The extract of Hibiscus syriacus inducing apoptosis by activating p53 and AIF in human lung cancer cells.

Natural products including plants, microorganisms and marine life provide rich resources for anticancer drug discovery. The root bark of Hibiscus syriacus has been used as an antipyretic, anthelmintic and antifungal agent in Asia. The antiproliferative effects of H. syriacus on human lung cancer cells were evaluated with bio-assays. The apoptotic activity was detected by Hoechst 33342 DNA staining and annexin V staining. The expression of caspases, p53, apoptosis induced factor (AIF), Bcl-2 and Bax were evaluated with Western blotting. The in vivo anticancer activity was evaluated using A549-xenograft model. The acetone extract of H. syriacus (HS-AE) exhibited a better cytotoxic effect on lung cancer cells than its methanol extract (HS-ME) or water extract (HS-WE). The IC(50) values of HS-AE on A549 (adenocarcinoma), H209 (squamous cell carcinoma) or H661 (large cell carcinoma) lung cancer cells ranged from 14 to 22 microg/ml after 48 hours of treatment. After 48 hours of exposure, HS-AE (15 microg/ml) induced A549 cell apoptosis to 48 +/- 3.6% of the control. Using Western blotting, HS-AE appears to suppress the expression of p53 and AIF. The results of the in vivo study showed that HS-AE suppresses growth in A549 subcutaneous xenograft tumors. These results indicate that HS-AE exerts significant and dose-dependent antiproliferative effects on cancer cells in vitro and in vivo, which prompts us to further evaluate and elucidate the bioactive component(s) of H. syriacus.

The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo.

The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.

In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A549 cells.

We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (BS-AE) 60 microg/ml has anti-proliferation activity and apoptotic effects on A549 non-small cell lung cancer (NSCLC). A novel lignan, isochaihulactone (4-benzo[1,3]dioxol-5-ylmethyl-3(3,4,5-trimethoxyl-benzylidene)-dihydro-furan-2-one), was isolated from BS-AE and identified from spectral evidence ((1)H NMR, (13)C NMR, IR, and MS) and by comparison with authentic synthetic standards. Isochaihulactone was cytotoxic (IC(50)=10-50 microM) in a variety of human tumor cell lines. In in vitro and in vivo microtubule assembly assays, it inhibited tubulin polymerization in a concentration-dependent manner. As determined by flow cytometry, isochaihulactone caused G2/M phase arrest and apoptosis in a time- and concentration-dependent manner. G2/M arrest was correlated with increased p21/WAF1 levels, downregulation of the checkpoint proteins cyclin B1/cdc2 and mobility shift of cdc25C. Moreover, isochaihulactone (30 and 50 mg/kg) inhibited the growth of non-small cell lung carcinoma A549 xenograft in nude mice. These findings indicate isochaihulactone is a promising new antimitotic anticancer compound with potential for clinical application in the future.

Anti-proliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo.

Nan-Chai-Hu, the root of Bupleurum scorzonerifolium, is a traditional Chinese herb used in treatment of liver diseases such as hepatitis and cirrhosis. We recently reported that the acetone extract of B. scorzonerifolium (BS-AE) could inhibit proliferation and induce apoptosis in A549 human lung cancer cells. We further examined its anti-proliferative mechanisms and in vivo anticancer effect. Our results showed that BS-AE had the ability to cause cell cycle arrest in G2/M phase, inducing tubulin polymerization, and activating caspase-3 and -9 in A549 cells. BS-AE-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk in majority. The result of in vivo study showed that BS-AE could suppress growth in A549 subcutaneous xenograft tumors. These results indicate that BS-AE exerts antiproliferative effects on A549 cells in vitro and in vivo, and prompted us to further evaluate and elucidate the chemical composition profile of BS-AE.

Requirement for ERK activation in acetone extract identified from Bupleurum scorzonerifolium induced A549 tumor cell apoptosis and keratin 8 phosphorylation.

We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (AE-BS) 60 microg/ml has anti-proliferation activity and apoptosis effects to A549 human lung cancer cells. They can also cause tumor cell arrest in G2/M phase. To better understand its target protein in A549 cell, two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry were applied. The modification of keratin 8 was identified. By immunoblot, the expression of phosphorylated keratin 8 at Ser-73 was increased from 2.0 to 3.0-fold after AE-BS treatment 24 to 48 hr respectively as compared with untreated A549 control cells. Furthermore, the A549 cells were pretreated with 50 microM PD98059, a specific inhibitor of the upstream regulator of ERK1/2, or with the p38 kinase inhibitor 20 microM SB203580 or JNK inhibitor 20 microM SP600125 for 30 min, followed by 24 h of incubation with AE-BS, PD98059 can inhibit K8-Ser-73 hyperphosphorylation and prevented cell apoptosis which was induced by AE-BS significantly. By immunoblot, AE-BS also can induce ERK 1/2 phosphorylation. In conclusion, our data indicate that the AE-BS induced tumor apoptosis in A549 cells was related to ERK 1/2 activation. The molecular mechanism of hyperphosphorylation of K8 on Ser-73 was associated with ERK 1/2 activation rather than JNK and p38 kinase. The apoptosis induced by AE-BS may be related to K8 phosphorylation.

Acetone extract of Angelica sinensis inhibits proliferation of human cancer cells via inducing cell cycle arrest and apoptosis.

Angelica sinensis (Oliv.) Diels, a traditional Chinese medicine, has been widely prescribed in treatment of gynecological diseases. Bio-based assays for extracts of Angelica sinensis showed that the acetone extract (AE-AS) had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells. The IC50 values of AE-AS on mentioned cancer cells ranged from 35 to 50 microg/ml after 24 h of treatment. After 72 h of exposure, AE-AS (40 microg/ml) significantly reduced A549 cell proliferation to 24 +/- 3.2% of control. In A549 cells, the cell cycle analysis showed that AE-AS induced a significant increase in the number of cells in G0/G1, with a concomitant decrease in the number of cells in S phase. AE-AS-induced chromatin changes and apoptosis of A549 cells were confirmed by Hoechst 33342 DNA staining and annexin V staining. A549 cells treated with AE-AS caused activation of caspase-9 and -3, and AE-AS-induced apoptosis could be inhibited by the broad-spectrum caspase inhibitor, z-VAD-fmk. The Western blot indicated the AE-AS-triggered apoptosis is mediated via suppression of Bcl-2 oncoprotein expression rather than p53 or Bax. Besides, AE-AS decreased the levels of cdk4 protein was observed. These results indicate that the AE-AS could induce G1/S arrest and activate the mechanism of apoptosis in human cancer cells. Extracts obtained from different methods of fractionation might possess distinct bioactivity. These results prompted us to further evaluate the in vivo anticancer effects and elucidate the chemical composition profile of AE-AS.

Acetone extract of Bupleurum scorzonerifolium inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing telomerase activity.

Bupleuri radix, a traditional Chinese herb, has been widely used to treat liver diseases such as hepatitis and cirrhosis. The acetone extract of Bupleurum scorzonerifolium (AE-BS) showed a dose-dependently antiproliferative effect on the proliferation of A549 human lung cancer cells. The IC(50) of AE-BS, i.e., the concentration required to inhibit proliferation of A549 cells, was 59 +/- 4.5 microg/ml on day 1. The IC(50) of AE-BS for WI38 human normal lung fibroblast cells, however, was significant higher than that for A549 cells (150 +/- 16 microg/ml, p< 0.01). After 72 hours of exposure, AE-BS (60 microg/ml) significantly reduced A549 cell proliferation to 33 +/- 3.2% of control. In TUNEL assay, A549 cells treated with AE-BS showed typical morphologic features of apoptosis, and the percentage of apoptotic cells was approximately 38 % on day 1. In the TRAP assay, AE-BS-treated cells demonstrated significantly lower telomerase activity on day 3. This result indicates that the AE-BS could suppress the proliferation of lung cancer cells via inhibition of telomerase activity and activation of apoptosis.