Comparative Study on the Content and Cytotoxicity of Pseudolaric Acid B in the Five Plant Parts of Pseudolarix kaempferi

Pseudolaric acids of Pseudolarix kaempferi (Pinaceae) have been known as diterpenoids with potent anti-fungal-, anti-microbial, and cytotoxic activities. In the present study, the five MeOH extracts were prepared from the five plant part (root bark, stem bark, leaf, the inner part of root, and cone) to find the relation between the concentration of pseudolaric acids and cytotoxicity. Pseudolaric acids B and C were isolated from the root bark of P. kaempferi to use them as standard compounds. The five extracts were tested on cytotoxicity against six cancer cell lines, A549 (lung), HCT116 (colon), MDA-MB-231 (breast), SNU638 (stomach), and SK-hep-1 (liver) by SRB assay, but against K562 (leukemia) by SRB- or MTT assay. HPLC quantification were performed on a Shisheido Capcell PAK C18 column (5 µm, 4.6 mm × 250 mm) using 254 nm wavelength. The cytotoxicity (IC₅₀, 0.36 µg/ml on K562 cell lines) of the root bark extract was potent and the content (101.1 mg/g extract) of pseudolaric acid B was very high in the root bark. These results suggest that the MeOH extract obtained from the root bark could be developed as the anti-cancer agent with a high quantity of pseudolaric acid B.

Pseudolaric acid B induces glioblastoma cell line U87 mitotic arrest and apoptosis / 中国病理生理杂志

AIM: To investigate the effects of pseudolaric acid B on the growth and apoptosis of glioblastoma cell line U87.METHODS: The cell morphological changes were observed under inverted microscope.The cell viability was evaluated by MTS assay.The cell cycle was analyzed by flow cytometry and Western blot.The cell apoptosis was de-tected by flow cytometry.The changes of apoptosis-related proteins cleaved PARP, caspase-3, procaspase-9 and caspase-8 were determined by Western blot.RESULTS: Pseudolaric acid B inhibited the viability of U87 cells, arrested U87 cells in mitosis.Apoptosis of U87 cells was induced by pseudolaric acid B.The caspase pathway was activated.CONCLUSION:Pseudolaric acid B induces glioblastoma cell line U87 mitotic arrest and apoptosis.

The Effect of Pseudolaric Acid on the Expression of VEGF in Hepatocellular Carcinoma Cells / 天津医药

Objective To investigate the effect of pseudolaric acid B(PAB) on the expression of vascular endothelial growth factor (VEGF) in human hepatoma BEL-7402 cells. Methods BEL-7402 cells were cultured with PAB (0, 0.5, 1.0, 2.0, 4.0 and 8.0μmol · L-1) for 6 h and stained with 0.4%trypan blue. The cell viability was observed and calculated under the microscope. The cell proliferation was detected by WST-8 analysis. The expression of VEGF mRNA in BEL-7402 cells was detected using RT-PCR method. The expression of VEGF protein in BEL-7402 cells was detected using Western blot assay. Results There was no significant difference in the cell proliferation of BEL-7402 cells between different PAB treat-ment groups and control group. With the increased concentration of PAB, the expressions of VEGF mRNA were significantly decreased in different PAB treatment groups (1.90±0.02,1.31±0.03,0.86±0.04,0.60±0.03 and 0.34±0.02) compared with that of control group (2.53±0.07, P＜0.01). The expressions of VEGF protein were significantly decreased in different PAB treat-ment groups (2.60±0.09, 2.42±0.07, 1.85±0.05, 0.92±0.05 and 0.60±0.04) compared with that of control group (3.06±0.15, P＜0.01). Conclusion PAB can inhibit the expressions of VEGF mRNA and protein in BEL-7402 cells.

Pharmacodynamic analysis and related mechanisms of pseudolaric acid B on contact hypersensitivity / 中国药理学通报

Aim To investigate the pharmacodynamic experiment and molecular mechanisms of a diterpenoid from cortex pseudolaricis, pseudolaric acid B ( PB ) , on immunoregulation. Methods The mouse models of contact hypersensitivity ( CHS) were induced by 2,4-dinitrofluorobenzene ( DNFB ) . Then , the ear swelling and spleen index were measured after administered o-rally with PB. The pathological changes such as in-flammatory cell infiltration in ear skin were observed by hematoxylin and eosin ( HE ) staining. Besides, the expression of peroxisome proliferater-activated receptorγ ( PPARγ) and the phosphorylation of Akt were ana-lyzed by Western blot. The activity of PPARγ was fur-ther detected by luciferase reporter gene assay. Results The results showed that PB could both alleviate the ear thickness, inhibit the spleen index, and reduce the inflammatory degree of their ear skin, which might be involved in inducing PPARγexpression and activation, associated with suppressing Akt signaling pathway. Conclusion It is suggested that PB might regulate PPARγ-related Akt pathway, which indicates the pos-sibility of developing PB as a novel immunoregulation agent for treating inflammatory-immune disease.

Inhibitory effect of pseudolaric acid b on proliferation of rat glioma c6 cells / 第二军医大学学报

Objective To observe the inhibitory effects of pseudolaric acid B (PAB) on the proliferation of rat glioma C6 cells in vivo and in vitro. Methods MTT assay was used to examine the inhibitory effect of 0, 0.1, 0.2, 0.4, 0.8,1.6,8, and 16 μg/ml PAB on C6 cells; the distribution of cell cycle was determined by flow cytometry. C6 cells were injected subcutaneously to establish glioma-bearing rat model and the tumor volume was measured. The expression of proliferating cell nuclear antigen (PCNA) was detected immunohistochemically in the implanted glioma. Results In vitro results showed that PAB inhibited C6 cell growth in a dose-dependent manner, with the IC50 being 0.03 μg/ml. It also arrested the cells in G2/M phase. In vivo results showed that the tumor size of PAB group was decreased compared with the control group; moreover, PCNA expression was also decreased by PAB. Conclusion PAB can inhibit rat glioma C6 cell growth, which might be associated with cell arrest in G2/M phase and down-regulation of PCNA expression.

Pseudolaric acid B inhibits growth of human gastric carcinoma cells in vitro / 基础医学与临床

Objective To investigate the effect of pseudolaric acid B(PLAB) on growth of human gastric carcinoma cells in vitro.Methods The expression of PPAR? was detected by RT-PCR;the effect of PLAB on cell growth was tested by MTT;Hoechst33342/PI and DNA gel electrolysis were employed to examine apoptosis;cell cycle was checked by flow cytometry.Results When treated with 0.1～10 ?mmol/L PLAB for 72,the proliferation of MGC803 cells was significantly inhibited.The proportion of MGC803 cells at G2 phase was significantly increased when treated with 10 ?mmol/L PLAB after 48 h,and showed an apparent G2 phase arrest.After treatement with PLAB for 72,typical apoptotic changes were observed.The expression of PPAR? was at a low level in MGC803 cells and up-regulated when treated with 10 ?mmol/L PLAB for 48 h(P

Involvement of JNK-initiated p53 accumulation and phosphorylation of p53 in pseudolaric acid B induced cell death

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.

Pseudolaric acid B induces apoptosis via activation of c-Jun N-terminal kinase and caspase-3 in HeLa cells

Pseudolaric acid B was isolated from Pseudolarix kaempferi Gordon (Pinaceae) and was evaluated for the anti-cancer effect in HeLa cells. We observed that pseudolaric acid B inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. HeLa cells treated with pseudolaric acid B showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. JNK inhibitor, SP600125, markedly inhibited pseudolaric acid B-induced cell death. In addition, Bcl-2 expression was down-regulated while Bax protein level was up-regulated. Caspase-3 inhibitor, z-DEVD-fmk, partially blocked pseudolaric acid B-induced cell death, and the expression of two classical caspase substrates, PARP and ICAD, were both decreased in a time- dependent manner, indicative of downstream caspase activation.

Pseudolaric acid B induced apoptosis and mitotic arrest circumventing Fas receptor pathway in MCF-7 cells / 中国药理学通报

Aim To study the mechanisms of Pseudolaric acid B(PAB)-induced MCF-7 cell apoptosis and mitotic arrest.Methods MTT assay was performed to assess the cell growth inhibition,contrast phase microscope was used to observe cellular morphologic alteration,and the change of DNA was detected by fluorescent microscopy.The distribution of cell cycle was determined by flow cytometric analysis of propidium iodide staining,and the protein expression was examined by Western blot analysis.Results PAB inhibited MCF-7 cell growth in a dose-and time-dependent manner.4 ?mol?L-1 PAB induced DNA condensation at 24 h.PAB cleaved PARP in a time-dependent manner.At 36 h,PAB up-regulated the expression of cdc 2 and nuclear cyclin B1.Fas antagonistic antibody UB2 had no effect on apoptosis,but agonistic antibody CH11 enhanced the apoptosis induced by PAB.UB2 exerted no effect on cell cycle arrest,and CH11 had the same action as UB2 except for reducing the mitotic arrest through enhancing apoptotic subdiploid peak.Conclusion PAB inhibited MCF-7 cell growth through mitotic arrest and apoptosis.Apoptosis and mitotic arrest were independent of Fas pathway.

Effects of pseudolaric acid B on the metastatic activity and invasiveness of HeLa cells and the expression of matrix metalloproteinase / 解放军医学杂志

Objective To investigate the effects of pseudolaric acid B(PAB) on the invasiveness and metastatic activity of cervical cancer HeLa cells in vitro and the possible mechanism.Methods HeLa cells were divided into 5 groups: blank control group,DDP 3.125?g/ml group,20?mol/L PAB group,10?mol/L PAB group and 5?mol/L PAB group.The effect of PAB on invasive ability of HeLa cells was observed with a Transwell cell culture chamber.The mRNA expression of MMP22 and MMP29 in HeLa cells was detected by RT-PCR and the protein expression was detected by Western blotting.Results The average number of invaded HeLa cells was 15.60?2.45 in 20?mol/L PAB group and 20.14?2.48 in 10?mol/L PAB group,which was significantly lower than those in control group(46.50?4.70) and PAB 5?mol/L group(42.44?5.80,P

Effect of pseudolaric acid B on proliferation of human LiBr cell line in vitro / 中草药

Object To study the inhibitory effects and mechanism of pseudolaric acid B (PAB) on the growth of LiBr cell line. Methods PAB in different concentrations was added into the medium in which LiBr cells were cultured. The changes of cell morphology were observed by phase contrast microscope. Cytotoxicity of PAB was detected by MTT. The number of apoptotic cell was calculated by double fluorescent staining. The level of p21 WAF1 protein was measured with immunocytochemistry. Results The growth of LiBr cell line was remarkably inhibited by PAB. The IC 50 values of PAB for LiBr cells was 2.5?10 -5 mol/L. The inhibitory rate and the apoptotic cell rate were related with the medicine concentration. The level of p21 WAF1 protein in LiBr cells treated with PAB was obviously increased. Conclusion PAB can effectively inhibit the proliferation of LiBr cell line.