Exploring the mechanism of daphne-type diterpenes against gastric cancer cells.

Gastric cancer is one of the common malignant tumors. It is reported that daphne-type diterpenes have inhibitory effects on gastric cancer cells, but the mechanism is still unknown. To explore the detailed mechanism of the anticancer effect of daphne-type diterpenes, we carried out an integrated network pharmacology prediction study and selected an effective component (yuanhuacine, YHC) for the following validation in silico and in vitro. The result showed that daphne-type diterpenes exerted an anti-tumor effect by targeting proto-oncogene tyrosine-protein kinase SRC as well as regulating the Ras/MAPK signaling pathway, which caused the apoptosis and mitochondrial damage in gastric cancer cells.

Daphnegiravone D from Daphne giraldii induces cell death by targeting ATR in Hep3B cells.

Ataxia telangiectasia and Rad3-related protein (ATR) plays a crucial role in cancer and has become a promising target for cancer therapy. Daphnegiravone D (DGD), which could induce apoptosis and oxidative stress in hepatocellular carcinoma (HCC) cells, but the detailed target protein was still unclear. The study provided that the possible target of DGD against HCC cells was determined by isobaric labels for relative and absolute quantification (iTRAQ) assay. In all changed proteins the fold change of ATR was particularly significant. The results from GO, KEGG and PPI analysis showed that DNA damage, cell cycle, apoptosis, DNA repair related pathways changed and ATR was exactly related to them. Moreover, the mRNA and protein of ATR were both decreased in a concentration-dependent manner, and the results of molecular docking also verified the binding. Additionally, cellular thermal shift assay (CETSA) suggested that DGD could directly target at ATR protein. Furthermore, the knockdown of ATR could increase apoptosis and reactive oxygen species (ROS) which induced by DGD. Since ATR inhibitors were generally used in combination with chemotherapy drugs (especially DNA damage drugs) in clinical trials, we investigated the combined application of DGD and oxaliplatin. The results showed that DGD combined with OXA also increased the apoptosis and ROS production of Hep3B cells over either drug alone. Taken together, this study revealed that DGD targeting ATR could be a promising therapeutic strategy for the treatment of liver cancer.

Enantiomeric 8-O-4'-type neolignans from Crataegus pinnatifida exhibit cytotoxic effect via apoptosis and autophagy in Hep3B cells.

Crataegus pinnatifida has been famous for its nutritional purpose. However, systematic investigation on the bioactive constituents is still lacking, although this fruit has been reported for its cytotoxic effect before. In this study, two pairs of new lignan enantiomers (1a/1b, 2a/2b), which isolated using chiral chromatographic column from the fruits of C. pinnatifida were studied. The absolute configurations of enantiomers were determined by comparison between the experimental electronic circular dichroism (ECD) and calculated ECD spectra. Among them, 1a/1b exhibited a better cytotoxic effect in hepatocellular carcinoma Hep3B cells with an IC50 value of 34.97 ± 2.74 and 17.42 ± 0.71 µM, respectively. In addition, 1b induced much more apoptotic, autophagic cells than 1a in Hep3B cells. Furthermore, the underlying mechanism was demonstrated that p38 activation could promote 1b-induced apoptosis and autophagy. Moreover, 1b-induced apoptosis was significantly decreased in the presence of autophagic inhibitor Bafilomycin A1 (Baf A1), suggesting that the induction of autophagy enhanced apoptotic cell death in 1b-treated cells. In general, these findings provide a valuable basis for further understanding the effect of 8-O-4' lignans in C. pinnatifida on cytotoxic effect.

Yuanhuatine from Daphne genkwa selectively induces mitochondrial apoptosis in estrogen receptor α-positive breast cancer cells in vitro.

Breast cancer is one of the most common cancers diagnosed among women worldwide. Estrogen receptor alpha (ERα) is a transcriptional factor that plays an important role in the development and progression of breast cancer. Yuanhuatine, a natural daphnane-type diterpenoid extracted from Daphne genkwa, was reported to exhibit significant cytotoxicity against breast cancer cells. However, the underlying mechanism is still unclear. In this study, we evaluated the cytotoxicity of yuanhuatine on two breast cancer cell lines that are ERα-positive and -negative. The results show that yuanhuatine inhibits the growth of ERα-positive cells (MCF-7) with much stronger inhibitory activity (IC50 = 0.62 µM) compared with positive control tamoxifen (IC50 = 14.43 µM). However, no obvious cytotoxicity was observed in ERα-negative cells (MDA-MB-231). Subsequent experiment also indicated that yuanhuatine markedly induced mitochondrial dysfunction, leading to apoptosis in MCF-7 cells. Molecular docking studies suggest the potential interactions between yuanhuatine and ERα. Immunofluorescence staining and Western blot analysis indicated that yuanhuatine down-regulated the expression of ERα in MCF-7 cells. MPP, a specific ERα inhibitor, significantly enhanced yuanhuatine-induced mitochondrial dysfunction and apoptosis in MCF-7 cells. On the contrary, the treatment with yuanhuatine causes no apoptosis in MM231 cells. Altogether, in vitro and in silico results suggested that ERα down-regulation was involved in yuanhuatine-induced mitochondrial dysfunction and apoptosis in ERα-positive breast cancer cells. Thus, yuanhuatine could be a potential candidate for treating ERα-positive breast cancer.

Sesquiterpenoids from the roots of Croton crassifolius.

Phytochemical investigation of Croton crassifolius roots afforded five sesquiterpenes (1-5), including two new sesquiterpenes 6S-hydroxy-cyperenoic acid (1) and crassifterpenoid A (5), together with three known compounds (2-4). The structures of the new compounds were determined by comprehensive spectroscopic methods, and their absolute configurations were determined by quantum chemical ECD calculation. Crassifterpenoid A (5) is the first germacrane-type sesquiterpene isolated from C. crassifolius, which enriched the diversity of chemical constituents in Croton crassifolius. In addition, the cytotoxicities of all compounds against human liver cancer lines HepG2 and Hep3B were determined, but none showed significant activity.

Coumarins from the bark of Juglans mandshurica exhibited anti-hepatoma activities via inducing apoptosis.

Hepatocellular carcinoma (HCC), the most common type of liver cancer, has high morbidity and mortality rates, and its prognosis is poor. The treatment options of HCC are limited by the lack of effective chemotherapy. Therefore, looking for effective drugs with little toxicity is very urgent. The aim of this study was to search for small molecule targeting on liver cancer from Juglans mandshurica, which has been used to treat cancers for a long time in China. Under the guide of anti-hepatoma activity, a new coumarin (1), together with eight reported analogs (2‒9), was isolated from the 75% EtOH extract. The structures of these compounds were determined by 1D and 2D NMR experiments. The absolute configuration of 1 was established by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. The in vitro cytotoxicity experiments on two liver cancer cell lines (HepG2 and Hep3B) showed that compounds 2 and 5 had moderate antitumor activities on both cell lines. And further studies of antitumor mechanisms by the observation of morphological changes and Western blot analyses exhibited that induction of apoptosis might be a possible way that inhibited cell growth.

Network pharmacology predicted HDAC6 as a potential target of flavones from Daphne giraldii on hepatocellular carcinoma.

Daphne giraldii Nitsche, belongs to Daphne genus, has been reported to exert anti-tumor activities. Our previous study suggested that flavones from Daphne giraldii have significant inhibitory effects on hepatocellular carcinoma (HCC) cells. However, the potential target of this type flavone was still unknown. In this study, 74 flavonoids compounds of Daphne giraldii and 41 potential targets of HCC were analyzed by the network, the most potential target was histone deacetylase 6 (HDAC6). Considering the cytotoxicity, compound 70 (Daphnegiravone D, DGD) was chosen for further confirmation. Molecular docking study revealed that DGD formed high binding affinity with HDAC6. Concomitantly, pharmacological studies indicated that DGD could inhibit the expression of HDAC6 in vitro and in vivo. In this study, network pharmacology along with experimental validation predicted and verified HDAC6 as one of potential targets of flavones, these investigations provide a new insight for further study of Daphne giraldii on HCC treatment.

Brusatol: A potential anti-tumor quassinoid from Brucea javanica.

Brusatol, a triterpene lactone compound mainly from Brucea javanica, sensitizes a broad spectrum of cancer cells. It is known as a specific inhibitor of nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. In this review, we provide a comprehensive overview on the antitumor effect and molecular mechanisms of brusatol in vitro and in vivo. This review also covers pharmacokinetics studies, modification of dosages forms of brusatol. Increasing evidences have validated the value of brusatol as a chemotherapeutic agent in cancers, which may contribute to drug development and clinical application.

Triterpenes from the fruit of Camptotheca acuminata suppress human hepatocellular carcinoma cell proliferation through apoptosis induction.

The fruit of Camptotheca acuminata, a kind of mainly medicinal plant, possesses good antitumor properties. In order to explore the bioactive compounds for the treatment of hepatocellular carcinoma, the study focused on the isolation of cytotoxic compounds from the fruit of Camptotheca acuminata, which led to the discovery of fourteen compounds, including one new triterpene, 3ß,20-dihydroxy-30α-methyl,17(29)-ß-epoxy-28-norlupane (1), together with thirteen known compounds (2-14). The structures of isolated compounds were demonstrated by spectroscopic methods including 1D and 2D NMR spectroscopy. Moreover, all triterpenes were evaluated for antiproliferative activities against two human hepatocellular carcinoma cell lines, HepG2 and Hep3B. Compound 3 showed the strongest cytotoxic activity against the HepG2 with IC50 value at 29.6 µM. Further study demonstrated that compound 3 exhibited cytotoxic activity through the induction of apoptosis.

Racemic neolignans from Crataegus pinnatifida: Chiral resolution, configurational assignment, and cytotoxic activities against human hepatoma cells.

Phytochemical investigation of the fruit of Crataegus pinnatifida led to the isolation of four pairs of dihydrobenzofuran neolignan enantiomers (1a/1b-4a/4b) including six new compounds (1a/1b, 2a/2b, 3a and 4a). The enantioseparations of the racemates were achieved successfully by chiral chromatographic column. Their structures were established by comprehensive spectroscopic analyses and the absolute configurations were determined by quantum mechanical calculation of electronic circular dichroism (ECD) spectra. All compounds were evaluated in vitro for their cytotoxicity using human hepatocellular carcinoma Hep3B and HepG2 cells. Among them, it was found that 2a had a selective cytotoxicity against Hep3B cells with IC50 value of 25.47 µM, while the IC50 value of its enantiomer 2b on Hep3B cells was 59.37 µM. These results implied that the absolute configurations of 2a and 2b possessed remarkable influences on their cytotoxicity. Further flow cytometry analysis indicated that 2a performed more significant effect on cell apoptosis compared with its enantiomer 2b.

Phenylpropanoid derivatives from the fruit of Crataegus pinnatifida Bunge and their distinctive effects on human hepatoma cells.

Ten undescribed phenylpropanoid derivatives including four pairs of enantiomers and two 8-9' linked neolignans, together with fifteen known ones were isolated from the fruit of Crataegus pinnatifida Bunge. Their structures were established by comprehensive spectroscopic analyses. Enantiomers were separated successfully by chiral chromatographic column and their absolute configurations were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. The in vitro cytotoxicity of the isolates were evaluated against two human hepatocellular carcinoma, HepG2 and Hep3B cells. Among them, (±)-crataegusanoid A, (±)-crataegusanoid B and crataegusanoid F exhibited moderate cytotoxicity. Interestingly, the different absolute configurations of (±)-crataegusanoid A and B demonstrated enantioselective cytotoxicity in HepG2 cells. Further flow cytometry analysis indicated that both (-)-crataegusanoid A and (-)-crataegusanoid B performed more significant effects on cell apoptosis, autophagy, and cell cycle progression compared with their enantiomers (+)-crataegusanoid A and (+)-crataegusanoid B. In addition, the results revealed that these two pairs of enantiomers induced protective autophagy in HepG2 cells.

Prenylated flavans from Daphne giraldii and their cytotoxic activities.

Nine new prenylated flavan compounds with multi-chiral centers including two pairs of epimers were isolated from the stem and root bark of Daphne giraldii. Their structures were established by extensive NMR and HR-ESIMS spectroscopic data analyses. The in vitro cytotoxicity experiments indicated that compound 6 showed the most significant cytotoxicity against Hep3B cells, with an IC50 value of 9.83 µM. Hoechst 33258 and Annexin V-FITC/PI staining suggested that 6 could induce apoptosis of Hep3B cells in a concentration-dependent manner. Further mechanism study indicated that the apoptosis was associated with the up-regulations of Bax, cl-PARP and a decrease in Bcl-2 expression.

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells.

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H2O2-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.

Daphnegiravone D from Daphne giraldii Nitsche induces p38-dependent apoptosis via oxidative and nitrosative stress in hepatocellular carcinoma cells.

Daphnegiravone D (DGD), a prenylated flavonoid from Daphne giraldii Nitsche, significantly inhibited cell growth of several cancer cell lines without cytotoxicity on human normal cells. Our previous study showed that DGD could induce apoptosis in hepatocellular carcinoma Hep3B and HepG2 cells, but the detailed mechanism was still unclear. The present study provides that DGD-induced oxidative and nitrosative stress contribute to apoptotic cell death in Hep3B and HepG2 cells. Furthermore, there is a positive loop between oxidative stress and p38 activation, similar result is observed between nitrosative stress and p38. N-Acetylcysteine (NAC), a reactive oxygen species scavenger, could relieve DGD-induced oxidative stress, but exerts little effect on nitrosative stress. In addition, carboxy-PTIO (PTIO, a well-known scavenger of reactive nitrogen species) down-regulates the induction of nitrosative stress without obvious effect on oxidative stress in DGD-treated cells. In conclusion, the induction of oxidative and nitrosative stress could enhance p38-mediated apoptosis in DGD-treated Hep3B and HepG2 cells. Moreover, we speculated that OS and NS could not ultimately affect each other in DGD-treated HCC cells. This study gives a new insight on the mechanism of DGD-induced apoptotic cell death via oxidative and nitrosative stress in HCC cells.

Phenylpropanoids from the fruit of Crataegus pinnatifida exhibit cytotoxicity on hepatic carcinoma cells through apoptosis induction.

Eight new phenylpropanoids (1a/1b, 2-4, 5a/5b and 6) including two pairs of enantiomers (1a/1b and 5a/5b), along with a known analogue (7) were isolated from the fruit of Crataegus pinnatifida. Their structures were elucidated using comprehensive spectroscopic methods. Compounds 1a/1b and 5a/5b were separated successfully by chiral chromatographic column. The absolute configurations of enantiomers were determined by comparison between the experimental and calculated electronic circular dichroism (ECD) spectra. The in vitro antitumor activities of the isolates were evaluated against two human hepatocellular carcinoma HepG2 and Hep3B cells. Five compounds (1a/1b, 2-4) exhibited more potent cytotoxicity and their structure-activity relationships were also discussed. Annexin V-FITC/PI staining using flow cytometry was carried out to examine cell apoptosis, and the results showed that compounds 3-4 with the presence of two methoxy groups substituted at C-3' significantly induced apoptosis in HepG2 cells.

Bioactivity-guided isolation of ß-Carboline alkaloids with potential anti-hepatoma effect from Picrasma quassioides (D. Don) Benn.

ß-Carboline alkaloids in Picrasma quassioides (D. Don) Benn. have been proven to possess inhibitory activity against various cancer cells. However, their effect on hepatocellular carcinoma and structure-activity relationships (SAR) have not been systematically reported. In this work, bioactivity-directed fractionation of P. quassioides led to the separation of active fraction A2-2. A total of 39 ß-carbolines, including 4 new ones (1-4), were obtained from the active fraction. Moreover, all the isolated compounds were identified in the active fraction A2-2 by LC-MS. The cytotoxicity on HepG2 and Hep3B cells of all compounds was screened by MTT assay, and the SAR were established. The SAR were also supported by the apoptosis ratio of HepG2 cells using flow cytometry analysis after treatment with potential compounds 1, 2, 9, 10, 12, 29, 36 and 38. It suggested that these active compounds caused death of hepatoma cells through apoptosis induction. In addition, further study revealed that compounds 12, 29, 36 significantly activated caspase-3 in HepG2 cells.

Eclalbasaponin I from Aralia elata (Miq.) Seem. reduces oxidative stress-induced neural cell death by autophagy activation.

Oxidative stress has been proposed to contribute to DNA damage and is involved in many neurodegenerative diseases. It has been reported that Aralia elata (Miq.) Seem. (A. elata) exhibits an anti-oxidative effect but the mechanisms underlying this protective effect are still unclear. In this study, six known triterpene saponins were isolated from the buds of A. elata, a well-known medicinal and edible plant in Northeast China. Subsequently, the anti-oxidative effects of all six triterpene saponins were screened by H2O2-induced damage in human neuronblastoma SH-SY5Y cells. Compound 6, also known as Eclalbasaponin I (EcI), was the most potent. Furthermore, the mechanism by which EcI combats H2O2-induced oxidative stress was investigated. The data suggested that EcI could down-regulate apoptosis induction and the generation of reactive oxygen species (ROS) induced by 200µM H2O2 in SH-SY5Y cells. Moreover, EcI increased the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxides (GSH-Px), reduced the levels of malondialdehyde (MDA) to restore the antioxidant defense system, and activated the nuclear factor E2-related factor (Nrf2)/heme oxygenase 1 (HO-1) pathway to combat oxidative stress. In addition, EcI also promoted autophagy during this process. Interestingly, the protective effect was remarkably reversed by autophagy inhibitors, bafilomycin A1 (Baf) or 3-Methyladenine (3-MA). These results demonstrate that autophagy is contribute to the protective effect of EcI. Collectively, our findings provide a new insight into the potential protective effect of EcI by focusing on the role of autophagy.