W.L. Wendy Hsiao

Thermoreversible Pluronic® F127-based hydrogel containing liposomes for the controlled delivery of paclitaxel: in vitro drug release, cell cytotoxicity, and uptake studies

Purpose To develop an in situ gel system comprising liposome-containing paclitaxel (PTX) dispersed within the thermoreversible gel (Pluronic® F127 gel) for controlled release and improved antitumor drug efficiency. Methods The dialysis membrane and membrane-less diffusion method were used to investigate the in vitro drug release behavior. Differential scanning calorimetry (DSC) thermal analysis was used to investigate the “micellization” and “sol/gel transition” process of in situ gel systems. In vitro cytotoxicity and drug uptake in KB cancer cells were determined by MTT, intercellular drug concentration, and fluorescence intensity assay. Results The in vitro release experiment performed with a dialysis membrane model showed that the liposomal gel exhibited the longest drug-release period compared with liposome, general gel, and commercial formulation Taxol®. This effect is presumably due to the increased viscosity of liposomal gel, which has the effect of creating a drug reservoir. Both drug and gel release from the in situ gel system operated under zero-order kinetics and showed a correlation of release of PTX with gel, indicating a predominating release mechanism of the erosion type. Dispersing liposomes into the gel replaced larger gel itself for achieving the same gel dissolution rate. Both the critical micelle temperature and the sol/gel temperature, detected by DSC thermal analysis, were shifted to lower temperatures by adding liposomes. The extent of the shifts depended on the amount of embedded liposomes. MTT assay and drug uptake studies showed that the treatment with PTX-loaded liposomal 18% Pluronic F127 yielded cytotoxicities, intercellular fluorescence intensity, and drug concentration in KB cells much higher than that of conventional liposome, while blank liposomal 18% Pluronic F127 gel was far less than the Cremophor EL® vehicle and empty liposomes. Conclusions A thermosensitive hydrogel with embedded liposome is a promising carrier for hydrophobic anticancer agents, to be used in parenteral formulations for treating local cancers.

Oleanolic acid isolated from Oldenlandia diffusa exhibits a unique growth inhibitory effect against ras-transformed fibroblasts.

AIMS Oldenlandia diffusa (Willd.) Roxb. (O. diffusa) is a commonly used traditional Chinese medicine for treating cancer. Its pharmacological activities and anti-cancer effects have been the focus of intense research in recent years. In the present study, we aim to investigate whether the five major compounds from O. diffusa possess a unique inhibitory activity against ras-transformed cells in a well-established cell model. MAIN METHODS The anti-cancer effects of O. diffusa were assessed in a co-culture system containing normal and transformed Rat 6 (R6) fibroblasts. In addition, a transwell assay was used to examine the interaction between the drugs and the co-cultivated cells. KEY FINDINGS Our data showed that among the samples tested, oleanolic acid (OA), but not the structural isomer ursolic acid (UA), inhibits the growth of ras oncogene-transformed R6 cells at a dosage that is not toxic to the co-cultivated normal fibroblasts. A significant inhibitory effect was also observed in the transwell experiments, indicating that the mode of action for OA-mediated growth inhibition of transformed cells does not require direct cell-to-cell contact between normal and ras-transformed cells. Data obtained from experiments conducted with the conditioned medium that was collected from normal R6 cells treated with OA also suggest that OA might cause normal cells to secrete inhibitory factor(s) against the transformed cells. The enhanced ability of OA to cause cytotoxicity in transformed cells in the presence of normal fibroblasts is also observed with the human hepatocellular carcinoma cell line, SMMC-7721. SIGNIFICANCE The present study demonstrates that OA may possess both cancer chemotherapeutic and chemopreventive activities. Thus, it may have great potential for clinical application as a novel anti-cancer drug.

Five new ocotillone-type saponins from Gynostemma pentaphyllum

Five new ocotillone-type saponins, gynosides A-E (1-5), along with 10 known dammarane-type saponins, were isolated from the aerial parts of Gynostemma pentaphyllum. The structures of these new compounds were determined by NMR analysis and acid hydrolysis. The structure and stereochemistry of gynoside A (1) were confirmed by X-ray crystallography.

The anticancer effect of oridonin is mediated by fatty acid synthase suppression in human colorectal cancer cells

BackgroundFatty acid synthase (FAS) inhibitors could be a therapeutic target in cancer treatment. However, only a few FAS inhibitors showing clinical potential have been reported. Oridonin is a diterpenoid isolated from Rabdosia rubescens. Although it has antiproliferative activity in cancers, little was known about its anticancer effect on colorectal cancer. In this regard, we aimed to investigate if oridonin could be a novel FAS inhibitor and its anticancer mechanism in human colorectal cancer cells.MethodsTwo human colorectal cancer cell lines SW480 and SW620 were used as models for this study.ResultsWe demonstrated that oridonin reduced viability and induced apoptosis in colorectal cancer cells. Knockdown of the expression of FAS in colorectal cancer cells by siRNA induced apoptosis. This led us to examine whether oridonin-induced apoptosis was mediated by FAS suppression in these cells. We found that oridonin effectively inhibited FAS and SREBP1 mRNA and protein expression in human colorectal cancer cells. In a transient reporter assay, oridonin also reduced transcriptional activity of the FAS promoter region containing the SREBP1 binding site. The FAS inhibition was paralleled by reduction in cellular palmitate and stearic acid. Upregulation of SREBP1 and FAS expression by insulin rescued these cells from oridonin-induced apoptosis.ConclusionThese results not only provide a novel molecular mechanism for the anticancer effect of oridonin in colorectal cancer, but also suggest oridonin could be a novel FAS inhibitor in cancer treatment. These results strengthen the scientific basis for the therapeutic use of oridonin in colorectal cancer.

Inhibition of the p38 and PKA signaling pathways is associated with the anti-melanogenic activity of Qian-wang-hong-bai-san, a Chinese herbal formula, in B16 cells.

ETHNOPHARMACOLOGICAL RELEVANCE Qian-wang-hong-bai-san (QW), a Chinese herbal formula, is traditionally used as a skin whitening agent in China. AIM OF STUDY In our previous screening assays, QW was identified as an effective tyrosinase inhibitor. In this study, we aim to investigate the underlying mechanism of the anti-melanogenic effect of QW in B16 cells. MATERIALS AND METHODS Cytotoxicity of QW in B16 cell line was examined by MTT assay. Cellular tyrosinase activity was determined based on the melanin content measured at 475 nm with a microplate spectrophotometer. Protein expression was analyzed by Western blotting and quantified by Quantity One. RESULTS QW dose-dependently inhibited tyrosinase activity and decreased melanin content at 48 h without significant cytotoxicity in B16 cells. Western blot analysis showed that QW treatment down-regulated the expression levels of phospho-p38, phospho-CREB, MITF, tyrosinase, TRP-1 and TRP-2 in a dose-dependent manner. At the same time, QW treatment for 48 h inhibited IBMX-induced elevation of cellular melanin content and tyrosinase activity. However, the attenuation of IBMX-mediated up-regulations of phospho-CREB and phospho-PKA was readily observed with 60 min of QW treatment. CONCLUSIONS The anti-melanogenic activity of QW in B16 melanoma cells can be attributed, at least in part, to the inhibition of the p38 MAPK and PKA signaling pathways. These findings shed new light on the molecular mechanisms of the skin-whitening property of QW.

Chemical and DNA authentication of taste variants of Gynostemma pentaphyllum herbal tea

Gynostemma pentaphyllum Makino (Gp) was once used as a sweetener in Japan and is now widely consumed as an herbal tea worldwide for lowering cholesterol levels. Two taste variants, bitter and sweet, of Gp exist in the commercial market, but they cannot be differentiated morphologically nor by existing chemical analytical methods. This has been creating a problem in quality control of Gp products. In the present study, using HPLC-DAD and HPLC-ESI-MS analysis, we found that the Gp saponins, not flavonoids, from the sweet and bitter variants have distinctly different profiles. In addition, the two variants share only 69.01% homology in the ribosomal ITS-1 region, suggesting a phylogenic gap between these two variants. The combinations of chemical profiling and phylogenic analysis clearly confirm, for the first time, the distinction between these two taste variants. This information has direct application in the authentication and quality assessment of the various Gynostemma tea products.

Proteomic study of pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome in rats.

Pyrrolizidine alkaloids (PAs) are a group of phytotoxins that can induce human liver injury, particularly hepatic sinusoidal obstruction syndrome (HSOS). To date, the molecular targets of PA-induced HSOS are largely unknown. In this study, retrorsine (RTS), a known hepatotoxic PA, was used as a representative PA for proteomic studies. Toxicological assessment demonstrated that 35 mg/kg RTS (designated as RTS-L) caused early lesions of HSOS at 24 h after dosing. A proteomic approach revealed 17 up-regulated and 31 down-regulated proteins in RTS-L-treated rats. Subsequently, bioinformatic analysis suggested that two proteins, carbamoyl-phosphate synthase (CPS1) (p < 0.05) and ATP synthase subunit beta (ATP5B) (p < 0.01) were associated with RTS-L intoxication. Using immunohistochemical staining, we further verified the down-regulation of CPS1 and ATP5B in RTS-L-treated rats. These findings indicated that CPS1 and ATP5B were altered in the RTS-induced early lesions of HSOS in rats, and therefore, these two proteins and their involved pathways might play important roles in the initiation of HSOS. To the best of our knowledge, our study using a proteomic approach combined with conventional toxicological assessment is the first systems toxicology study on PA-induced HSOS. The results of this study provide novel findings on protein profiles in response to PA exposure, which can serve as a starting point to further investigate potential protein targets and their interactions with PAs to induce HSOS.

Selective G2/M arrest in a p53Val135-transformed cell line induced by lithium is mediated through an intricate network of MAPK and β-catenin signaling pathways

AIMS Lithium is a common mood stabilizer to treat bipolar disorder. It has a narrow window of therapeutic action and its mechanism of action and possible side effects are still not fully understood. Lithium is a potent inhibitor of glycogen synthase kinase 3β (GSK-3β). Previous studies indicated that lithium can induce cell cycle arrest by stabilization of p53. In order to further elucidate the signaling mechanism of lithium-induced cell cycle arrest and its potential pharmacological effect on p53 transformed cell lines, we studied the effect of lithium on the rat fibroblast cell line R6 and a p53(Val135) transformed cell line R6T2 (hereafter referred to as T2). MAIN METHODS We monitored the effects of lithium on cell cycle progression by FACS analysis and the activation of MAPK signaling pathways by Western blot using anti-phospho-MAPK antibodies in R6 and T2. KEY FINDINGS We report here lithium can induce G2/M arrest in T2 independent of β-catenin signals. Lithium increases phosphorylation of extracellular signal-regulated kinases (ERKs) leading to the up-regulation of p53 levels and subsequent G2/M arrest. Lithium also induced phosphorylation of p38 MAPK, consequently downregulated p53 and alleviated G2/M cell cycle arrest. We further showed the gate-keeping role of p53 in the lithium-induced G2/M arrest in the T2 cell line. SIGNIFICANCE Our results reveal a novel mechanism underlying the differential response of the transformed and normal R6 to lithium-induced G2/M cell cycle arrest and delineate the multiplicity of signaling pathways dictating the cell fate in responding to cell stress signals.

Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in Apc Min/+ mice

Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing. Study showed that GpS treatment significantly reduced the number of intestinal polyps in a preventive mode. More importantly, GpS feeding strikingly reduced the sulfate-reducing bacteria lineage, which are known to produce hydrogen sulfide and contribute to damage the intestinal epithelium or even promote cancer progression. Meanwhile, GpS also boosted the beneficial microbes. In the gut barrier of the ApcMin/+ mice, GpS treatment increased Paneth and goblet cells, up-regulated E-cadherin and down-regulated N-cadherin. In addition, GpS decreased the pro-oncogenic β-catenin, p-Src and the p-STAT3. Furthermore, GpS might also improve the inflamed gut epithelium of the ApcMin/+ mice by upregulating the anti-inflammatory cytokine IL-4, while downregulating pro-inflammatory cytokines TNF-β, IL-1β and IL-18. Intriguingly, GpS markedly stimulated M2 and suppressed M1 macrophage markers, indicating that GpS altered mucosal cytokine profile in favor of the M1 to M2 macrophages switching, facilitating intestinal tissue repair. In conclusion, GpS might reverse the hosts inflammatory phenotype by increasing beneficial bacteria, decreasing sulfate-reducing bacteria, and alleviating intestinal inflammatory gut environment, which might contribute to its cancer preventive effects.

Tumor Grafting Induces Changes of Gut Microbiota in Athymic Nude Mice in the Presence and Absence of Medicinal Gynostemma Saponins

Recent findings have revealed that gut microbiota plays a substantial role in modulating diseases such as autism, rheumatoid arthritis, allergies, and cancer that occur at sites distant to the gut. Athymic nude mice have been employed for tumorigenic research for decades; however, the relationships between the gut microbiome and host’s response in drug treatment to the grafted tumors have not been explored. In this study, we analyzed the fecal microbiome of nonxenograft and xenograft nude mice treated with phytosaponins from a popular medicinal plant, Gynostemma pentaphyllum (Gp). Analysis of enterobacterial repetitive intergenic consensus (ERIC)-PCR data showed that the microbiota profile of xenograft mice departed from that of the nonxenograft mice. After ten days of treatment with Gp saponins (GpS), the microbiota of the treated mice was closer to the microbiota at Day 0 before the implantation of the tumor. Data obtained from 16S pyrosequencing of fecal samples reiterates the differences in microbiome between the nonxenograft and xenograft mice. GpS markedly increased the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. This study, for the first time, characterizes the properties of gut microbiome in nude mice responding to tumor implant and drug treatment. We also demonstrate that dietary saponins such as GpS can potentially regulate the gut microbial ecosystem by increasing the number of symbionts. Interestingly, this regulation of the gut ecosystem might, at least in part, be responsible for or contribute to the anticancer effect of GpS.