-MS/MS. Combined with metabolomics analysis and network pharmacology, we predicted the potential targets, which further were verified by Western Blot, RT-qPCR, and Immunohistochemistry assays. Finally, SeeSAR software was applied to predict the potential active components of MTE against PCa. RESULTS: A total of 21 components in MTE were confirmed by HPLC-CAD-QTOF-MS/MS analysis. MTE inhibited the proliferation and colony formation of PCa cells. A total of 20 metabolites closely related to glycerophospholipid metabolism, glycolysis/gluconeogenesis, and tricarboxylic acid (TCA) cycle were significantly changed in PC3 cells treated with MTE. The network pharmacology analysis revealed that MTE suppressed the growth of PC3 cells might by regulating the ErbB2-GSK3ß-HIF1α signaling axis. Furthermore, we also confirmed that stimulation of MTE significantly inhibited the phosphorylation of ErbB2 at Tyr877 and the activities of its downstream signal transducers (GSK3ß and HIF1α) in PCa, as well as the mRNA levels of critical factors (IDH2, LDHA, and HIF1A) in the tricarboxylic acid (TCA) cycle. Molecular docking further suggested that Tenacissimoside E, cryptochlorogenic acid, and scopoletin might be the active ingredients of MTE for PCa treatment. CONCLUSION: This study proposed that MTE exerts a potential anti-tumor effect in PCa through inhibiting ErbB2-GSK3ß-HIF1α signaling axis, which may be related to the TCA cycle.

Pharmacokinetics, plasma protein binding, and metabolism of a potential natural chemosensitizer from Marsdenia tenacissima in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight et Arn is a medicinal plant mainly distributed in southwest China. It is used in folk medicine for the treatment of tumors and is synergistic with chemotherapies. In our previous study, 11α-O-2-methybutyryl-12ß-O-tigloyl-tenacigenin B (MT2), a main steroid aglycone isolated from the total aglycones of M. tenacissima, significantly enhanced the in vivo antitumor effect of paclitaxel in mice bearing human tumor xenografts, showing its potential as a chemosensitizer. However, the pharmacokinetic characteristics, plasma protein binding rate, and metabolic profile of MT2 remain unclear. AIM OF THE STUDY: To elucidate the pharmacokinetic characteristics, plasma protein binding rate, and metabolic profile of MT2 in rats. MATERIALS AND METHODS: MT2 in rat plasma and phosphate-buffered saline was quantified using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, while the MT2 metabolites in rat liver microsomes were analyzed using UPLC-triple time-of-flight MS/MS. RESULTS: For intravenously administered MT2, the maximum plasma concentration and the area under the plasma concentration-time curve indicated dose dependency, while the elimination half-life time, the mean residence time, apparent volume of distribution and total apparent clearance values remained relatively unchanged in both the 5 mg/kg and 10 mg/kg groups. For orally administered MT2, the bioavailability was 1.08-1.11%. In rat plasma, MT2 exhibited a protein binding rate of 93.84-94.96%. In rat liver microsomes, MT2 was metabolized by oxidation alone or in combination with demethylation, and five MT2 metabolites were identified. CONCLUSION: MT2 has low oral bioavailability and a high plasma protein binding rate in rats. After administration, MT2 is transformed into oxidative metabolites in the liver. To achieve a high blood concentration of MT2, it should be administered intravenously. These findings would serve as a reference for further MT2-based pharmacological study and drug development.

Distributions of eight bioactive components in rat tissues administered Marsdenia tenacissima extract orally detected through UPLC-MS/MS.

Marsdenia tenacissima (Roxb.) Wight et Arn. (M. tenacissima) is considered an anticancer medicine in traditional Chinese medicine, which is extensively used in clinical application since it has great therapeutic effects. Currently, although a number of articles have examined M. tenacissima in terms of its pharmacology and quality control, few have investigated the in vivo mechanism of M. tenacissima active ingredients. Previously, we have studied the pharmacokinetics of eight active ingredients after oral administration of M. tenacissima extracts in rat plasma. This study constructed a new scientific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach to simultaneously quantify the contents of tenacissosides B, G, H and I, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid and caffeic acid in rats orally administered M. tenacissima extract. The proposed approach was successfully used for investigating the distributions of those eight analytes in rat tissues, with digoxin being used as an internal control. The Eclipse Plus C18 RRHD column was used for determination at a column temperature of 30°C. The mobile phase system consisted of acetonitrile and water (supplemented with 0.1% formic acid) under optimal gradient elution conditions. Afterwards, this approach was validated according to the requirements for the analysis of biological samples developed by the US Food and Drug Administration, including precision, accuracy, stability and matrix effects. Based on tissue distribution analysis, those eight analytes showed rapid distribution within all the tested tissues. With regard to organic acid distribution, it followed the order stomach > liver > kidney > small intestine > lung > spleen > heart, whereas the four steroids followed the order stomach > lung > spleen > small intestine > liver > kidney > heart. The present study lays the theoretical foundation for the use and development of M. tenacissima in clinical practice.

Nitric oxide, a communicator between tumor cells and endothelial cells, mediates the anti-tumor effects of Marsdenia Tenacissima Extract (MTE).

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight & Arn is a well-known traditional Chinese medicine for treating cancer. The anti-tumor effects of the water soluble component of M. tenacissima (MTE, M. Tenacissima Extract) have been intensely studied. However, the roles of microenvironmental cells in mediating the anti-tumor actions of MTE remain to be defined. AIM OF THE STUDY: To determine the roles of nitric oxide (NO) released by endothelial cells (ECs), an important component of tumor microenvironment, in regulating the anti-cancer effects of MTE, and to explore the underlying mechanisms. MATERIALS AND METHODS: Co-culture system of ECs and A549 non-small cell lung cancer (NSCLC) cells was established for determining the interactions of ECs and lung cancer cells. Nitro-L-arginine methyl ester hydrochloride (L-NAME) was used to inhibit the production of NO. Cell viability was examined using cell counting kit 8 and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. NO assay and Western blot were used to determine the involved signaling pathway. Primary lung microenvironmental cells (PLMCs) were cultured to examine the roles of NO released from the lung microenvironment in regulating the anti-cancer effects of MTE. A subcutaneous xenograft model was established to determine the involvement of NO in effects of MTE against NSCLCs in vivo. RESULTS: In the co-culture system of ECs and A549 NSCLC cells, MTE (30 mg/mL) treatment reduced viability of lung cancer cells. However, when L-NAME (a nitric oxide synthase (NOS) inhibitor, 300 µM) was introduced into the co-culture system, the NSCLC-inhibiting effects of MTE were significantly suppressed. By contrast, addition of L-NAME (300 µM) did not affect the anti-cancer efficiency of MTE when ECs were not present. Mechanistically, MTE enhanced endothelial production of NO via stimulating PKA-endothelial nitric oxide synthase (eNOS) signaling. Elevated levels of NO inhibited proliferation and promoted apoptosis of the A549 NSCLC cells. Importantly, PKA-eNOS-NO signaling was effective in mediating the anti-cancer effects of MTE, when lung cancer cells were co-cultured with PLMCs. Finally, oral administration of MTE to the subcutaneous xenograft mice significantly suppressed tumor growth, while elevated NO productions. Plasma NO was also revealed to be negatively correlated with the tumor weight. CONCLUSIONS: ECs significantly contributed to anti-cancer effects of MTE by elevating production of NO, in a PKA-dependent manner. The present study revealed a novel anti-cancer mechanism of MTE through regulating the function of ECs, an important component of tumor microenvironment.

Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2).

Biosynthesis of Zinc oxide nanoparticles (ZnONPs) from natural plants stands as a promising nanodrug delivery system in cancer therapeutics. Marsdenia tenacissima (M.t), a Chinese medicinal plant has been extensively used as clinical remedy for treating several types of cancer. In this present study, ZnONPs were synthesized from Marsdenia tenacissima and its anti cancer potency was assessed against in vitro laryngeal cancer cell line Hep-2. The biosynthesized Marsdenia tenacissima Zinc Oxide Nanoparticles [M.t-ZnONPs] was characterized using UV-visible Spec, SEM, TEM and EDAX analysis. The cytotoxic and apoptotic inducing potential of M.t-ZnONPs was assessed by MTT assay and staining such as DCFH-DA, AO/EtBr, Rhodamine 123, DAPI and comet assay. The anticancer potential of M.t-ZnONPs was analysed by Real time PCR analysis of proapoptotic, antiapoptotic and caspases proteins. Our present findings showed characteristic and morphological representation of synthesized M.t-ZnONPs by UV-visible Spec, SEM, TEM and EDAX analysis. M.t-ZnONPs exhibits its cytotoxicity by inhibiting the viability of Hep-2 cells and IC50 value was obtained by MTT assay. The results of apoptotic staining techniques in M.t-ZnONPs treated Hep-2 cells confirm with excess ROS generation, disruption of mitochondrial membrane potential and nuclear damage. The apoptotic inducing potential of M.t-ZnONPs was also evidenced by upregulation of proapoptotic proteins Bax, Caspase 3 & 9 and downregultion of antiapoptotic protein Bcl-2 by RT-PCR analysis. Finally, these results suggested that biosynthesized M.t-ZnONPs is an effective anticancer agent which induces apoptosis in Hep-2 laryngeal cell line and thus conclude that M.t-ZnONPs, a valid anticancer strategy in treating various cancer.

Anticancer activity of green synthesised gold nanoparticles from Marsdenia tenacissima inhibits A549 cell proliferation through the apoptotic pathway.

Gold nanoparticles (AuNPs) as the most excellent anticancer theranostic nanoparticles were synthesized through efficient, simple, and green synthesis method using Marsdenia tenacissima plant extracts and they are widely characterized by several techniques including ultraviolet-visible (UV) spectroscopy, atomic force microscopy (AFM), energy-dispersive X-ray spectrometers (EDS), transmission electron microscopy (TEM), and Fourier transform infrared (FT-IR) spectroscopy. From the AuNPs synthesized by M. tenacissima extracts, it was discovered that particle size around 50 nm, which is admirable nano dimension, was achieved by plant-mediated synthesis. After characterization of these nanoparticles, they performed as in vitro anticancer activity against lung cancer cell lines (A549). MTT assay revealed that AuNPs produce toxicity based on the dose-dependent A549 cells growth inhibition. AuNPs treatment activates caspase expression and down-regulates the anti-apoptotic protein expression in A549 cells. Our results point out that the AuNPs from M. tenacissima extract are apposite stabilizing agents, which serve as an effective anticancer agent against lung cancer cell lines (A549).

Synthesis and characterization of gold nanoparticles from Marsdenia tenacissima and its anticancer activity of liver cancer HepG2 cells.

Nowadays, the synthesis and characterization of gold nanoparticles (AuNPs) from plant based extracts and effects of their anticancer have concerned an important interest. Marsdenia tenacissima (MT), a conventional Chinese herbal medicine, has long been used for thousands of years to treat tracheitis, asthma, rheumatism, etc. In this present study, we optimize the reaction of parameters to manage the nanoparticle size, which was categorized by high-resolution transmission electron microscopy (HR-TEM). A different characterization method, for example, UV-visible spectroscopy (UV-vis), fourier-transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) were performed to consider the synthesized AuNPs getting from the MT leaf extract. The MT-AuNPs were analyzed for their cytotoxicity property against HepG2 cells by MTT analysis. The apoptosis was evaluated by using reactive oxygen species (ROS), migration assay, mitochondrial membrane potential (MMP) and apoptotic protein expression. Interestingly, the findings of our study observed the cytotoxicity effect of synthesized MT-AuNPs at a concentration of 59.62 ± 4.37 µg after 24 hrs treatment. Apoptosis was induced by the MT-AuNPs with enhanced ROS, changed MMP and inhibit the migration assay. Finally, the apoptosis was confirmed by the considerable up-regulation of Bax, caspase-9 and caspase-3, while the anti-apoptotic protein expressions of Bcl-2 and Bcl-XL were down-regulated. Although, in this studies, we evaluated the characterization, synthesis and anticancer action of gold nanoparticles from MT (MT-AuNPS) helpful for liver cancer therapeutics.

Screening potential antagonists of epidermal growth factor receptor from Marsdenia tenacissima via cell membrane chromatography model assisted by HPLC-ESI-IT-TOF-MS.

Marsdenia tenacissima, or Tongguanteng in Chinese, is a traditional Chinese herb and has a broad application in clinical practice for its pharmacological effects of treating asthma, pneumonia, tonsillitis, pharyngitis tumors, etc. However, few studies have reported the screening of the active components of this medicine for tumor therapy. In this work, a two-dimensional analytical system was developed to screen antagonists of epidermal growth factor receptor (EGFR) from M. tenacissima. A fraction was retained on the EGFR cell membrane chromatography (CMC) column, separated and identified as tenacissoside G (TG), tenacissoside H (TH) and tenacissoside I (TI) by two-dimensional HPLC-IT-TOF-MS. Molecular docking and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay were carried out to assess the activity of TS (including TG, TH and TI). Molecular docking results showed that the binding mode of TS on EGFR is similar to that of gefitinib. The MTT assay demonstrated that gefitinib and TS (especially TI) could inhibit the growth of EGFR highly expressed cell lines in a dose-dependent manner in the range of 5-50 µmol/L. In conclusion, the two-dimensional EGFR/CMC-HPLC-IT-TOF-MS system could be a useful approach in drug discovery from traditional Chinese medicines for searching for potential antitumor candidates.

Tenacigenin B ester derivatives from Marsdenia tenacissima actively inhibited CYP3A4 and enhanced in vivo antitumor activity of paclitaxel.

ETHNOPHARMACOLOGICAL RELEVANCE: Marsdenia tenacissima (Roxb.) Wight et Arn is a vine distributed in southwest area of China and used in folk medicine for treatment of tumors. Recent decades of studies on this plant reveal its synergistic effects with certain anticancer drugs in cancer therapy. In our previous study, an extract ETA which contains total aglycones made from M. tenacissima significantly enhanced antitumor activity of paclitaxel in tumor-bearing mice. However, the effective constituents in ETA and the underlying mechanisms remain unclear. AIM OF THE STUDY: Reveal the active components in ETA as well as the molecular mechanism in enhancing antitumor efficacy of paclitaxel. MATERIAL AND METHODS: Main constituents in ETA were purified by chemical methods. Effects of the purified constituents on metabolic activity of CYP450 enzymes were evaluated in human liver microsomes. Ability of the constituents to enhance antitumor activity of paclitaxel were investigated in nude mice bearing HeLa tumors. Pharmacokinetic study was performed in SD rats. Molecular docking was carried out for investigation of drug-protein interactions. RESULTS: Three main C21 steroidal aglycones, 11α-O-tigloyl-12ß-O-acetyl-tenacigenin B (MT1), 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B (MT2) and 11α-O-2-methylbutanoyl-12ß-O-acetyl-tenacigenin B (MT3), together with tenacigenin B (MT4) was prepared from ETA. Among them, MT1, MT2 and MT3 strongly inhibit the metabolic activity of CYP3A4. MT2 also showed inhibitory effects on CYP2C8, CYP2B6 and CYP2C19. In HeLa tumor xenografts, MT1, MT2 and MT3 (30 mg/kg) did not affect tumor growth themselves, but significantly enhanced paclitaxel-induced growth inhibition. In addition, coadministration of MT2 with paclitaxel resulted in significant reduction of liver CYP2C8. In pharmacokinetic study, MT2 significantly increased the blood concentration of paclitaxel with increased AUC value by 2.2-5.3 folds. Molecular docking analysis suggested hydrophobic interaction modes of tenacigenin B derivatives with CYP3A4, and also the essential roles of the C-11 and C-12 ester groups for effective interaction with CYP3A4. CONCLUSION: Our study proves that, 11α-O-tigloyl-12ß-O-acetyl-tenacigenin B, 11α-O-2-methylbutanoyl-12ß-O-tigloyl-tenacigenin B and 11α-O-2-methylbutanoyl-12ß-O-acetyl-tenacigenin B, which are the main constituents of ETA, are active inhibitors of CYP3A4 with potential to increase therapeutic efficacy of anticancer drugs that are substrates of CYP3A4. Tenacigenin B derivatives with C-11 and C-12 ester group substitutions, or at least a large part of them, are active components in ETA and M. tenacissima to enhance in vivo antitumor efficacies of paclitaxel.

Pregnane glycosides from the bark of Marsdenia cundurango and their cytotoxic activity.

Seven new pregnane glycosides (1-7) and eight known compounds (8-15) were isolated from the bark of Marsdenia cundurango (Asclepiadaceae). The structures of 1-7 were determined by spectroscopic analysis, including two-dimension NMR spectroscopy, chemical transformations, and chromatographic analysis of the hydrolyzed products. The isolated compounds 1-15 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 normal human lung cells, including apoptosis-inducing activity of a representative pregnane glycoside in HL-60 cells.

Ethyl acetate fraction in ethanol extract from root of.

OBJECTIVE: To evaluate the anti-tumor activity of ethyl acetate fraction (EFA), extracted with ethanol from the root of ""Dai-Bai-Jie"" in A549 cancer cells and its underlying mechanism. METHODS: ""Dai-Bai-Jie"" was extracted with 95% ethanol-aqueous (DBJ-1), 50% ethanol-aqueous (DBJ-2), and water (DBJ-3) by reflux method. 95% ethanol-aqueous extract was separated byethyl acetate (EFA) and n-butyl alcohol (DBJ-5), consecutively. The SRB method was used to evaluate the cytotoxic activity. Annexin V-FITC staining was applied to observe the apoptosis and analyze the cell cycle activated by EFA in A549 tumor cell. Western blot was used to detect the apoptosis/related proteins expressions. A549 tumor cellsbearing nude mice model was employed to measure the tumor volume, mice weight, and tumor inhibition ratio in order to verify the antitumor activity in vivo. RESULTS: DBJ-1 and EFA showed better cytotoxic activity on A549 tumor cells with IC50 25 and 3.5 ¦Ìg/mL, respectively. EFA can exhibit the proliferation, arrest cell cycle at G0/G1 phase, and induce apoptosis in A549 tumor cells in vitro. The mechanisms of apoptosis induced by EFA may be associated with decreasing Bcl-2 protein expression and increasing p53, Bax, Caspase-3, and Caspase-8 proteins expression. EFA also possessed significant anti-tumor efficacy in nude mice, and little toxicity was observed in the host. CONCLUSION: EAF could induce A549 tumor cells apoptosis and G0/G1 cell cycle arrest. A549 tumor cells apoptosis induced by EAF may be associated with the decrease in the ratio of Bcl-2 and Bax mRNA levels, and increase in the expression of p53, Caspase-3, and Caspase-8 proteins.

Cytotoxicity of anti-tumor herbal Marsdeniae tenacissimae extract on erythrocytes.

Marsdeniae tenacissimae extract (MTE) has been used as an adjuvant medicine for cancer therapy for a long time. Although massive studies demonstrated its considerable anti-cancer effect, there is no research on its influence on erythrocytes, which are firstly interacted with MTE in the circulation. To investigate the influence of MTE on erythrocytes, we used a flow cytometer to detect the MTE-treated alternations of morphology, calcium concentration, and reactive oxygen species (ROS) level in erythrocytes. We used hemolysis under different osmotic solutions to evaluate the fragility of erythrocytes. Data showed that MTE treatment dose-dependently increased the ratio of erythrocyte fragmentation (P<0.001) and shrinking, and elevated the forward scatter (FSC) value (P<0.001) and calcium accumulation (P<0.001). MTE induced ROS production of erythrocytes under the high glucose condition (P<0.01) and consequently caused a rise in fragility (P<0.05). These results suggest that MTE induces cytotoxicity and aging in erythrocytes in a dose-dependent manner, and presents the possibility of impairment on cancer patients' circulating erythrocytes when MTE is used as an anti-cancer adjuvant medicine.

Reversal of multidrug resistance by Marsdenia tenacissima and its main active ingredients polyoxypregnanes.

ETHNOPHARMACOLOGICAL RELEVANCE: Multidrug resistance (MDR) of cancer is often associated with the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (P-gp), multidrug resistance-associated protein-1 (MRP-1) and breast cancer resistance protein (BCRP or ABCG2), in cancer cells, which facilitates the active efflux of a wide variety of chemotherapeutic drugs out of the cells. Marsdenia tenacissima is a traditional Chinese medicinal herb that has long been clinically used for treatment of cancers, particularly in combinational use with anticancer drugs. Polyoxypregnanes (POPs) are identified as main constituents of this herb, and three of them have been reported to exhibit P-gp modulatory effect and thus reverse MDR. Therefore, it is of great necessity to investigate more POPs that have potential to reverse transporters-mediated MDR. AIM OF THE STUDY: We aimed to identify POPs as the chemical basis responsible for circumventing ABC transporters-mediated MDR by M. tenacissima. MATERIALS AND METHODS: The MDR reversal effects of M. tenacissima crude extract together with a series of isolated POPs were evaluated on several MDR cancer cell lines that overexpress P-gp, MRP1 or ABCG2. The activities of P-gp, MRP1 and ABCG2 were determined by the flow cytometry-based substrate efflux assay. Molecular docking of POPs to a three-dimensional human P-gp homology structure was also performed. RESULTS: The crude extract of M. tenacissima was firstly found to circumvent P-gp-mediated MDR. Then, 11 polyoxypregnane compounds (POPs) isolated from this herb were found to overcome P-gp-, MRP1- and/or ABCG2-mediated MDR. Further mechanistic study delineated that the reversal of MDR by these POPs was due to significant increase in the intracellular concentrations of the substrate anticancer drugs via their inhibition of different ABC transporter-mediated efflux activities. Furthermore, molecular docking revealed that POPs with P-gp modulatory effect bound to P-gp and fitted well into the cavity between the alpha and beta subunit of P-gp via forming hydrogen bonds. In addition, several key structural determinants for inhibition of P-gp, MRP1 or ABCG2 by POPs were illustrated. CONCLUSIONS: Our findings advocated the rational use of M. tenacissima to enhance efficacies of conventional anticancer drugs in tumors with ABC drug transporters-mediated MDR. Furthermore, 11 POPs were found to contribute to MDR reversal effect of M. tenacissima via inhibition of different ABC efflux transporters.

Marsdenia tenacissimae extraction (MTE) inhibits the proliferation and induces the apoptosis of human acute T cell leukemia cells through inactivating PI3K/AKT/mTOR signaling pathway via PTEN enhancement.

Marsdenia tenacissimae extraction (MTE) as a traditional Chinese herb has long been used to treat some diseases such as tumors in China. However, the potential effectiveness of MTE in leukemia has not yet been fully understood, and the related molecular mechanism is still unknown. In the present study, we aimed to evaluate the effects of MTE on the proliferation and apoptosis of Jurkat cells (T-ALL lines) and lymphocytes from T-ALL (T-cell acute lymphoblastic leukemia) patients. Firstly, CCK8 assays and flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of Jurkat cells by arresting cell cycle at S phase. Secondly, Annexin V-FITC/PI-stained flow cytometry and TUNEL staining assays showed that MTE promoted the apoptosis of Jurkat cells. Mechanistically, MTE enhanced PTEN (phosphatases and tensin homolog) level and inactivated PI3K/AKT/mTOR signaling pathway in Jurkat cells, which mediated the inhibition of cell proliferation by MTE and MTE-induced apoptosis. Finally, MTE significantly inhibited the proliferation and promoted the apoptosis of lymphocytes from T-ALL patients, compared with lymphocytes from healthy peoples. Taken together, these results reveal an unrecognized function of MTE in inhibiting the proliferation and inducing the apoptosis of T-ALL cells, and identify a pathway of PTEN/PI3K/AKT/mTOR for the effects of MTE on leukemia therapy.