Search

Abstract: Previously, we demonstrated that LYG-202, a newly synthesized flavonoid with a piperazine substitution, exhibited obvious antitumor activity in vivo and in vitro. The exact mechanism of this new compound remains unclear. In the present study, we examined the effects of LYG-202 on reactive oxygen species (ROS) production and the downstream signaling pathway in the apoptosis of human hepatocellular carcinoma HepG2 cells. Pretreatment with NAC (N-acetylcysteine), a ROS production inhibitor, partly inhibited the apoptosis induced by LYG-202 via blocking the ROS generation. Further data revealed that LYG-202 induced ROS accumulation followed by a decrease in mitochondrial membrane potential (MMP), release of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) to cytosol, which induced apoptosis of the cells. Moreover, the mitogen-activated protein kinases (MAPK), the downstream effect of ROS accumulation including c-Jun N-terminal kinase (JNK) and p38 MAPK, could be activated by LYG-202. Taken together, the generation of ROS might play an important role in LYG-202-induced mitochondrial apoptosis pathway, which provided further support for LYG-202 as a novel anticancer therapeutic candidate. [Copyright &y& Elsevier]

Abstract: Gambogic acid (GA) has been wildly studied to show potent anti-tumor effects in vivo and in vitro. We have confirmed that GA stabilized and activated p53 through down-regulating the expression of MDM2 in variety of cancer cell lines. However, GA-induced p53 activation could be partially reversed by caffeine, a PI3k inhibitor. Therefore, questions of whether GA induces post-translational modifications of p53 and subsequent activation of p53; and if that is the case, which upstream signaling pathway(s) is (are) responsible for that are proposed. Here, the relationship between p53 activation and its post-translational modifications was investigated in the human cancer cell lines HepG2 and A549 in response to GA or adriamycin treatment. GA induces p53 phosphorylation at sites Ser15 and Ser20 in a concentration- or time-dependent way, which was a direct result of DNA damage, as γ-HA2X foci and ‘comet’ DNA fragments were detected. GA induces p53 phosphorylation through activation of an ATM- and Rad3-related pathway, and GA-induced phosphorylation of Chk1 is also involved. Upon treatment with GA, ATR activation is clearly associated with p53 phosphorylation, as well as activation of its target gene p21Waf/CIP1. Furthermore, we found the dephosphorylation of Cdk1 at Thr161 induced by GA was abrogated, followed by a remarkable disruption of G2/M arrest when the cells were pre-incubated with caffeine. Interestingly, the sensitivity to caffeine enhanced the cytotoxicity of GA as well. Taken together, these data showed an important role of the DNA damage response mediated by ATR-Chk1 in p53/p21Waf/CIP1 activation and downstream G2/M arrest during GA treatment. [Copyright &y& Elsevier]

Abstract: 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 p21Waf1/CIP1 expression in p53 null cancer cells, which was associated with GA-mediated impairing of the interaction between MDM2 and p21Waf1/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. [Copyright &y& Elsevier]

Abstract: We recently established that asparanin A, a steroidal saponin extracted from Asparagus officinalis L., is an active cytotoxic component. The molecular mechanisms by which asparanin A exerts its cytotoxic activity are currently unknown. In this study, we show that asparanin A induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Following treatment of HepG2 cells with asparanin A, cell cycle-related proteins such as cyclin A, Cdk1 and Cdk4 were down-regulated, while p21WAF1/Cip1 and p-Cdk1 (Thr14/Tyr15) were up-regulated. Additionally, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3, caspase-8 and caspase-9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, where Bax was also up-regulated. We also found that the expression of p53, a modulator of p21WAF1/Cip1 and Bax, was not affected in asparanin A-treated cells. Collectively, our findings demonstrate that asparanin A induces cell cycle arrest and triggers apoptosis via a p53-independent manner in HepG2 cells. These data indicate that asparanin A shows promise as a preventive and/or therapeutic agent against human hepatoma. [Copyright &y& Elsevier]

Angiogenesis is associated with the progression of multiple myeloma (MM). Wogonin is an active mono-flavonoid with remarkable antitumor activity. However, its impact on MM-stimulated angiogenesis remains largely unknown. Here, we demonstrated that wogonin decreased expression and secretion of pro-angiogenic factors in MM cells via c-Myc/HIF-1α signaling axis, reducing MM-stimulated angiogenesis and MM cell proliferation in vivo. Overexpression of c-Myc in MM cells disrupted the balance between VHL SUMOylation and ubiquitination, and thus inhibited proteasome-mediated HIF-1α degradation. Impaired function of VHL ubiquitination complex in c-Myc-overexpressing cells was fully reversed by wogonin treatment via increasing HIF-1α-VHL interaction and promoting HIF-1α degradation. Collectively, our in vitro and in vivo studies reveal for the first time that wogonin represses MM-stimulated angiogenesis and tumor progression via c-Myc/VHL/HIF-1α signaling axis.

Objective: This study aims to investigate the potential benefit of combination therapy with magnetic nanoparticles of Fe3O4 (Fe3O4-MNP) and gambogic acid (GA) on SMMC-7721 cells., Methods: The inhibition of proliferation of SMMC-7721 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was calculated and analyzed by flow cytometry, and the expressions of the apoptosis-related protein were detected by Western blot., Results: GA enhanced the cytotoxicity of SMMC-7721 cells in a dose-dependent manner. The Fe3O4-MNP itself had no obviously inhibitory effect, but it could enhance the effect of GA on proliferation of SMMC-7721 cells. The apoptotic rate of SMMC-7721 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The expression levels of caspase-3 and caspase-8 after co-treatment of GA and Fe3O4-MNP were higher than that exposed to either GA or Fe3O4-MNP alone, while the levels of bcl-2 were downregulated., Conclusion: Fe3O4-MNP can promote GA-induced apoptosis of SMMC-7721 cells, which may be related to the downregulation of Bcl-2 and upregulation of caspase-3.

Objective: To explore the expression of PXR (Pregnane X receptor) in several malignant hematological cell lines, and to investigate the reversal effect of Gambogic acid (GA) on multi-drug resistance (MDR) of K562/A02 cell line and its reversal mechanism., Methods: Transcription of PXR was detected by real-time PCR in several malignant hematological cell lines. The growth inhibition rate of K562/A02 in different experimental groups was assayed by MTT method, and the expression of PXR protein was measured by Western blot., Results: PXR gene transcription could be detected in several hematological malignancy cell lines, and it was significantly higher in K562/A02 cell line, compared with the other cell lines used in this experiment. Low-dose GA could enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression. PXR gene transcription and protein expression in GA and DNR+GA groups decreased as compared with control group and the DNR group, suggesting that low-dose GA can down-regulate PXR gene transcription and protein expression., Conclusion: PXR gene transcription can be detected in several hematological malignancy cell line, which is significantly higher in K562/A02 cell line, as compared with the other cell lines used in this experiment. Low-dose GA can enhance cell growth inhibition rate, increasing the effect of chemotherapy, which may be associated with down-regulation of PXR expression.

Picroside II, separated from Chinese herbal medicine, is an active compound with neroprotective activity. Molecularly imprinted polymers (MIPs) have high affinity toward template molecules synthesized by molecularly imprinted technology for its specific combined sites, which can overcome the shortcomings of traditional separation methods, such as complex operation and low efficiency. In this paper, MIPs were prepared by precipitation polymerization with picroside II as the template molecule, 1-vinylimidazole (1-Vinyl) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linker. The morphology of MIPs was characterized by scanning electronmicroscope (SEM) and its static adsorption capacity was measured by the scatchard equation. The results showed that picroside II MIPs have spherical shape, and most of them are uniform in size. Furthermore, the maximum binding capacity (Q(max)) of MIPs is 3.02 mg x g(-1), higher than that of non-imprinted polymers (NIPs). This result indicated that picroside II MIPs with good morphology and high targeted affinity toward the template molecules can be prepared by precipitation polymerization, which can be used to separate picroside II and its analogies from extract of Chinese herbal medicine. In addition, this method has the advantages of good environment and simple operation, which might offer a novel method for the efficient separation of picroside II in the traditional herbal medicines.

The aim of this study was to explore the effect of gambogic acid (GA) on MDS SKM-1 cell proliferation, apoptosis and their possible mechanism. Cell proliferation was determined by MTT method. The apoptosis percentage and cell cycle regulation of SKM-1 cells were analyzed by flow cytometry. Morphological features were observed by light microscopy. The mRNA expression of bcl-2 and bax were detected by RT-PCR. The results showed that GA could inhibit the proliferation of SKM-1 cells in a dose- and time-dependent manner (IC50 was 0.37 µg/ml at 48 h), increase the apoptotic percentage of SKM-1 cells, and arrest cell cycle at the G0/G1. The expression of bax mRNA was up-regulated while that of bcl-2 mRNA was down-regulated in SKM-1 cells treated with GA for 48 h. It is concluded that GA can induce apoptosis, which may be related to its effect of arresting cells at phase of G0/G1 and down-regulating bcl-2/bax ratio.

Gamboge, the resin of Garcinia hanburyi has had a long history of use as the traditional dye as well as a complementary and alternative medicine. The antitumor activities of gamboge have been well demonstrated by inhibiting the growth and progression of cancer cells both in vitro and in vivo. In order to further clarify the mode of action of gamboge, there are three key questions needed to be answered, including what's in gamboge? How do the chemical components from gamboge work on cancer cells? How do biological systems work on the chemical components from gamboge after administration? In this review, we summarize the explorations of the answers toward these questions according to the recent progress in both of chemistry and biology research of gamboge. In addition, the implication in the future research and discovery of the caged G. xanthones as anticancer agents is also discussed.

Gambogic acid (GA), a natural product, was identified as a promising antitumor agent. To further explore the structure-activity relationship of GA and discover novel GA derivatives as antitumor agents, 19 novel GA derivatives modified at C(34) were synthesized and evaluated against A549, BGC-823, U251, HepG2, and MB-231 cancer cell lines by cellular assays. Among them, 15 compounds were found to be more potent than GA against some cancer cell lines. Notably, compound 3 possessed potent inhibitory activities against five cell lines with IC(50) values ranging between 0.24 and 1.09 μM. Compounds 9 and 18 were seven to eightfold more active than GA against A549 cell line. Chemical modification at C(34) of GA by introducing of hydrophilic aliphatic amines resulted in increased activity and improved drug-like properties. These findings will enhance our understanding of the SAR of GA and can lead to the discovery of novel GA derivatives as potential antitumor agents., (Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.)

The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational medicinal modifications on gambogic acid will improve its physicochemical properties and drug-like characters. To investigate the structure-activity relationship of gambogic acid and also to find rational modification position on its chemical skeleton, we designed, synthesized, and characterized 16 derivatives of gambogic acid that were modified at C(39). The structure-activity relationships (SARs) were discussed. The anti-proliferation data were accquired through MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assays of A549, BGC823, U251, HepG2, and MDA-MB-231 cancer cell lines. Most of the synthesized compounds showed strong inhibitory effects. The SAR study revealed that derivatives with aliphatic amino moieties at C(39) were more potent than those with other substituents. The C(39) position can undergo different kinds of chemical modifications without leading to loss of activity. Compounds 4 and 6 can serve as potential lead compounds for further development of new anticancer drugs., (Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.)

5-Fluorouracil (5-FU) is a principal drug for the treatment of colorectal cancer. Due to its low response and high toxicity, synergistic effects of 5-FU in combination with other drugs have been widely researched. This study investigated whether oroxylin A improved the sensitivity of HT-29 human colon cancer cells to 5-FU. A correlation between COX-2 inhibition by oroxylin A and a synergistic effect of 5-FU on the growth of HT-29 cells was observed, and a COX-2 pathway for this effect was recognized; oroxylin A evidently elevated the level of reactive oxygen species in HT-29 cells, which subsequently inhibited COX-2 expression and enhanced the susceptibility of HT-29 cells to 5-FU. Likely also related to COX-2 inhibition, oroxylin A decreased PGE(2) levels in HT-29 cells. The synergistic effect of 5-FU induced by oroxylin A was also found in the suppression of Bcl-2 and in the activation of P53, Bax, PARP, and procaspase-3 proteins in HT-29 cells. Ultimately, a combination of 5-FU with oroxylin A significantly reduced the growth of HT-29 tumors in nude mice compared with treatment with 5-FU or oroxylin A alone. In conclusion, a combination of 5-FU and oroxylin A has a significant synergistic effect in the inhibition of HT-29 cell proliferation in vitro and controls HT-29 tumor growth in vivo. This synergistic effect may be mainly related to COX-2 inhibition by oroxylin A in HT-29 cells.

A tightly regulated catabolic process named autophagy involves the degradation of intracellular components via lysosomes. Here we investigate the antitumor effect of E Platinum, a newly synthesized derivative of oxaliplatin, in vivo and in vitro. E Platinum exhibits growth inhibition of various tumor cells in a dose-dependent manner, but the mechanism underlying it is unclear. Based on theory introducing autophagy, we preliminarily investigate whether autophagy could contribute to the antitumor activity of E Platinum. Our results showed that autophagy induced by 12.5 μM E Platinum in gastric carcinoma BGC-823 cells was significantly characterized by the FITC-fluorescent microtubule associated protein 1 light chain 3 (MAP-LC3), lysosomal-rich/acidic compartments visualized with Lysotracker red (LTR-red) and an accumulation of numerous large autophagic vesicles within the cytoplasm, but not in the control cells. Meanwhile treatment of cells with 12.5 μM E Platinum resulted in conversion of water soluble LC3 (LC3-I) to lipidated and autophagosome-associated form (LC3-II) as well as increasing expression of autophagy protein Beclin 1. Activation of predominant lysosomal aspartic protease, LAMP-1 and cathepsin D, was demonstrated. Moreover, RNA interference targeting Beclin 1, inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine significantly suppressed the above process as well as the BGC-823 cells growth inhibition triggered by 12.5 μM E Platinum. Studies of mechanism revealed that E Platinum suppressed activation of mTOR and p70S6K by decreasing phosphorylation of Akt, ERK1/2, JNK and p38 involved in mitogen-activated protein kinase signaling. We supported new evidences for E Platinum as a promising antitumor agent, involving with autophagy induction., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

This study was purposed to investigate the reversal effect of gambogic acid (GA) on multidrug resistance of K562/A02 cells and its mechanism. The IC(50) (half maximal inhibitory concentration) of adriamycin (ADM) was evaluated by MTT. Cell apoptosis was detected by flow cytometry. Morphological changes of K562/A02 cells were observed by fluorescent microscopy with DAPI staining. The expressions of Survivin and P-gp were determined by Western blot. The results showed that the IC(50) of ADM on K562 and K562/A02 cell proliferation were (1.42 ± 0.07) µg/ml and (28.42 ± 1.40) µg/ml respectively. GA ≤ 0.0625 µmol/L had no inhibitory effect on proliferation of K562 and K562/A02. 0.0625 µmol/L GA could enhance the sensitivity of K562/A02 cells to ADM (P < 0.05) and the reversal multiples was 1.53. The apoptotic rate was raised after treating with ADM combined with 0.0625 µmol/L GA for 48 h (P < 0.05). Morphological differences were typical and obvious between cells of control and treated groups under fluorescence microscopy using DAPI staining. After treating K562/A02 cells with ADM combined with 0.0625 µmol/L GA for 48 h, the expressions of Survivin and P-gp were down-regulated at protein levels. It is concluded that GA can enhance the sensitivity of K562/A02 cells to ADM, which may be related to increasing cell apoptosis and down-regulating expressions of Survivin and P-gp.

In this study, oroxylin A showed strong reversal potency in BEL7402/5-FU cells and the reversal fold (RF) reached 4.69. Simultaneously, rhodamine-123 accumulation assay and flow cytometry analysis demonstrated oroxylin A could increase drug accumulation. When combined with oroxylin A, 5-FU showed inducing apoptosis effect more seriously in DAPI staining experiment. Moreover, the mRNA and protein expression of multi-drug resistance gene (MDR1) were also decreased by oroxylin A. Further experiments exhibited that oroxylin A can downregulate P-gp expression through inhibiting nuclear factor-κB (NF-κB) signaling pathway, which might be the mechanism of reversal resistance of oroxylin A. In summary, oroxylin A could be a good candidate for the development of new MDR reversal agent and its reversal mechanism probably due to the suppression of P-gp expression via inhibiting NF-κB signaling pathway., (Copyright © 2011 Wiley Periodicals, Inc.)

Objectives: This study investigated the antiproliferative and apoptotic activities of CPUYJ039, a newly synthesized benzimidazole-based kinesin spindle protein (KSP) inhibitor, on HCT116 cell lines., Methods: KSP-inhibitory activity was tested using the malachite-green method. The in-vitro cell proliferation inhibitory activity of the sample was measured using WST reagent. Flow-cytometric evaluation of cellular DNA content was performed. Apoptotic cells were quantified by annexin V-FITC-PI double staining. To confirm that the cytotoxic activity was a consequence of KSP inhibition, microtubule morphology and DNA segregation were observed by double staining of microtubules and DNA. The expression of Bcl-2 and Bax in CPUYJ039-treated HCT116 cells was detected by Western blotting., Key Findings: CPUYJ039 was evaluated and proved to have potent inhibitory activities in the KSP ATPase and HCT116 cell proliferation assays. CPUYJ039 inhibited the proliferation of HCT116 cells in a dose- and time-dependent manner and markedly induced G2/M phase cell-cycle arrest with characteristic monoastral spindles and subsequent cell death in HCT116 cells, which was associated with an increase of the Bax/Bcl-2 ratio., Conclusions: These results suggest that CPUYJ039 may be a novel inducer of apoptosis in HCT116 cells with potent KSP inhibitory activity., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Kinesin spindle protein (KSP) inhibitors are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the design, synthesis and biological evaluation of a novel series of 1,4-dihydroquinolin-4-ones and 1,2,3,4-tetrahydroquinazolin-4-ones using de novo design method. The synthesized compound was evaluated and proved to have potent inhibitory activities in the KSP ATPase. Compounds 15j and 15p show potent inhibitory activities in cell proliferation assays. Preferred compound 15j markedly induced G2/M phase cell cycle arrest with characteristic monoastral spindles and subsequent cell death in A549 cells. In vivo evaluation of 15j on the growth of transplantable S180 sarcoma in mice suggested its therapeutic potential for further development., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A new synthetic flavonoid DHF-18, synthesized with a piperazine substitution, has been recently found to show potent anti-tumor activities both in vivo and in vitro. In this study, we demonstrated that DHF-18 significantly inhibited tumor growth in mice inoculated with Heps hepatoma cells without evident toxicity. After the treatment of 40mg/kg DHF-18, the inhibitory rate of tumor weight was 53.69%. To investigate whether apoptosis induction contributed to the anti-tumor effects of DHF-18, DAPI (diamidino-phenyl-indole) staining and Annexin V/PI (Propidium iodide) double staining were performed in our tests. The data showed that DHF-18 could induce the apoptosis cell death in HepG2 cells. Moreover, the apparent increase of intracellular reactive oxygen species levels and the reduction of mitochondria ΔΨm were both observed in HepG2 cells after DHF-18 treatment. Meanwhile, the transposition of apoptotic inducing factor (AIF) from mitochondria to nuclei, the release of cytochrome c from mitochondria and the activation of caspase-3, -9 were also detected, indicating that DHF-18 may induce apoptosis through a mitochondrial-mediated pathway. Additionally, DHF-18 decreased the expression of Bcl-2 protein, whereas the levels of Bax and Bak were found to increase after DHF-18 treatment. Moreover, the activation of caspase-8, the increase of TNF-R1 (Tumor necrosis factor receptor) and Bid were found. Taken together, our results suggested that DHF-18 may induce HeG2 cells apoptosis through a mitochondrial-dependent and independent pathway., (Copyright © 2010. Published by Elsevier B.V.)

Traditional Chinese medicines have been recognized as a new source of anticancer drugs or chemotherapy adjuvant to enhance the efficacy of chemotherapy and to ameliorate the side effects. Wogonin (WOG) has a potential for therapeutic use in the treatment of antitumor and chemoprophylaxis. 5-Fluorouracil (5-FU) is a key systemic chemotherapy drug and widely use in the treatment of solid tumors. In this study, we found that combination of WOG and 5-FU inhibited the viability of MGC-803 cells in a concentration-dependent manner and exhibited a synergistic anticancer effect (CI<1) when 5-FU was used at relatively low concentrations. The pro-apoptotic activity of two-drug combination was much stronger than single. Furthermore, WOG could decrease the mRNA levels of dihydropyrimidine dehydrogenase (DPD), the metabolic enzymes of 5-FU. WOG could inhibit the NF-kappaB nuclear translocation and I-kappaB phosphorylation. Moreover, combined treatment caused significantly growth inhibition of human tumor xenografts. In addition, WOG markedly enhanced the antitumor activity of low dose 5-FU (i.p. 10mg/kg/day), however there is no toxicity and influence on diet consumption in experimental animals. Taken together, our data's showed that WOG increased 5-FU retention for a prolonged catabolism by modulating 5-FU metabolic enzymes and sensitized the MGC-803 cells to 5-FU induced apoptosis by inhibiting the NF-kappaB nuclear translocation. The anti-gastric cancer effect of two-drug combination was much stronger than that of WOG or 5-FU alone. These results may be relevant to design new clinical therapeutic strategies against gastric cancer in future., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Aim: To investigate the synergistic inhibitory effects of the combination of 5-fluorouracil (5-FU) with the natural flavanoid oroxylin A on human hepatocellular carcinoma cells HepG2 in vitro and on transplanted murine hepatoma 22 (H22) tumors in vivo and the preliminary mechanisms., Methods: The inhibitory effects of 5-FU combined with the natural flavanoid oroxylin A in vitro were detected by MTT assay and the effects in vivo were investigated by transplanted H22 mice model. DAPI staining and Annexin V/propidium iodide (PI) double staining were used to detect the cell morphological changes and apoptosis. The mRNA levels of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in HepG2 cells after oroxylin A and 5-FU combination treatment were observed by quantitative real-time PCR. Western blotting assay was used to reveal the expressions of apoptotic-inducing proteins P53, cleaved PARP, COX-2, Bcl-2, and pro-caspase3., Results: Oroxylin A in combination with 5-FU presented synergistic effect (CI<1) on HepG2 cells in vitro when the inhibitory rate was higher than 7.5%. The inhibitory rate on H22 murine solid tumor in vivo in the combination group was higher than monotherapy. 5-FU combined with oroxylin A exerted stronger apoptotic induction in HepG2 cells than either single drug treatment. Quantitative real-time PCR discovered the downregulation of TS mRNA and DPD mRNA in HepG2 cells after combination treatment. Western blotting assay revealed oroxylin A enhanced 5-FU-induced apoptosis in HepG2 cells by elevating the expressions of apoptotic-inducing proteins P53 and cleaved PARP and decreasing the expression of apoptotic-inhibitory proteins COX-2, Bcl-2, and pro-caspase3., Conclusion: The anti-hepatocellular carcinoma effects in vitro and in vivo of 5-FU and oroxylin A combinations were synergistic and oroxylin A increased the sensitivity of HepG2 cells to 5-FU by modulating the metabolic enzymes of 5-FU and apoptotic-related proteins.

Wogonin, a naturally occurring flavonoid, has been shown to have tumor therapeutic potential both in vitro and in vivo. To better understand its anticancer mechanism, we examined the effect of wogonin on human cervical carcinoma HeLa cells. In this study, we observed that G1 phase arrest was involved in wogonin-induced growth inhibition in HeLa cells. Over a 24 h exposure of HeLa cells to 90 micromol x L(-1) wogonin, the promoters of G1-S transition, including cyclin D1/Cdk4 and pRb, decreased within 12 h and E2F-1 depleted in the nucleus at the same time. As the G1 phase arrest developed, p53 and the Cdk inhibitor p21Cip1 elevated both at protein and mRNA levels. Furthermore, the up-regulation of p21Cip1 induced by wogonin was dramatically inhibited by siRNA-mediated p53 gene silencing. Collectively, our data suggested that wogonin induced G1 phase arrest in HeLa cells by modulating several key G1 regulatory proteins, such as Cdk4 and cyclin D1, as well as up-regulation of a p53-mediated p21Cip1 expression. This mechanism of wogonin may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.

Oroxylin A is a flavonoid isolated from the root of Scutellaria baicalensis Georgi. Our previous work demonstrated that the anti-tumor activity of oroxylin A was mainly attributed to its apoptosis inducing effect in cells. The present study explores the exact molecular mechanism of oroxylin A-induced apoptosis in tumor cells. We showed that oroxylin A-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. We also investigated which mitochondrial channels, PTP or MAC or both, were involved in the permeabilization of the mitochondrial outer membrane after treatment with oroxylin A. The results showed that oroxylin A-induced apoptosis in a PTP-independent manner; therefore, we focused our attention on MAC. As Bax is an essential constituent of MAC in certain systems, we examined the activation, subcellular location, oligomeric structure of Bax in HepG2 cells treated with oroxylin A. Moreover, our results showed that overexpression of Bcl-2 inhibited oroxylin A-induced apoptosis. In summary, we have demonstrated that opening of MAC, but not PTP, played a key role in oroxylin A-induced activation of mitochondrial apoptotic pathway in HepG2 cells., (2009 Elsevier Ireland Ltd.)

To identify and compare the features of stem like cells in human glioblastoma cell lines U251, U87MG, A172 with primary cultured glioblastoma stem cells, the ratio of CD133+ cells, the ability of tumor sphere formation, and self-renewing capacity of U251, U87MG, A172 cells in serum free medium plus EGF, bFGF and B27 supplement were detected. The results suggested that there might be more cancer stem like cells in U251 cells compared with others. CD133+ cells enriched in SP cells and in U251 cells cultured with the serum free medium. They expressed the neural stem cell markers CD133 and Nestin, but lacked of neuronal and astrocyte marker MAP2, beta-III tubulin and GFAP. They could apparently generate both neurons and glial cells after serum retrieved in vitro. Gli1, Bmi1, Notch2 and PTEN were also found expressed highly in them. Moreover, CD133+ cells were more resistant to hypoxia, irradiations and some chemotherapeutics than CD133-cells. So we suggested that glioblastoma stem like cells were existed in CD133+ cells in U251 cell line with characteristics of self-renew and generation of an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may be exploited in the development of novel cancer therapeutic drugs.

Because c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heterocycle amine-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, synthesized and evaluated as multiple targets agents in cancer therapy. All compounds were evaluated by two related enzyme inhibition assays and an anti-proliferation assay in vitro. The results showed that most compounds inhibited c-Src and iNOS well. The best compound 33 inhibited both enzymes with the IC50 values of 0.0484 micromol x L(-1) and 34.5 micromol x (-1), respectively. Some of the compounds also showed moderate anti-proliferation activities at 10 micromol x L(-1) against colon cancer HT-29 and liver cancer HepG2 cell lines.

Gambogic acid (GA) has been known to have antitumor activity in vitro and in vivo. In the present study, we investigated the anti-invasive effects of GA in MDA-MB-231 human breast carcinoma cells. The results indicated that GA significantly inhibited the adhesion, migration, and invasion of the cells in vitro tested by the heterotypic adhesion assay, wound migration assay, and chamber invasion assay. Results of Western blotting and immunocytochemistry analysis showed that GA could suppress the expressions of matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) in MDA-MB-231 cells. Furthermore, gelatin zymography revealed that GA decreased the activities of MMP-2 and MMP-9. Additionally, GA exerted an inhibitory effect on the phosphorylation of ERK1/2 and JNK, while it had no effect on p38. Taken together, our results demonstrated the anti-invasive property of GA for the first time and indicated it could serve as a promising drug for the treatment of cancer metastasis.

Wogonin, a natural monoflavonoid, has been shown to have tumor therapeutic potential in vitro and in vivo. Recently many studies have focused on the induction of apoptosis of tumor cells by wogonin. In this study, we found that wogonin could induce differentiation and G1 phase arrest of human U-937 leukemia cells. The growth of U-937 cells incubated with wogonin was inhibited in a time- and concentration-dependent manner. After treatment with wogonin, U-937 cells exhibited the characteristics of mature granulocytes, such as increased cytoplasmic-to-nuclear ratio, enhanced prominence of cytoplasmic granules, membrane ruffling, a higher NBT-reducing ability, and an increased expression of CD11b. Moreover, wogonin could induce G1 phase arrest and influenced the expression of associated proteins. For example, the expression of phorsphorylated protein kinase C (PKC) delta, p21 increased, while that of cyclin D1/cyclin-dependent kinase (CDK) 4, p-Rb decreased. The upregulation of p21 could be reversed by rottlerin, an inhibitor of PKCdelta. Taken together, wogonin induced U-937 cells to undergo granulocytic differentiation and G1 phase arrest via PKCdelta phosphorylation-induced upregulation of p21 proteins.

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.

Because both c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heteroarylamino-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, prepared, and evaluated for blocking multiple signaling pathways in cancer therapy. All compounds were evaluated by two related enzyme inhibition assays and an anti-proliferation assay in vitro. The results showed that most compounds could inhibit both enzymes, and several of them showed potent inhibition activity against different cancer cell lines. The best compound 20 (CPU-Y020) showed the IC(50) values of 6.58 and 7.61microM toward colon cancer HT-29 and liver cancer HepG2 cell lines.

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.

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 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.

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.

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.

Aim: 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., Methods: 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., Results: 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., Conclusion: 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.

Aim: To study the inhibitory effect of general gambogic acids (GGA) on transplantation tumor SMMC-7721 in experimental animal model and SMMC-7721 cells in vitro., Methods: Anti-tumor activity of GGA in the experimental transplantation tumor SMMC-7721 was evaluated by relative tumor growth ratio. Cell morphology was observed with inverted microscope and electron microscope. Cell proliferation was measured by MTT assay and the telomerase activity was determined by PCR., Results: In vivo study indicated that GGA (2, 4, and 8 mg/kg, iv, 3 times per week for 3 weeks) displayed an inhibitory effect on the growth of transplantation tumor SMMC-7721 in nude mice compared with the normal saline group (P<0.01). At the concentrations of 0.625-5.0 mg/L, GGA remarkably inhibited the proliferation of SMMC-7721 cells in vitro. GGA 2 mg/L dramatically changed morphology of SMMC-7721 cells and inhibited the telomerase activity in SMMC-7721 cells., Conclusion: GGA had inhibitory effect on the growth of SMMC-7721, which might be related to its inhibition of telomerase activity.

Aim: To investigate the antitumor effect of recombinant L-asparaginase on the growth of several tumors such as P388, L1210, hepatocellular carcinoma (Heps), K562, P815, and sarcoma 180 (S180)., Methods: Tumor cells (K562, L1210, and P815) were cultured in vitro and the morphology of those cells was observed with inverse microscope and transmission electron microscope. MTT assay was performed to measure the cell proliferation and inhibition rate. DNA content was assayed by flow cytometry. According to protocols of transplant tumor research, mice were transplanted with tumor cells L1210, P388, Heps, and S180. The survival rate and weight of tumor were observed after the treatment of test drugs., Results: The antitumor effects of L-asparaginase were observed in vitro with tumor cells K562, L1210, and P815 (P <0.01). In vivo experiments showed that ip administration of L-asparaginase significantly increased the survival rate and life span of mice with P388 or L1210 tumor cells (P <0.01). Tumor growth induced with Heps was also significantly suppressed. Furthermore, significant suppression of tumor growth was observed in mice induced with Heps and S180 by iv administration of L-asparaginase., Conclusion: Recombinant L-asparaginase markedly inhibited tumors tested in this study and the results strongly suggest that recombinant L-asparaginase has great potential for clinical treatment of these tumors.