Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Bioorganic and Medicinal Chemistry Letters

Volumn 16, Issue 17, 2006, Pages 4575-4580

  Cheng, Guang ^a   Zhang, Yun ^b   Zhang, Xiang ^a   Tang, Hai Feng ^a   Cao, Wei Dong ^a   Gao, Da Kuan ^a   Wang, Xi Ling ^a  

^a FOURTH MILITARY MEDICAL UNIVERSITY   (China)

^b FOURTH MILITARY MEDICAL UNIVERSITY   (China)

Author keywords

Apoptosis; Bax; Bcl 2; Human glioblastoma; Tubeimoside V (1); U87MG cell

Indexed keywords

ANTINEOPLASTIC AGENT; LIPOCORTIN 5; PHOSPHATIDYLSERINE; PLANT EXTRACT; PROTEIN BAX; PROTEIN BCL 2; TUBEIMOSIDE V; UNCLASSIFIED DRUG;

ANTINEOPLASTIC ACTIVITY; APOPTOSIS; ARTICLE; BOLBOSTEMMA PANICULATUM; CANCER CELL CULTURE; CELL CYCLE; CELL PROLIFERATION; CHEMOTHERAPY; CONCENTRATION RESPONSE; CONTROLLED STUDY; CYTOTOXICITY; DNA DETERMINATION; DOSE RESPONSE; ELECTRON MICROSCOPY; EXPOSURE; FLOW CYTOMETRY; FLUORESCENCE; GLIOBLASTOMA; HUMAN; HUMAN CELL; IC 50; MEDICINAL PLANT; PLANT TUBER; POLYMERIZATION; PROTEIN EXPRESSION; WESTERN BLOTTING;

ANTINEOPLASTIC AGENTS; APOPTOSIS; CELL LINE, TUMOR; CELL SHAPE; CUCURBITACEAE; GLIOBLASTOMA; HUMANS; MICROSCOPY, ELECTRON; MOLECULAR STRUCTURE; PROTO-ONCOGENE PROTEINS C-BCL-2; SAPONINS; TRITERPENES;

BOLBOSTEMMA PANICULATUM;

EID: 33746196497     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.06.020     Document Type: Article

References (29)

1. Boudreau C.R., Yang I., and Liau L.M. Surg. Neurol. 64 (2005) 286
2. Glinski B., Dymek P., and Skolyszewski J. J. Neurooncol. 36 (1998) 159
3. Fine H.A., Dear K.B., Loeffler J.S., Black P.M., and Canellos G.P. Cancer 71 (1993) 2585
4. Curtin J.F., King G.D., Candolfi M., Greeno R.B., Kroeger K.M., Lowenstein P.R., and Castro M.G. Curr. Top. Med. Chem. 5 (2005) 1151
5. Kasai R., Miyakoshi M., Matsumoto K., Nie R.L., Zhou J., Morita T., and Tanaka O. Chem. Pharm. Bull. 34 (1986) 3974
6. Yu L., Yu T., and Ma R. Carcinogenesis 16 (1995) 3045
7. Yu T.X., Ma R.D., and Yu L.J. Acta Pharmacol. Sin. 22 (2001) 463
8. Yu L.J., Ma R.D., Wang Y.Q., Nishino H., Takayasu J., He W.Z., Chang M., Zhen J., Liu W.S., and Fan S.X. Int. J. Cancer 50 (1992) 635
9. Tang H.F., Yi Y.H., Zhang S.Y., Sun P., LI L., and Zhou D.Z. Chin. Chem. Lett. 16 (2005) 479
10. 3 cells per well were seeded in 96-well plates. Different concentrations of this were then added to the cells 24 h after seeding. The cells were continuously treated for 72 h and then the MTT assay was performed as described.
11. Cell cycle analysis. U87MG cells were trypsinized, counted, centrifuged, and fixed in ethanol at certain time points after the treatment. Then cells were washed twice in PBS and centrifuged. Pellet was resuspended in a solution of RNase (0.02 mg/ml, Sigma, USA) and propidium iodide (PI, 0.02 mg/ml, Sigma, USA), and incubated at 4 °C for 30 min. Fluorescence of stained cells was measured for approximately 10,000-20,000 cells. Data were collected and analyzed. Results were expressed as a plot of fluorescence intensity vs cell number.
12. Hoechst staining. For apoptosis assays, apoptosis was analyzed after staining of the cells with Hoechst 33342 (Sigma-Aldrich) and subsequent fluorescence microscopy. In brief, cells were incubated with Hoechst 33342 at a final concentration of 1.5 μM for 10 min. Cell morphology was then determined by fluorescence microscopy (Zeiss Axiovert 200, Carl Zeiss, Germany). Cells were analysed with 40× magnification and documented using a CCD camera device (Zeiss Axiocam MR).
13. Leist M., and Jaattela M. Nat. Rev. Mol. Cell Biol. 2 (2001) 589
14. Lovborg H., Nygren P., and Larsson R. Mol. Cancer Ther. 3 (2004) 521
15. Electron microscope. The U87MG cell line was plated in 35 mm dishes and allowed to incubate overnight. Aliquots of Tubeimoside V (1) (10.0 μg/ml) were added into the culture dish for 24 h. At the end of incubation, cell samples were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), postfixed in 2% buffered osmium tetroxide for 2 h and dehydrated in ethanol. Specimens for transmission electron microscopy were embedded in epon. Thin sections were cut on an ultramicrotome and double stained with uranyl acetate and lead citrate. Electron micrographs were taken on an electron microscope (JEM-2000EX, JEOL, Japen) operating at 80 kV.
16. Obrero M., Yu D.V., and Shapiro D.J. J. Biol. Chem. 277 (2002) 45695
17. Hao L.L., Mei Q.B., Zhang B.L., Jia M., Li X.Q., and Zhang F. Acta Pharmacol. Sin. 25 (2004) 1509
18. 2). Thereafter, 5 μl of annexin V-FITC (BD Pharmingen) and 10 μl of propidium iodide (50 μg/ml) were added to 100 μl of cell suspension and incubated for 15 min at room temperature protected from light. Finally 400 μl of binding buffer was added to the samples and handled ice-cold until they were analyzed on FACSCalibur (Becton-Dickinson) flow cytometer. Ten thousand cells were analysed per sample.
19. Kuo M.L., Huang T.S., and Lin J.K. Biochim. Biophys. Acta 1317 (1996) 95
20. Liu D., Yang B., Cao R., and Huang Z. Bioorg. Med. Chem. Lett. 15 (2005) 4467
21. Western blot analysis. Protein extracts of U87MG cells were prepared by lysing cells in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris HCl, pH 8), 10 mM EDTA, and 1 mM PMSF (Sigma, USA) for 30 min at 4 °C. Samples were then centrifuged for 15 min at 15,000g. Protein concentration in supernatant was determined. For each sample, 60 μg of protein was loaded on 12.5% SDS-polyacrylamide gel, electrophoresed, and transferred to nitrocellulose membrane (0.22 μm, Protran, Schleicher and Schuell). Membrane was blocked for 1 h at the room temperature with blocking buffer (TBS containing 0.1% Tween 20 (Sigma, USA) and 5% milk). Primary antibodies (applied for 1 h at room temperature, or overnight at 4 °C) were: anti-Bcl-2 (mouse monoclonal Ab-1, Oncogene), anti-Bax (rabbit polyclonal N-20, Santa Cruz), and anti-actin (mouse monoclonal C-2, Santa Cruz). They were diluted 1:1000, except: anti-actin (1:500). Thereafter, membranes were incubated for 1 h with HRP-labeled secondary antibodies (Amersham Pharmacia Biotech, Sweden): sheep anti-mouse (diluted 1:2500) and donkey anti-rabbit (diluted 1:5000), and then developed by an ECL system according to the manufacturer's instructions (Amersham).
22. Shackelford R.E., Kaufmann W.K., and Paules R.S. Environ. Health Perspect. 107 (1999) 5
23. Engeland M.V., Ramaekers F.C., Schutte B., and Reutelingsperger C.P. Cytometry 24 (1996) 131
24. Hockenbery D. J. Cell Sci. 18 (1994) 51
25. Bernardi P., Broekemeier K.M., and Pfeiffer D.R. J. Bioenerg. Biomembr. 26 (1994) 509
26. Reed J.C., and Paternostro G. Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7614
27. Tsujimoto Y. J. Cell Physiol. 195 (2003) 158
28. Filomeni G., Aquilano K., Rotilio G., and Ciriolo M.R. Cancer Res. 63 (2003) 5940
29. Nakagawa H., Tsuta K., Kiuchi K., Senzaki H., Tanaka K., Hioki K., and Tsubura A. Carcinogenesis 22 (2001) 891