Jing-Jing Rong

Microtubule depolymerization and phosphorylation of c-Jun N-terminal kinase-1 and p38 were involved in gambogic acid induced cell cycle arrest and apoptosis in human breast carcinoma MCF-7 cells

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.

Wogonin potentiates the antitumor effects of low dose 5-fluorouracil against gastric cancer through induction of apoptosis by down-regulation of NF-kappaB and regulation of its metabolism

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 datas 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.

Gambogic acid down-regulates MDM2 oncogene and induces p21Waf1/CIP1 expression independent of p53.

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.

Gambogic acid‐induced degradation of mutant p53 is mediated by proteasome and related to CHIP

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. J. Cell. Biochem. 112: 509–519, 2011.

Involvement of p53 in oroxylin A‐induced apoptosis 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.

MAC related mitochondrial pathway in oroxylin A induces apoptosis in human hepatocellular carcinoma HepG2 cells

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.

Synergistic effect of 5-fluorouracil with gambogic acid on BGC-823 human gastric carcinoma

The design of novel targeted or combination therapies may improve treatment options for gastric cancer. In this study, we determined the inhibitory effects of 5-fluorouracil (5-FU) combined with gambogic acid (GA) on BGC-823 human gastric carcinoma cells in vitro and in vivo and investigated the underlying mechanisms. 5-FU combined with GA inhibited the viability of BGC-823 human gastric cells in a concentration-dependent manner. The pro-apoptotic activity of the two-drug combination was much stronger than single. Furthermore, the results showed GA could regulate the metabolic enzymes of 5-FU. GA decreased the mRNA levels of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD), while increased the mRNA level of orotate phosphoribosyltransferase (OPRT). Moreover, combined treatment caused significantly growth inhibition of human tumor xenografts in vivo. Taken together, our data showed that GA attenuated 5-FU-induced apoptosis by modulating metabolic enzymes of 5-FU and the antigastric cancer effect of two drugs combination was much stronger than that of GA or 5-FU alone.

Different apoptotic effects of wogonin via induction of H2O2 generation and Ca2+ overload in malignant hepatoma and normal hepatic cells

Wogonin, a major active constituent of Scutellaria baicalensis, possesses potent anticancer activities both in vivo and in vitro. This paper describes the different apoptotic effects of wogonin in HepG2 and L02 cells and the possible mechanism for the differences. Through DAPI staining, Annexin‐V/PI double‐staining assay, JC‐1 detection and the expressions of the key apoptotic proteins, we find that wogonin prefers to induce apoptosis in HepG2 cells through the mitochondrial pathway, while has much less effects on L02 cells. Moreover, overexpression of Bcl‐2 can block wogonin‐induced apoptosis in HepG2 cells. To illustrate the specific selective mechanism of wogonin in apoptosis induction, H2O2, ·O  2− and Ca2+ are measured by 2′,7′‐dichlorfluorescein‐diacetate, dihydroethidium and Flou‐3 AM assay, respectively. The results show that the different apoptotic effects of wogonin in HepG2 and L02 cells are due to the different regulations to the redox balance of reactive oxygen species and the Ca2+ release from endoplasmic reticulum. IP3R‐sensitive Ca2+ channels are the key targets of the wogonin‐increased H2O2. Besides, the activation of PLCγ1 plays as a bridge between H2O2 signal molecules and Ca2+ release. Taken together, wogonin preferentially kills hepatoma cells by H2O2‐dependent apoptosis triggered by Ca2+ overload. The results reveal that wogonin is a competitive anticancer drug candidate for the malignant hepatoma therapy. J. Cell. Biochem. 111: 1629–1641, 2010.

LYG-202, a new flavonoid with a piperazine substitution, shows antitumor effects in vivo and in vitro

LYG-202 is a newly synthesized flavonoid with a piperazine substitution. We investigated the antitumor effect of LYG-202 in vivo and in vitro. We show that, LYG-202 significantly decreases tumor growth in mice inoculated with S180 sarcoma cells, compared with the control group. Meanwhile, the viabilities of various kinds of tumor cells were inhibited by LYG-202 with IC(50) values in the range of 4.80 to 27.70 microM. Then apoptosis induced by LYG-202 in HepG2 cells was characterized by DAPI staining and Annexin V/PI double staining and degradation of PARP was observed. Activation of the caspase cascade for both the extrinsic and intrinsic pathways was demonstrated, including caspase-8, -9, and -3. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that LYG-202 exhibited strong antitumor effect in vivo and in vitro, involving with apoptosis induction.

DHF-18, a new synthetic flavonoid, induced a mitochondrial-mediated apoptosis of hepatocarcinoma cells in vivo and in vitro

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.