Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC.

NCPMF-60 induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

We recently established that NCPMF-60, a newly synthesized flavonoid, is an active cytotoxic component. The molecular mechanisms by which NCPMF-60 exerts its cytotoxic activity are currently unknown. In this study, we show that NCPMF-60 induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. After treatment of HepG2 cells with NCPMF-60, cell cycle-related proteins, such as cyclin B1, cyclin H, CDK7, and p-CDK1 (Thr161), were downregulated, whereas p21 and p-CDK1 (Thr14/Tyr15) were upregulated. The activity of CDK1/cyclinB complex was also inhibited by NCPMF-60. In addition, we observed poly(ADP-ribose) polymerase cleavage and activation of caspase 3 and caspase 9. The expression ratio of Bax/Bcl-2 was increased in the treated cells, in which Bax was also upregulated. We also found that the expression of p53 and its phosphorylation at Ser15 accumulated after the treatment of NCPMF-60. Moreover, upregulation of p21, p53-upregulated modifier of apoptosis, and Bax, three p53-target gene products, and the downregulation of Bcl-2 and MDM2, were observed in NCPMF-60-treated cells. However, p53 is not the only regulator in the stimulation of NCPMF-60 on p21 transcriptional level and posttranscriptional level. These results suggested that NCPMF-60 indeed activated the p53 pathway, which may contribute to its induction of cell cycle arrest and apoptosis in HepG2 cells. Collectively, our findings show that cell cycle arrest and apoptosis induced by NCPMF-60 was associated with the activation of p53 pathway and the inhibition of CDK-activating kinase activity in HepG2 cells.

MAC-related mitochondrial pathway in oroxylin-A-induced 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.

Asparanin A induces G(2)/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

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 G(2)/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 p21(WAF1/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 p21(WAF1/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.

Apoptosis induction of oroxylin A in human cervical cancer HeLa cell line in vitro and in vivo.

Oroxylin A is a flavonoid that is found in the roots of Scutellaria baicalensis Georgi. Here, we investigated the antitumor effect of oroxylin A in human cervical cancer HeLa cell line in vitro and in vivo. We found that after inoculated with the HeLa cells the mice treated with oroxylin A showed a significant decrease of tumor volumes and tumor weight compared with the control. Meanwhile, the growth inhibition of oroxylin A on HeLa cells were observed by MTT assay and the value of IC(50) was 19.4+/-0.7 microM after treatment for 48h. Upon our previous research, the inhibition by oroxylin A might be through apoptosis. Then apoptosis induced by oroxylin A in HeLa cells was characterized by DAPI staining and Annexin V/PI double staining, and degradation of PARP (poly-ADP-ribose polymerase) was both found in HeLa cells and tumor tissue. Next, activation of the caspase cascade for both the extrinsic and intrinsic pathways were demonstrated in vivo and in vitro, including caspase-8, -9 and -3. We also found that the expression of Bcl-2 protein decreased, which leading to an increase of the Bax/Bcl-2 ratio. Our results showed that oroxylin A exhibited strong antitumor effect in HeLa cell line and apoptosis induction involved in it.