Search

Introduction Osteoarthritis is the most prevalent articular disease in the elderly. We aimed to explore the role of cordycepin (COR) in the progression and development of osteoarthritis and its correlation with TGF-β activity and autophagy. Methods Sprague Dawley rats were induced by anterior cruciate ligament transection (ACLT) to establish knee osteoarthritis model. To investigate the role of COR in knee osteoarthritis, rats were injected with 5, 10, and 20 mg/kg of COR before joint surgery. After surgery, paw withdrawal mechanical threshold (PWMT) was performed. HE staining and Alcian blue staining were carried out to detect cartilage damage. ELISA was used to detect the level of TGFβ in the serum. Protein expression was analyzed by Western blotting. Results In this study, we found that the PWMT of rats with osteoarthritis induced by ACLT was decreased significantly, accompanied by obvious histological and cartilage damage. After different doses of COR treatment, the PWMT of osteoarthritis rats induced by ACLT was increased in a dose-dependent manner. In addition, compared with the control group, COR treatment also reversed the effect of ACLT on cartilage injury in rats. Furthermore, the level of TGF-β in serum of ACLT rats was increased significantly, which may be related to the overexpression of TGF-β R1. However, the increase of serum TGF-β level in ACLT rats was reversed by COR treatment in a dose-dependent manner. It is worth noting that TGF-β overexpression reduced the proportion of autophagy-related protein LC3-II/I, thus inhibiting autophagy. In order to further confirm the effect of TGF-β on autophagy, TGF-β was overexpressed or the autophagy inhibitor 3-MA was applied. The results showed that TGF-β overexpression and 3-MA treatment reversed the effect of COR on autophagy. Conclusion In summary, our findings declared that COR alleviated ACLT-induced osteoarthritis pain and cartilage damage by inhibiting TGF-β activity and inducing autophagy in rat model with knee osteoarthritis.

Methotrexate (MTX) pharmacokinetics has substantial inter-individual variability and toxicity. In children with medulloblastoma treated with high-dose methotrexate (HD-MTX), the pharmacokinetic properties of methotrexate have not been established. A total of 660 serum samples from 105 pediatric patients with medulloblastoma were included in a population pharmacokinetic (PPK) analysis of methotrexate by using the nonlinear mixed-effects modeling method. The basic one-compartment population pharmacokinetic model was established by NONMEM software and the first-order conditional estimation (FOCE) method, and the final covariate model was obtained by the stepwise regression method. Weight (WT), creatinine clearance (CrCL), and whether the treatment was combined with dexamethasone (DEX) were covariates that had significant effects on the clearance rate (CL) of the model. The pharmacokinetic equation of CL in the final covariate model was as follows: CLi = 9.23× (1 + 0.0005× (θCrCL -105.78)) × (1 + 0.0017× (θWT -16)) × eηcl,i (L/h), IF (θDEX ) CLi = 1.19× CLi (L/h). The estimation accuracy of all pharmacokinetic parameters were acceptable (relative standard error < 14.74%). The goodness-of-fit diagram and bootstrap tests indicated that the final PPK model was stable with acceptable predictive ability. The PPK model may be useful for determining personalized medication levels in pediatric medulloblastoma patients undergoing HD-MTX therapy.

Massive mafic sheets were recently recognized intruding the Neoproterozoic strata in Fuyang area, eastern Jiangnan orogen. Geochronological, geochemical, and isotopic studies were carried out to understand their mantle source, crust–mantle interaction, and tectonic setting. LA-ICP-MS U-Pb zircon data indicate that the sheets were generated at 808 ± 7 Ma. The mafic sheets consist of two groups: high Ti and low Ti. They are enriched in light rare earth elements (LREE; 3.3–5.3 ppm) and show negligible Eu anomalies (δEu = 0.77–1.12). They also have strong large-ion lithophile element (LILE; Sr, K, Rb, Ba) enrichment, moderately strong high-field-strength element (HFSE) enrichment (except for Nb-Ta depletion), and positive eNd(t) (5.1–9.1). Geochemical and isotopic data indicate that the mafic sheets were generated from a depleted asthenospheric mantle source. The high-Ti mafic sheets have higher HFSE contents and less Nb-Ta depletion than the low Ti series, indicating a lower degree of partial melting...

Objective To clone, express and purify C12orf49 recombinant protein. To prepare rabbit anti-C12orf49 protein polyclonal antibody in order to further elucidate its biological function. Methods PCR was used to amplify the gene C12orf49 in vitro. pET-32a (+)-C12orf49, the recombinant protein prokaryotic expression vector, was transformed into E. coli. IPTG was used as the inductive agent to obtain C12orf49 recombinant protein, and the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Specific polyclonal antibody was derived from rabbits that immunized by recombinant protein. ELISA and Western blot were used to test its titer and specificity, respectively. MTT cell proliferation experiment was carried out to observe effect of the protein on proliferation of HepG2 cells. Results The C12orf49 recombinant protein was expressed in a large quantity. Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000. And the antibody also had a good specificity, confirmed by Western blot. C12orf49 recombinant protein may had a advanced effect on the proliferation of HepG2 cells. Conclusions Using C12orf49 recombinant protein, we can obtain the polyclonal antibody with great titer and good specificity. Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.

The influence of the rice husks powder (RHP) content and its particle size distribution on the composite’s tensile strength, fracturing elongation ratio, flexural strength and flexural elastic modulus has been investigated. Respective water absorption and thermal properties of PP composites incorporated with different proportion of RHP have also been analyzed. The microstructure of fractured surfaces was further observed in scanning electron microscopy (SEM). The results showed that the composites with RHP of 245 μm have higher mechanical properties. The tensile strength and fracturing elongation ratio decrease with the increase of RHP content, and reach peak values in 30% RHP content. Water absorption and volume expansion ratio of the composite increase with the increasing of RHP content. Flexural strength and flexural modulus decrease after water absorption. When PHR content is low, the RHP particles are well distributed and the interface of RHP and PP is smooth. When PHR content is higher, the RHP particles tend to agglomerate, leading to poorer interface and lower mechanical properties, the composite failed with brittle fracture.

As an oncoprotein, mutant p53 is a potential tumor-specific target for cancer therapy. Most mutated forms of the protein are largely accumulated in cancer cells due to their increased stability. In the present study, we demonstrate that mutant p53 protein stability is regulated by gambogic acid (GA). Following GA exposure, protein levels of mutant p53 decreased while the mRNA levels were not affected in MDA-MB-435 cells, which indicate that GA down-regulates mutant p53 at post-transcription level. Co-treatment with GA and cycloheximide, a protein synthesis inhibitor, induced a decrease of half-life of mutant p53 protein. These findings indicated that the reduction of mutant p53 by GA was due to the destabilization and degradation of the protein. Furthermore, inhibition of proteasome activity by MG132 blocked GA-induced down-regulation of mutant p53, causing mutant p53 accumulation in detergent-insoluble cellular fractions. Further studies revealed that mutant p53 was ubiquitinated and it was chaperones related ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) rather than MDM2 involved in the degradation of mutant p53. In addition, GA prevented Hsp90/mutant p53 complex formation and enhanced interaction of mutant p53 with Hsp70. Depletion of CHIP stabilized mutant p53 in GA treated cells. In conclusion, mutant p53 may be down-regulated by GA through chaperones-assisted ubiquitin/proteasome degradation pathway in cancer cells.

Oroxylin A, a naturally occurring monoflavonoid extracted from Scutellariae radix, exhibits anticancer activity and induces apoptosis in human hepatocellular carcinoma HepG2 cells according to our previous data. In this study, we investigate whether p53 is involved in oroxylin A-triggered viability inhibition and apoptosis induction in cancer cells. In a panel of different cancer cell lines, more potent inhibitory effects of oroxylin A were observed in wtp53 cells than those in mtp53 or p53-null cells. Moreover, p53-siRNA-transfected HepG2 cells showed lower levels of apoptosis induced by oroxylin A than control-siRNA-transfected cells. Likewise, after oroxylin A treatment, p53-null K-562 cells displayed promoted apoptosis rate when transfected with wtp53 plasmid. Western blot and real-time RT-PCR assay revealed that oroxylin A markedly upregulated p53 protein expression in HepG2 and p53-overexpressing K-562 cells, but had no influence on p53 mRNA synthesis. Furthermore, after co-treatment with cycloheximide, oroxylin A still exerted a little effect on p53 expression. The negative regulator of p53, MDM2 protein was detected, and downregulated expression was observed. In the presence of MG132, an inhibitor of proteasome-mediated proteolysis, no change in p53 expression was obtained. Additionally, the antioxidant N-acetyl-L-cysteine could obviously abrogate p53 stabilization triggered by oroxylin A. Therefore, it is summarized that oroxylin A stabilized p53 expression and induced apoptosis at the posttranslational level via downregulating MDM2 expression and interfering MDM2-modulated proteasome-related p53 degradation. This indicated that oroxylin A could be served as a potential, novel agent candidate for cancer therapy. © 2009 Wiley-Liss, Inc.

Cancer cell invasion is one of the crucial events in local spreading, growth, and metastasis of tumors. The present study investigated the antiinvasive and antimetastatic action of gambogic acid (GA) in MDA-MB-435 human breast carcinoma cells. GA caused a concentration-dependent suppression of cell invasion through Matrigel and significantly inhibited lung metastases of the cells transplanted in vivo. The potent effects of GA have been attributed to its ability to reduce the expression of matrix metalloproteinases (MMP) 2 and 9 in vitro and in vivo both at the protein and mRNA levels, which were associated with protein kinase C (PKC) signaling pathway as supported by the diminished antiinvasive effect of GA in the presence of specific activator of the pathway. Collectively, our data demonstrated that GA exhibited antiinvasion properties on highly invasive cancer cells via PKC mediated MMP-2/9 expression inhibition. This indicated that GA can be served as a potential novel therapeutic candidate for the treatment of cancer metastasis.

Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence forming another his-tag structure, was approved by the State Food and Drug Administration of China (SFDA) in 2005 for the treatment of non-small-cell lung cancer. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we examined the anti-invasive activities of endostar in vitro. The results showed that endostar suppressed MDA-MB-435 cell adhesion to the fibronectin-coated substrate in a concentration-dependent manner. It could inhibit the wound healing migration of MDA-MB-435 cells and invasion of MDA-MB-435 cells through reconstituted ECM (matrigel). Zymography revealed that endostar decreased the secretion of MMP-2 and MMP-9. Endostar could also inhibit the expressions of MMP-2 and MMP-9 in MDA-MB-435 cells. Additionally, endostar exerted an inhibitory effect on the phosphorylation of ERK1/2. Collectively, these data provided a molecular basis for the anti-invasive effects of endostar.

Gambogic acid, the major active ingredient of gamboge, has been shown to exhibit anti-cancer activity both in vivo and in vitro. However, its potential activity in tumor metastasis remains unclear. In the present study, we found that gambogic acid strongly inhibited the adhesion of highly metastatic mouse melanoma B16-F10 cells in vitro. Gambogic acid also exhibited significant anti-metastasis activity on the development of in vivo artificial metastases (i.e. following tail vein i.v. injection of the B16-F10 melanoma tumor cells in C57BL/6 mice). Flow cytometric analysis and Western blot showed that gambogic acid inhibited the cell surface expression of alpha(4) integrin, while exhibited negligible effects on the expression of alpha(5) and beta(1) integrin. Thus we concluded for the first time that gambogic acid could inhibit the adhesion and migration of B16-F10 cells in vitro and in vivo, in which down-regulation of alpha(4) integrin expression was involved.

Gambogic acid (GA), an ingredient isolated from Garcinia hanburyi, has potent anticancer activity both in vitro and in vivo. In the present study, we examined the effects of GA on intracellular microtubules and reconstituted microtubules in vitro. Immunofluorescence microscopy revealed that 2.5 muM GA caused microtubule cytoskeleton disruption and microtubule depolymerization in human breast carcinoma MCF-7 cells, thereby reducing the amount of polymer form of tubulin and increasing the amount of monomer form of tubulin. We further confirmed that GA could depolymerize microtubule associated protein (MAP)-free microtubules and MAP-rich microtubules in vitro. Thus we suggested that GA-induced G2/M phase cell cycle arrest may be attributed to its depolymerization of microtubules. We also revealed that phosphorylation levels of p38 and c-Jun N-terminal kinase-1 (JNK-1) were increased markedly by GA, resulting in apoptosis of MCF-7 cells. Taken together, our results suggested that GA depolymerized microtubules and elevated the phosphorylation levels of JNK1 and p38, which caused G2/M cell cycle arrest and apoptosis in MCF-7 cells.

Aim To study the chronic toxicity of gambogic acid (GA), the major active ingredient of gamboges, a brownish to orange resin extracted from the Garcinia hanburyi (family Guttiferae ) in Southeast Asia, using Sprague–Dawley rat as an animal model and provide further theoretical support for clinical applications of this promising natural anticancer agent. Methods GA was administered orally at dosages of 120, 60 and 30 mg/kg once every other day for a total of 13 weeks. Then we carried out the chronic toxicity studies including general body parameters, hematological, serum biochemistry, histopathological, and viscera examination. Results The results from the studies demonstrated that rats treated with high dose (120 mg/kg) of GA for a long time can lead to the damage on the kidney and liver. An innocuous dose was established to be 60 mg/kg after administration to rats for a total of 13 weeks at a frequency of one administration every other day. This dose was approximately 18.0 (body weight) or 9.6 (body surface area) times higher then that of the dose (200 mg/60 kg, every other day) used for human trials. Conclusions The studies demonstrated that the toxicity targets in the rats were the kidney and liver. These results provide further theoretical support for clinical applications of this promising natural anticancer agent.

Previous studies have firmly demonstrated that wogonin, a naturally occurring monoflavonoid extracted from the root of the Chinese herb medicine Scutellaria baicalensis, could effectively inhibit the proliferation of several cancer cell lines. However, little is known about the effect of wogonin on differentiation induction of leukemic cells. Here we investigate the potential role of wogonin in the proliferation and differentiation of NB4, a human promyelocytic leukemia cell line derived from a patient with acute promyelocytic leukemia. Our results indicated that wogonin significantly suppressed the proliferation and efficiently induced the differentiation of NB4 cells. NB4 cell growth was inhibited by 55-60% after treatment with 50 microM wogonin for a period of 5 days. The results of the nitroblue tetrazolium (NBT) reduction test (with 67.13% positive cells by 50 microM wogonin for 5 days), Giemsa staining (with 67.24% positive cells by 50 microM wogonin for 5 days), and the expression of mature-related cell-surface differentiation antigens CD11b and CD14 (with 70.94% CD11b(+) and 5.82% CD14(+) cells by 50 microM wogonin for 5 days) demonstrated an increase in the differentiation-inducing action of wogonin on the NB4 cells, which was accompanied by an increase in mRNA and protein expression of phospholipids scramblase 1 (PLSCR1). Meanwhile, the level of phosphorylated PKC delta (Ser643) was dramatically increased in wogonin treated NB4 cells. Interestingly, wogonin treatment displayed little effect on the apoptosis of NB4 cells. Taken together, the results reported here demonstrated that wogonin could promote the granulocytic differentiation of NB4 cells by up-regulating the expression of PLSCR1 gene.

We previously reported that gambogic acid (GA), a natural product, was an effective telomerase inhibitor by repressing hTERT promoter. In this study, posttranscriptional regulation of the telomerase hTERT by GA was investigated in BGC-823 human gastric carcinoma cells. The telomerase activity was detected by PCR-TRAP assay. RT-PCR assay and Western blot were performed to examine the repression of telomerase hTERT and c-Myc after GA or c-Myc-specific siRNA treatment. The results indicated that GA repressed telomerase activity and hTERT transcriptional activity via down-regulation of c-Myc expression in BGC-823 human gastric carcinoma cells. We further observed that hTERT transcriptional activity was not completely blocked by c-Myc-specific siRNA, suggesting that additional factors are involved in the repression of telomerase activity. The results of Western blot and immunoprecipitation assay revealed that GA inhibits the phosphorylation of Akt. The further results also confirmed that celecoxib, an inhibitor of Akt phosphorylation, could significantly repressed telomerase activity alone and enhance the repression of telomerase activity combined with GA. These data suggested that GA inhibits the posttranslational modification of hTERT by inhibiting the phosphorylation of Akt. Collectively, we suggest that GA represses telomerase activity not only by repressing hTERT transcriptional activity via c-Myc but also by posttranslational modification of hTERT via Akt.

Previous studies revealed that wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, possessed anticancer activity both in vitro and in vivo. However, the molecular mechanism of its potent anticancer activity remains poorly understood and warrants further investigations. In this study, we found for the first time that wogonin inhibited the growth and tumor angiogenesis of human gastric carcinoma in nude mice. We explored the inhibitory effect of wogonin on angiogenesis stimulated by vascular endothelial growth factor (VEGF) in vitro. Wogonin suppressed the VEGF-stimulated migration and tube formation of human umbilical vein endothelial cells (HUVECs). It also restrained VEGF-induced tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). This inhibition of receptor phosphorylation was correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that wogonin might be a promising antitumor drug.

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia. The present study aims to demonstrate that gambogic acid (GA) has potent anticancer activity for glioblastoma by in vitro and in vivo study. Rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood-brain barrier (BBB). To reveal an involvement of the intrinsic mitochondrial pathway of apoptosis, the mitochondrial membrane potential and the western blot evaluation of Bax, Bcl-2, Caspase-3, caspase-9 and cytochrome c released from mitochondria were performed. Angiogenesis was detected by CD31 immunochemical study. The results showed that the uptake of GA by rBMEC was time-dependent, which indicated that it could pass BBB and represent a possible new target in glioma therapy. GA could cause apoptosis of rat C6 glioma cells in vitro in a concentration-dependent manner by triggering the intrinsic mitochondrial pathway of apoptosis. In vivo study also revealed that i.v. injection of GA once a day for two weeks could significantly reduce tumor volumes by antiangiogenesis and apoptotic induction of glioma cells. Collectively, the current data indicated that GA may be of potential use in treatment of glioblastoma by apoptotic induction and antiangiogenic effects.

Previous studies revealed that gambogic acid (GA), the major active ingredient of gamboge, a brownish to orange resin exuded from Garcinia hanburryi tree in Southeast Asia, possessed significant anticancer activity both in vitro and in vivo. In this study, we explored the high antiangiogenic activities of GA for the first time. GA inhibits the VEGF-stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) as well as microvessel sprouting from rat aortic rings in vitro. Moreover, GA inhibits vessel growth in matrigel plugs and CAM in vivo and transplanted tumor in mice. The results also indicated that GA decreases VEGF production of cultured tumor cells and inhibits VEGF-induced tyrosine phosphorylation of KDR/Flk-1. This inhibition of receptor phosphorylation is correlated with a significant decrease in VEGF-triggered phosphorylated forms of ERK, AKT and p38. Taken together, these findings strongly suggest that GA might be a structurally novel angiogenesis inhibitor.

The traditional Chinese medicine Scutellaria radix has been used for thousands of years, mainly for the treatment of inflammatory conditions including hepatitis. The major active constituent, wogonin (WG), isolated from S. radix has attracted increasing scientific attention in recent years due to its potent biological activities. However, pharmacologic studies have primarily been focused on wogonin's anti-inflammatory and anti-cancer activities. In this study, we have investigated wogonin's anti-hepatitis B virus (HBV) activity both in vitro and in vivo. In the human HBV-transfected liver cell line HepG2.2.15, wogonin effectively suppressed the secretion of the HBV antigens with an IC(50) of 4 microg/ml at day 9 for both HBsAg and HBeAg. Consistent with the HBV antigen reduction, wogonin also reduced HBV DNA level in a dose-dependent manner. Duck hepatitis B virus (DHBV) DNA polymerase was dramatically inhibited by wogonin with an IC(50) of 0.57 microg/ml. In DHBV-infected ducks wogonin dosed i.v. once a day for 10 days reduced plasma DHBV DNA level with an ED(50) of 5mg/kg. The in vivo anti-HBV effect of wogonin in ducks was confirmed by Southern blotting of DHBV DNA in the liver. Histopathological evaluation of the liver revealed significant improvement by wogonin. In addition, in human HBV-transgenic mice, wogonin dosed i.v. once a day for 10 days significantly reduced plasma HBsAg level. Immunohistological staining of the liver confirmed the HBsAg reduction by wogonin. In conclusion, our results demonstrate that wogonin possesses potent anti-HBV activity both in vitro and in vivo. Currently, wogonin is under early development as an anti-HBV drug candidate.

We previously reported that wogonin, a flavonoid compound, was a potent apoptosis inducer of human hepatoma SMMC-7721 cells and murine sarcoma S180 cells. In the present study, the effect of oroxylin A, one wogonin structurally related flavonoid isolated from Scutellariae radix, on human hepatocellular carcinoma cell line HepG2 was examined and molecular mechanisms were also investigated. Oroxylin A inhibited HepG2 cell proliferation in a concentration- and time-dependent manner measured by MTT-assay. Treatment with an apoptosis-inducing concentration of oroxylin A caused typical morphological changes and apoptotic blebbing in HepG2 cells. DNA fragmentation assay was used to examine later apoptosis induced by oroxylin A. FACScan analysis revealed a dramatic increase in the number of apoptotic and G(2)/M phase arrest cells after oroxylin A treatment. The pro-apoptotic activity of oroxylin A was attributed to its ability to modulate the concerted expression of Bcl-2, Bax, and pro-caspase-3 proteins. The expression of Bcl-2 protein and pro-caspase-3 protein was dramatically decreased after treatment with oroxylin A. These results demonstrated that oroxylin A could effectively induce programmed cell death and suggested that it could be a promising antitumor drug.

Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of GA caused an irreversible arrest in the G(2)/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr(15) (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H) activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr(161) CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G(2)/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.

The molecular mechanisms of wogonin-induced apoptosis of human hepatoma SMMC-7721 cells are reported. Wogonin treatment resulted in significant inhibition of SMMC-7721 cells in a time-dependent and concentration-dependent manner. Typical morphological changes and apoptotic blebbing in SMMC-7721 cells were observed after treatment with 1x10(-4) mol/l wogonin for a period of 0-48 h. Flow cytometry and Annexin-V/propidium iodide double-staining experiments revealed a dramatic increase in the number of apoptotic and G0/G1 phase cells after wogonin treatment. The proapoptotic activity of wogonin is attributed to its ability to modulate the expression of bcl-2 and bax proteins. It is observed that the expression of bax protein is dramatically increased whereas the synthesis of bc1-2 protein is significantly decreased when cells are treated with wogonin. The results presented in this paper suggested an important relationship between gene regulation and wogonin-induced apoptosis, and indicated the possibility of developing naturally occurring monoflavonoids as novel anticancer agents for better management of human cancers.

The anticancer effects of wogonin on murine sarcoma S180 both in vitro and in vivo were investigated, and its pro-apoptotic molecular mechanism was further studied. Wogonin treatment resulted in significant inhibition of S180 cells in a concentration-dependent manner detected by MTT assay. The IC50 value for 48 h was (7.37+/-1.53) x 10(-5) M. Typical morphological changes and apoptosis bleb phenomenon in S180 cells exposed to wogonin were distinctly observed by the inverted light microscope and the fluorescence microscope, respectively. According to protocols of transplanted tumor research, mice were transplanted with tumor cells S180. The weight of tumor and the peripheral leucocyte count were observed after the treatment of wogonin. The significant suppression of tumor growth was observed, and the peripheral leucocyte count of S180-bearing mice remained no significant changes compared with control group. After the treatment of 40 mg/kg wogonin, the inhibitory rate of tumor weight was 53.01%. Additional DNA fragmentation assay showed that wogonin induced apoptosis on murine sarcoma S180 tissue. RT-PCR results indicated that the increasing mRNA levels of bax and p53 and the decreasing mRNA level of bcl-2 were induced by wogonin. Western-blot assay showed that the increasing protein level of bax and the decreasing protein level of bcl-2 were induced by wogonin. Collectively, wogonin could induce apoptosis in murine sarcoma S180 thereby inhibiting the tumor growth both in vitro and in vivo. The pro-apoptotic effects might be related to the improvement of mRNA level of p53, the improvement of mRNA and protein levels of bax, and the reduction of mRNA and protein levels of bcl-2.

The clinical efficacy of acupuncture and moxibustion for post-stroke constipation was systematically reviewed. By computerized and manual retrieval of clinical research literature regarding acupuncture and moxibustion for post-stroke constipation, the randomized control trials (RCTs) that met the inclusive criteria were collected. Cochrane systematic review method was used and Revmen 5.2 software was adopted to perform this Meta analysis. Totally 8 articles were included, involving 610 cases of post-stroke constipation. As a result, the total effective rate and cured rate of acupuncture and moxibustion for post-stroke constipation were significantly superior to those of the control group [total effective rate: OR = 2.10, 95% CI (1.25, 3.54), Z = 2.78, P = 0.005; cured rate: OR = 2.37, 95% CI (1.57, 3.58), Z = 4.10, P0.0001]. This result indicated that acupuncture was effective for post-stroke constipation and had some advantages compared with other therapies. But the quality of included RCTs was low, and high-quality, large-sample and multi-center RCTs were needed to perform further verification.

Hybrid cells derived from stem cells play an important role in organogenesis, tissue regeneration and cancer formation. However, the fate of hybrid cells and their range of function are poorly understood. Fusing stem cells and somatic cells induces somatic cell reprogramming, and the resulting hybrid cells are embryonic stem cell-like cells. Therefore, we hypothesize that fusion-induced hybrid cells may behave like ES cells in certain microenvironments. In this study, human hepatic cells were induced to apoptosis with H2O2, and then co-cultured with hybrid cells that had been derived from mouse ES cells and human hepatic cells using a transwell. After co-culturing, the degree of apoptosis was evaluated using Annexin-V/PI double-staining analysis, flow cytometry and Western-blot. We observed that H2O2-induced cell apoptosis was inhibited by co-culture. In addition, the activity of injury-related enzymes (GSH-Px, LDH and SOD) and the level of albumin release in the co-culture system trended toward the level of normal undamaged hepatic cells. The stably increased levels of secretion of ALB in the co-culture system also confirmed that co-culture with hybrid cells helped in recovery from injury. The fate of the hybrid cells was studied by analyzing their gene expression and protein expression profiles. The results of RT-PCR indicated that during co-culturing, like ES cells, hybrid cells differentiated into hepatic lineage cells. Hybrid cells transcripted genes from both parental cell genomes. Via immunocytochemical analysis, hepatic directional differentiation of the hybrid cells was also confirmed. After injecting the hybrid cells into the mouse liver, the GFP-labeled transplanted cells were distributed in the hepatic lobules and engrafted into the liver structure. This research expands the knowledge of fusion-related events and the possible function of hybrid cells. Moreover, it could indicate a new route of differentiation from pluripotent cells to tissue-specific cells via conditional co-culture. Electronic Supplementary Material Supplementary material is available for this article at 10.2478/s11658-010-0018-0 and is accessible for authorized users.

Gambogic acid (GA), the natural compound extracted from gamboges, has recently been established as a potent anti-tumor agent. Although it was proved that GA enhances p53 protein level through inhibition of MDM2 in p53 wild-type cancer cells, the mechanisms of MDM2 inhibition especially with the absence of p53 are not fully understood. Herein we further studied the MDM2 regulation by GA and propose novel explanations of its unrecognized mechanism. Regardless of p53 status, GA reduced MDM2 expression in a concentration- and time-dependent manner. Moreover, the inhibitory effects were exhibited at both transcriptional and posttranslational levels. We found that P1 and P2 promoter of MDM2 were both responsive to GA, resulting in decreased Mdm2 RNA level. At the posttranslational level, GA promoted the autoubiquitination of MDM2, followed by proteasome-mediated degradation. Additionally, GA increased p21(Waf1/CIP1) expression in p53 null cancer cells, which was associated with GA-mediated impairing of the interaction between MDM2 and p21(Waf1/CIP1). Furthermore, the apoptosis, cytotoxicity and G2/M cell cycle arrest induced by GA were detected in both p53 wild-type and p53 null cancer cells. In vivo anti-tumor activity of GA was also confirmed in H1299 xenografts. It is concluded that GA down-regulates the MDM2 oncogene and exerts the anti-tumor activity independent of p53, and therefore provide more evidences for its therapeutic application.

In this study, we investigated the correlation between p53 and bcl-2 in gambogic acid (GA)-induced apoptosis.MTT assay was employed to evaluate MCF-7 cell viability after GA treatment. Cell morphological changes were observed follow-up under the inverted microscope after GA withdrawal. To observe the cell apoptosis, DAPI staining was used. Meanwhile, p53 small interfering RNA (si-RNA) was adopted to knock p53 down. All expressions of p53 and bcl-2 were evaluated by Western blot analysis.MTT assay showed that GA inhibited MCF-7 cell growth effectively in a time-dependent manner. With 0.5 h GA treatment, p53 was significantly increased, whereas bcl-2 was reduced potently with 6 h GA treatment. After GA withdrawal, p53 expressions were maintained in high levels. Furthermore, bcl-2 decreasing was attenuated after co-treatment with PFT alpha, a p53 transcription blocker. Same result was observed after p53 knock-down by p53 si-RNA.Gambogic acid induced human breast cancer cells MCF-7 apoptosis by reducing bcl-2 expression via p53.

Macranthoside B (MB) is a hederagenin saponin extracted from the flower bud of Lonicera macranthoides. In this study, we defined the anticancer effect of MB both in vitro and in vivo using cell proliferation assays and xenograft tumor growth assays. Our data indicate that MB inhibits the proliferation of various kinds of cancer cells with IC(50) values in the range of 10-20 microM. Moreover, the volume and weight of xenograft tumors in nude mice treated with 5mg/kgMB were decreased remarkably compared to those of the vehicle control group. Furthermore, DAPI staining and flow cytometry analysis with Annexin V/PI double staining revealed that more apoptotic cells were observed following MB treatment. In addition, degradation of PARP (poly-ADP-ribose polymerase), and activation of the caspase cascade for intrinsic pathways were observed. We also found that the expression of Bcl-2 protein decreased and the protein level of Bax increased which leading to an increase of the Bax/Bcl-2 ratio. Our results showed that MB exhibited strong anti-tumor effect and mitochondrion-mediated apoptosis induction involved in it.

Multiple lines of evidence have demonstrated that gambogic acid (GA) is an efficient apoptosis inducing agent. However, the mechanisms of GA induced apoptosis have been controversial, despite the tremendous effort made during recent years. Here we report a novel mechanism through which GA induces cell apoptosis. Instead of dealing with tumor cells directly, GA first activates inactive T lymphocytes, which in turn triggers cancer cell apoptosis. This is supported by the observation that GA inhibited tumor growth and extended the survival time of mice bearing H22 tumor. cDNA microarray analysis indicated that 22.92% of the 48 genes that were affected with GA treatment were immune related genes. RT-PCR assay revealed that GA up-regulated MHC-II and TCR transcriptions, implicating that GA activates T lymphocytes to induce tumor cell apoptosis in vivo. HE staining showed that T lymphocytes penetrated into tumor tissues after GA administration. Western blotting revealed that GA enhanced CD4+ and CD8+ expressions. Annexin-V/PI double-staining and DNA ladder assays confirmed that GA induced tumor cell apoptosis. In summary, this report demonstrated, for the first time, that GA mainly activates T lymphocytes to induce cancer cell apoptosis in H22 transplanted mice.

Gambogic acid (GA) is the major active ingredient of gamboge, a brownish resin exuded from Garcinia hanburryi tree in Southeast Asia. In this study, we compared the different apoptotic induction of GA on human normal embryonic hepatic L02 cells and human hepatoma SMMC-7721 cells by detecting growth inhibition, observing morphological changes, and the expressions of the relative apoptotic proteins (Bax, Bcl-2 and caspase-3). The results indicated that GA could selectively induce apoptosis of SMMC-7721 cells, while had relatively less effect on L02 cells. To illustrate the distinct selective antitumor mechanism of GA, we further study its distribution in cultured cells and in tumor-bearing mice. The results indicated that SMMC-7721 cells have higher GA binding activity than L02 cells. The retention time of GA in grafted tumor was longer than in liver, renal and other organs. Collectively, the selective anticancer activity of GA could be due to its significant apoptotic inducing effects as well as its higher distribution and longer retention time in tumor cells compared to the normal cells. So GA might be a kind of highly effective anticancer drug candidate with low toxicity to normal tissue.

Acute and chronic toxicity of gambogic acid, a promising novel anticancer agent, was determined using albino mice and Beagle dogs as model animals. Histopathological examination and viscera parameter investigation were also carried out after autopsy. The LD50 of gambogic acid was found to be 45 approximately 96 mg/kg and the 95% confidence limit was determined to be 43.18 approximately 48.45 mg/kg. The results from the chronic toxicity studies demonstrated that the toxicity targets in the experimental animals were liver and kidney. The innocuous dose was established to be 4 mg/kg after administration to dogs for a total of 13 weeks at a frequency of one injection every other day. This dose (4 mg/kg) was approximately 9.6 (body weight) or 5.1 (body surface area) times the dosage (25 mg/60 kg, every other day) recommended for human trials. Our results provide the theoretical foundation for clinical applications of this promising natural anticancer agent and will likely bring about considerable economic and social benefits.

Objectives: To investigate the effects and potential mechanisms of gambogic acid (GA), a naturally occurring anticancer agent, on the expression and regulation of telomerase in human gastric carcinoma cells. Methods: GA-induced inhibition of cell proliferation was evaluated by the commonly employed MTT assay on two human gastric carcinoma cell lines, MGC-803 and SGC-7901. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplication protocol-polymerase chain reaction and reverse transcription-polymerase chain reaction, respectively. The hTERT promoter activity was measured by luciferase assay. The expression of c-MYC, an apoptotic gene that modulates the expression of hTERT promoter, was quantified by Western blotting. Results: The proliferation of human gastric carcinoma cell lines, MGC-803 and SGC-7901, was significantly inhibited with GA treatment. Both telomerase activity and hTERT mRNA expression were notably decreased in cells treated with GA. The activity of hTERT promoter and the expression of c-MYC were also remarkably decreased in GA-treated cells. Conclusion: This study demonstrated that GA treatment of human gastric carcinoma cell lines, MGC-803 and SGC-7901, significantly reduced the expression of c-MYC in a time- and concentration-dependent manner accompanied with the down-regulation of the hTERT transcription and the ultimate reduction in telomerase activity. Our results indicate that the hTERT is a target of c-MYC activity and identify a feasible mechanism of GA’s potent anticancer activity.

We determined the in vivo and in vitro antitumor activities of gambogic acid (GA) and one of the possible mechanisms for its inhibitory activities. In vivo antitumor activity of GA was evaluated by the relative tumor growth ratio (T/C) in nude mice, and in vitro inhibition of SPC-A1 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue exclusion assay. Telomere repeats amplification protocol (TRAP)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to quantitatively detect telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) mRNA, respectively. Results from our in vivo study showed that transplantable tumor growth remained suppressed for up to 21 d with minimal animal weight loss in nude mice treated with gambogic acid (i.v.). Proliferation of SPC-A1 cells cultured in vitro was significantly inhibited (p

The selective induction of apoptosis of gambogic acid (GA) on MGC-803 cells and its probable molecular mechanism were studied. GA greatly inhibited (24, 48, 72 h) the growth of MGC-803 cells (by MTT); the IC(50) value was 0.96 microg/ml at 48 h. Meanwhile, no influence was observed on body weight, number of WBC (white blood cells) in blood or karyote in marrow of rats after GA was injected intravenously. We conclude that GA does not affect normal cells, but that it can induce apoptosis in tumor cells selectively and there were marked morphological changes. A great quantity of apoptotic cells and increasing G(2)/M phase cells were observed by flow cytometry, and a significant percentage of early apoptotic cells were observed by Annexin-V/PI double staining assay. The increase of bax gene and the decrease of bc1-2 gene expressions were detected by immunohistochemistry. Activation of bax and suppression of bc1-2 may contribute to the apoptosis mechanism.

To investigate the anticancer effect of a traditional Chinese medicine gambogic acid (GA) in human gastric cancer line BGC-823 and further study the mechanism of apoptosis induction of GA.Low differential human gastric cancer line BGC-823 were treated with GA at different doses and different times, the inhibitory rates were detected by MTT assay. Apoptosis induced by GA in BGC-823 cells was observed by Annexin-V/PI doubling staining flow cytometry assay. And T/C (%) was chosen to detect the inhibition of GA on human gastric adenocarcinoma BGC-823 nude mice xenografts. Apoptosis on nude mice xenografts was observed by Annexin-V/PI doubling staining flow cytometry assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by GA, the changes on the expression of bcl-2 and bax genes were detected by RT-PCR.After incubation with GA, low differential human gastric cancer line BGC-823 was dramatically inhibited in a dose-dependent manner. After these cells was exposed to GA for 24, 48 and 72 h, the IC(50) value were 1.02+/-0.05, 1.41+/-0.20 and 1.14+/-0.19 micromol/L, respectively. Apoptosis in BGC-823 cells induced by GA was observed by Annexin-V/PI doubling staining flow cytometry assay. The apoptotic population of BGC-823 cells was about 12.96% and 24.58%, respectively, when cells were incubated with 1.2 micromol/L GA for 48 and 72 h. T/C (%) of human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts was 44.3, when the nude mice were treated with GA (8 mg/kg). Meanwhile, apoptosis induced by GA was observed in human gastric carcinoma adenocarcinoma BGC-823 nude mice xenografts. The increase of bax gene and the decrease of bc1-2 gene expressions were found by RT-PCR.The inhibition of GA on human gastric cancer line BGC-823 was confirmed. This effect connects with the inducing apoptosis in BGC-823 cells and the molecular mechanism might be related to the reduction of expression of apoptosis-regulated gene bcl-2, and the improvement of the expression of apoptosis-regulated gene bax. The result was also confirmed in vivo.