Study of novel ginsenoside metabolites targeting HSP70 as anti-prostate cancer drugs.

Ginsenoside 20 (R)-25-methoxy-dammarane-3 ß, twelve ß, 20 triol (AD-1) is a promising new drug for the treatment of prostate cancer, but its bioavailability is low. This study investigated the effects of the main metabolites PD and M6 of AD-1 on prostate cancer cell PC3. The in vitro experimental results showed that the IC50 values of PC3 cells treated with PD and M6 were 65.61 and 11.72, respectively. Both PD and M6 inhibited the migration of PC3 cells, and the cell cycle was blocked in the G1 phase. The apoptosis rates of cells following M6 treatment at concentrations of 7.5, 15, and 30 µM were 13.4 %, 17.5 %, and 41.4 %, respectively, which stimulated the expression of apoptosis protein and significantly increased intracellular ROS levels. In xenograft models, PD and M6 have been reported to significantly inhibit tumor growth. We used a genome-wide mRNA expression profile to study the effects of PD and M6 on gene expression in PC3 cancer cells. PD and M6 induced downregulation of HSP70 subtypes HSPA1A and HSPA1B. RT-PCR confirmed that the significant down-regulation of HSP70 subtype expressions was consistent with the results of Transcriptome analysis. Moreover, M6 significantly downregulated the expression of AR, which was further proved by Western blot analysis. In summary, our research findings provide a scientific basis for interpreting the significant activity of AD-1 in prostate cancer, and for the research and development of PD and M6 as novel HSP70 inhibitors.

Glucose levels affect MgaSpn regulation on the virulence and adaptability of Streptococcus pneumoniae.

Streptococcus pneumoniae can regulate virulence gene expression by sensing environmental changes, which is key to its pathogenicity. The global transcription regulator MgaSpn of Streptococcus pneumoniae regulates virulence genes expression by directly binding to the promoter regions, but its role in response to different environments remains unclear. In this study, we found that glucose levels could affect phosphocholine content, which was mediated by MgaSpn. MgaSpn can also alter its anti-phagocytosis ability, depending on the availability of glucose. In addition, transcriptome analysis of wild-type D39s in low and high glucose concentrations revealed that MgaSpn was also involved in the regulation of carbon metabolism inhibition (carbon catabolite repression; CCR) and translation processes, which made S. pneumoniae highly competitive in fluctuating environments. In conclusion, MgaSpn is closely related to the virulence and environmental adaptability of Streptococcus pneumoniae.

Novel ginsenoside derivatives induce apoptosis in HepG-2 cells via the MDM2-p53 signaling pathway.

In this study, 6-aryl-5-cyanopyrimidines and quinazolinones were introduced into panaxadiol (PD) to synthesize 25 new panaxadiol derivatives. The cytotoxicity of these compounds against three cancer cells and one normal cell was examined by methylthiazoletetrazolium (MTT) cytotoxicity experiment. The findings demonstrated that PD cyanopyrimidine derivatives have superior anti-proliferative effects over quinazoline derivatives. Among them, PM14 had the strongest inhibitory activity on the proliferation of human hepatoma cell lines (HepG-2), the IC50 value was 2.13 ± 0.20 µM. 4',6-diamidino-2-phenyl-indole (DAPI) staining showed that PM14 made HepG-2 nucleus shrink and had obvious apoptosis. Mechanistic studies confirmed that the derivative PM14 activates p53 signaling pathway by reducing the expression of MDM2 protein, and further induces an increase in the intracellular Bax/Bcl-2 ratio, down-regulated the expression of Caspase-3, up-regulated Cl-Caspase-3 expression, eventually leading to cell apoptosis. This lays the foundation for subsequent development of derivatives with stronger anti-proliferative activity.

In Vivo Metabolites of Panaxadiol Inhibit HepG-2 Cell Proliferation by Inducing G1 Arrest and ROS-Mediated Apoptosis.

In this study, 10 metabolites were obtained by collecting and extracting fecal samples after oral administration of panaxadiol (PD). Of these 10 metabolites, M7 (3ß,21ß,22α-hydroxy-24-norolean-12-ene), M8 (21ß,22α-hydroxy-24-norolean-12-ene-3-one), M9 (3ß,30α-hydroxy-24-norolean-22,30-epoxy-12-ene), and M10 (3ß,21ß-hydroxy-24-norolean-12-ene) were new compounds. MTT screening of the isolated compounds revealed that the inhibitions of cancer cells by M2, M4, M7, M8, and M10 were significantly stronger than that by the mother drug M0, with the activity of M2 being the most significant. Further, we investigated the anticancer mechanism of M2. The results showed that M2 significantly increased the level of ROS in cells; regulated the expressions of Bax, Bcl-2, and Cyt-C through the mitochondrial pathway; triggered the caspase cascade; and induced apoptosis. M2 could also induce G1 phase arrest and significantly regulate cell cycle-related proteins. In conclusion, the experimental results provide data for further study on the metabolic mechanism of PD in vivo and the potential of developing new anti-cancer drugs.

Design, Synthesis and Cytotoxicity Evaluation of Novel Indole Derivatives of Panaxadiol.

Based on the well-known cytotoxicity of indole compounds, we used the 'Fisher indole synthesis' method to introduce appropriately substituted indole rings into panaxadiol (PD), generating eighteen novel Panaxadiol indole derivatives. Six human cancer cell lines (A549, HepG-2, HCT-116, SGC-7901, MDA-MB-231, PC-3 cells) and one normal ovarian cell lines (IOSE144) were designed to evaluate the anti-proliferative activity of the PD derivatives. The results showed that the majority of PD derivatives showed enhanced anti-proliferative activity, when compared with PD, with P-Methylindolo-PD exhibiting the highest cytotoxicity. In A549 cells, IC50 value was 5.01±0.87 µM, which is roughly 12 times higher than the activity of PD and 5 times that of 5-FU. Moreover, cell morphology analysis and Annexin V-FITC/PI assays exhibited that P-Methylindolo-PD could induce A549 cell apoptosis (55.7 % of apoptotic cells at 20 µM). Moreover, molecular docking experiments were performed to explore the molecular mechanism underlining the binding of P-Methylindolo-PD to the active site of EGFR. The results support that P-Methylindolo-PD might be a promising lead compound for further studies.

Gender-Related Differences in Tissue Distribution, Excretion, and Metabolism Studies of Panaxadiol in Rats and Anti-inflammatory Study.

In this study, we evaluated gender differences in PD excretion, tissue distribution, and metabolism in rats. In addition, we also evaluated its anti-inflammatory activity and mechanism. The results showed that the concentrations of PD in the stomach, small intestine, and large intestine were the highest. The Cmax of female rats was significantly higher than that of male rats. With regard to genital tissues, the Cmax of PD in the uterus and ovary was higher than that in the testis. In the excretion test, gender had no significant effect on the excretion of PD. Its total excretion in rats was about 30%. Therefore, we speculated 12 phase I metabolites. In the anti-inflammatory test, PD showed no cytotoxic effect on macrophage RAW 264.7 and significantly reduced the production of NO and expressions of interleukin 6, interleukin 1, and tumor necrosis factor-α. Further analyses demonstrated that PD activated the MAPK signaling pathway by reducing the phosphorylated levels of p38 and ERK.

Panaxadiol as a major metabolite of AD-1 can significantly inhibit the proliferation and migration of breast cancer cells: In vitro and in vivo study.

Previous studies have shown that 20 (R)-25-methoxyl-dammarane-3ß, 12ß, 20 triol (AD-1) can inhibit various cancer cell lines. This study aimed to explore the effect and mechanism of AD-1 metabolite M2 (Panaxadiol; PD) on breast cancer cells of nude mice. Five AD-1 metabolites were isolated and identified using various chromatographic techniques. PD was the main component. In vitro results showed that PD could inhibit the proliferation and migration of MDA-MB-231 cells by inducing G1-phase arrest. In addition, PD down-regulated the expression of Cyclin D1, cdk2, cdk4, cdk6, P-p38, and MMP9, and up-regulated p21 and p27. In vivo results showed that PD could effectively reduce the volume, weight, and migration of breast cancer Transcriptomics analyzed 491 differentially expressed genes by GO and KEGG enrichment. RT-PCR verification confirmed that the significant down-regulation of MMP9 was consistent with transcriptomics results. In further research showed that PD regulated the protein expression of P-p38 and MMP9 in MAPK pathway. In summary, in vivo and in vitro studies showed that PD significantly inhibit the occurrence and development of breast cancer, possibly through the MAPK pathway.

MgaSpn is a negative regulator of capsule and phosphorylcholine biosynthesis and influences the virulence of Streptococcus pneumoniae D39.

Global transcriptional regulators are prevalent in gram-positive pathogens. The transcriptional regulators of the Mga/AtxA family regulate target gene expression by directly binding to the promoter regions, that results in the coordinated expression of virulence factors. The spd_1587 gene of Streptococcus pneumoniae strain D39 encodes MgaSpn, which shares sequence similarity with global transcriptional regulators of the Mga/AtxA family. In this study, we demonstrated that MgaSpn regulates the biosynthesis of the capsule and phosphorylcholine, which play key roles in disease severity in S. pneumoniae infections. MgaSpn directly binds to the cps and lic1 promoters and affects the biosynthesis of the capsule and phosphorylcholine. MgaSpn binds to two specific sites on the promoter of cps, one of which contains the -35 box of the promoter, with high affinity. Consistently, low-molecular-weight capsule components were observed in the mgaSpn-null mutant strain. Moreover, we found that phosphorylcholine content was notably increased in the unencapsulated mgaSpn mutant strain. The mgaSpn null mutant caused more severe systemic disease than the parental strain D39. These findings indicate that the pneumococcal MgaSpn protein can inhibit capsule and phosphorylcholine production, thereby affecting the virulence of S. pneumoniae.

Inactivation of Transcriptional Regulator FabT Influences Colony Phase Variation of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic pathogen that can alter its cell surface phenotype in response to the host environment. We demonstrated that the transcriptional regulator FabT is an indirect regulator of capsular polysaccharide, an important virulence factor of Streptococcus pneumoniae. Transcriptome analysis between the wild-type D39s and D39ΔfabT mutant strains unexpectedly identified a differentially expressed gene encoding a site-specific recombinase, PsrA. PsrA catalyzes the inversion of 3 homologous hsdS genes in a type I restriction-modification (RM) system SpnD39III locus and is responsible for the reversible switch of phase variation. Our study demonstrated that upregulation of PsrA in a D39ΔfabT mutant correlated with an increased ratio of transparent (T) phase variants. Inactivation of the invertase PsrA led to uniform opaque (O) variants. Direct quantification of allelic variants of hsdS derivatives and inversions of inverted repeats indicated that the recombinase PsrA fully catalyzes the inversion mediated by IR1 and IR3, and FabT mediated the recombination of the hsdS alleles in PsrA-dependent and PsrA-independent manners. In addition, compared to D39s, the ΔfabT mutant exhibited reduced nasopharyngeal colonization and was more resistant to phagocytosis and less adhesive to epithelial cells. These results indicated that phase variation in the ΔfabT mutant also affects other cell surface components involved in host interactions. IMPORTANCE Streptococcus pneumoniae is a major human pathogen, and its virulence factors and especially the capsular polysaccharide have been extensively studied. In addition to virulence components that are present on its cell surface that directly interact with the host, S. pneumoniae undergoes a spontaneous and reversible phase variation that allows survival in different host environments. This phase variation is manipulated by the recombination of allelic hsdS genes that encode the sequence recognition proteins of the type I RM system SpnD39III locus. The recombination of hsdS alleles is catalyzed by the DNA invertase PsrA. Interestingly, we found the opaque colony morphology can be reversed by inactivation of the transcriptional regulator FabT, which regulates fatty acid biosynthesis. Inactivation of FabT leads to a significant decrease in capsule production and systematic virulence, but these phase variations do not correlate with the capsule production. This phase variation is mediated via the upregulated invertase PsrA in the ΔfabT mutant. These results identify an unexpected link between the specific phase variations and FabT that strongly suggests an underlying mechanism regulating the DNA invertase PsrA.

Novel ginsenoside derivatives have shown their effects on PC-3 cells by inducing G1-phase arrest and reactive oxygen species-mediate cell apoptosis.

In this study, piperazine groups were introduced into ginsenoside to enhance its ability to induce Reactive Oxygen Species (ROS) production and apoptosis in cancer cells. In total, 27 ginsenoside piperazine derivatives were synthesized and tested for their anti-proliferative activity in cancer cell lines by MTT assay. The results showed that compounds 4a, 4g, 4f, 4i, 5g, 5i, 6a, 6g, 6f and 6i had significant inhibitory effects on cancer cell growth. Compound 6g showed the strongest anti-proliferative effect on PC-3 cells with an IC50 of 1.98 ± 0.34 µM. Compound 6g could also induce G1-phase arrest and apoptosis in PC-3 cells, with apoptosis rates of 8.1%, 41% and 56.1% observed at 5, 10 and 20 µM, respectively. Compound 6g also significantly enhanced the intracellular fluorescence of ROS sensitive substrates, with a fluorescence intensity ratio of 23.1% observed in treated cells, indicative of ROS production. Following N-acetylcysteine treatment, apoptotic rates of the cancer cell lines decreased from 38.9% to 7.3%, and the expression of Cl-PARP, Cl-Caspase-3 and Cl-Caspase-9 also decreased, confirming that compound 6g induced apoptosis through ROS induction. Compound 6g also stimulated the translocation of Bax from the cytoplasm to the mitochondria, which enhanced Cytochrome C (Cyt C) release, and increased the expression of the apoptotic markers Cl-PARP, Cl-Caspase-3, and Cl-Caspase-9 in PC-3 cells. Taken together, these data reveal the anti-cancer effects of compound 6g that enhance ROS production, and then induce apoptosis through mitochondrial pathway.

Rational design, synthesis and biological evaluation of triphenylphosphonium-ginsenoside conjugates as mitochondria-targeting anti-cancer agents.

In anti-cancer therapy, targeting a single gene or a single metabolic pathway usually cannot effectively cure cancer, while targeting cellular mitochondria might be effective based on the role of mitochondria in the occurrence and development of cancer. Anti-cancer study on ginsenosides AD-1, AD-2 and PD have proved that they have broad spectrum anti-cancer activities in vitro and in vivo. However, they are not active at sufficiently low concentration, and their lower selectivity and cell permeability hindered therapeutic applications. In the present study, AD-1, AD-2 and PD are incorporated with triphenylphosphonium at the OH group in C-3 position through different length of alkyl chains, with the aim of targeting mitochondria and improving the efficacy and selectivity of parent compounds. Biological studies suggested that most of the conjugates had enhanced anti-proliferative activity, in particular, conjugate 1f had an IC50 value of 0.76 µM against MCF-7 cells while showed a high degree of selectivity to MCF-7 cells. In addition, 1f was obviously increased accumulation in the mitochondria, and induced apoptosis, elevated reactive oxygen species (ROS) level and caused mitochondrial membrane potential collapse in MCF-7 cells. Further study revealed that ROS-related mitochondrial translocation of p53 was also involved in 1f-induced mitochondrial apoptotic pathway. The results demonstrated that 1f could be a promising lead for the development of mitocans. These findings also provide a reference for the development of ginsenoside for mitochondria-targeted anti-cancer drugs.

Novel panaxadiol triazole derivatives induce apoptosis in HepG-2 cells through the mitochondrial pathway.

In this study, we introduced 1, 2, 4-triazole groups into panaxadiol (PD) to obtain 18 panaxadiol triazole derivatives. Five cancer cells and one normal cell were evaluated for cytotoxicity by MTT assay. The results showed that most of the derivatives could inhibit cancer cell proliferation, and the anti-proliferative activity of compound A1 was the most significant. For HepG-2 cells, the IC50 value was 4.21 ± 0.54 µM, which was nearly 15 times higher than the activity of PD. Further studies showed that compound A1 could induce apoptosis in HepG-2 cells, and could enhance the expression of Cl-caspase-3, Cl-caspase-9 and Cl-PARP. Moreover, Western blot analysis showed that after treating HepG-2 cells with compound A1, the expression of p53 protein was increased and the ratio of Bax/Bcl-2 was gradually increased. The cytoplasmic Bax is then translocated to the mitochondria, causing the release of Cyt c protein. Therefore, the results indicate that compound A1 induces apoptosis through the mitochondrial pathway and can be used the potential to develop new anti-proliferative agents.

Optimization of flash extraction, separation of ginsenosides, identification by HPLC-FT-ICR-MS and determination of rare ginsenosides in mountain cultivated ginseng.

In this paper, we used the flash extraction method (FEM) to extract ginsenosides from mountain cultivated ginseng (MCG), optimized the FEM process by response surface methodology (RSM), and separated 23 kinds of ginsenosides from MCG, including rare ginsenoside Rg3, 20(R/S)-Rg2, Rk3, 20(S)-Rh2, 20(R)-Rh1, F1 and Rg6. Among them, notoginsenoside R1 was isolated from MCG for the first time. Additionally, we established an HPLC-FT-ICR-MS method to accurately identify 20 ginsenosides in MCG, and quantitatively analyzed the differences in the content of rare ginsenosides in MCG and Garden-Cultivated Ginseng (CG) by HPLC-UV. The results showed that the chemical components of MCG and CG were similar, but the ginsenoside content of MCG was double that of CG. Notably, the content of ginsenoside 20 (S)-Rh2 and 20 (R)-Rh1 had the largest difference, and the content in MCG was 33 and 24 times higher than that in CG, respectively. Through quantitative analysis, we clarified the reason why the activity of MCG is stronger than that of CG, which provided a theoretical basis for clinical application and further research of MCG.

Synthesis and Cytotoxicity Evaluation of Panaxadiol Derivatives.

In this study, 13 panaxadiol (PD) derivatives were synthesized via reactions with aromatic compounds and amino acids. Following this, the cytotoxicity of these compounds was evaluated against four cancer cell lines (human hepatoma cells HepG-2, human lung cancer cells A549, human breast cancer cells MCF-7, and human colon cancer cells HCT-116) and one normal cell lines (human gastric epithelial cells GES-1). The results showed that the panaxadiol derivatives 3, 12, and 13 showed significant inhibition of cellular proliferation against cancer cells compared with PD, and the panaxadiol derivative 12 had the lowest IC50 value for A549 (IC50 =18.91±1.03 µm). For MCF-7 cells, most compounds exhibited good inhibition of cellular proliferation, and the panaxadiol derivative 13 showed the strongest inhibitory effect (IC50 =8.62±0.23 µm), which significantly increased the cytotoxicity of PD and was stronger than the positive control (mitomycin). For normal cells, all compounds exhibited low or no toxic effects; thus, these derivatives can be used to develop novel antiproliferative agents.

Semi-synthesis and anti-tumor activity of novel 25-OCH3-PPD derivatives incorporating aromatic moiety.

Previously we have reported that 25-OCH3-PPD could suppress the reproduction of cancer cells and cause apoptosis without obvious toxicity. Herein, we aimed to enhance its bioactivity by introducing aromatic groups to its dammarane-type skeleton. These synthesized derivatives were tested for their inhibitory activities against five cancer cell lines. Of them, compounds 3a, 14a and 18a had the strongest antiproliferative activities against tumor cells (IC50 < 15 µM, 5-fold to 10-fold increases than 25-OCH3-PPD). Especially compound 14a displayed the most potent activity against DU145, MCF-7 and HepG2 cells (IC50 = 6.7 ±â€¯0.8, 4.3 ±â€¯0.8 and 5.8 ±â€¯0.6 µM, respectively). Structure-activity relationships demonstrated that having aromatic ester at the C3 position could improve the bioactivity. The data provided new insights into exploring novel antiproliferative lead compounds.

Synthesis and Anticancer Activity Evaluation of Hydrolyzed Derivatives of Panaxnotoginseng Saponins.

To increase the antitumor activity of ginsenosides and acetylsalicylic acid, acid hydrolysis products of Panaxnotoginseng saponin were used as raw materials to be combined with salicylic acid to obtain ginsenoside salicylic acid derivatives. All derivatives were assessed for anti-cancer activity. A total of 20 target compounds were designed and synthesized. The cytotoxic activity on five cancer cell lines, including human colon cancer (HT-29), gastric cancer (BGC-823), cervical cancer (Hela), human breast cancer (MCF-7), human lung cancer cells (A549), and two normal cancer cell lines (human gastric epithelial cells (GES-1), and human ovarian epithelial cells (IOSE144)) was evaluated following treatment with the compounds. The results showed that all compounds inhibited the growth of cancer cells. Compounds 1a, 3a, 7a, 1b, 2b, 3b and 8b showed strong anticancer activity. For MCF-7 cells, compound 3b showed the strongest inhibitory activity, IC50 = 2.56 ± 0.09 µM. In the cytotoxicity test, all compounds showed low toxicity or no toxicity (IC50 > 100 µM). In addition, a cell cycle distribution assay and wound healing assay demonstrated that compound 3b specifically inhibited MCF-7 proliferation and migration ability. Our results indicate that compound 3b represents a promising compound for further cancer studies.

Synthesis and anti-tumor evaluation of panaxadiol halogen-derivatives.

In the current work, 13 novel panaxadiol (PD) derivatives were synthesized by reacting with chloroacetyl chloride and bromoacetyl bromide. Their in vitro antitumor activities were evaluated on three human tumor cell lines (HCT-116, BGC-823, SW-480) and three normal cells (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2) by MTT assay. Compared with PD, the results demonstrated that compound 1e, 2d, 2e showed significant anti-tumor activity against three tumor cell lines, the IC50 value of compound 2d against HCT-116 was the lowest (3.836µM). The anti-tumor activity of open-ring compounds are significantly better than the compounds of C-25 cyclization. Compound 1f, 2f, 2g showed the strong anti-tumor activity. The IC50 value of compound 2g against BGC-823 and SW-480 were the lowest (0.6µM and 0.1µM, respectively). Combined with cytotoxicity test, the IC50 value of compound 1e, 2d, 2e are greater than 100. the open-ring compounds (1f, 2f, 2g) showed a strong toxicity. The toxicity of 1f is lower than 2f and 2g. These compounds may be useful for the development of novel antiproliferative agents.