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. 2010 Oct 15;127(8):1758-68.
doi: 10.1002/ijc.25210.

Flavokawain B, a kava chalcone, induces apoptosis via up-regulation of death-receptor 5 and Bim expression in androgen receptor negative, hormonal refractory prostate cancer cell lines and reduces tumor growth

Yaxiong Tang  1 , Xuesen Li, Zhongbo Liu, Anne R Simoneau, Jun Xie, Xiaolin Zi
Affiliations

Flavokawain B, a kava chalcone, induces apoptosis via up-regulation of death-receptor 5 and Bim expression in androgen receptor negative, hormonal refractory prostate cancer cell lines and reduces tumor growth

Yaxiong Tang et al. Int J Cancer. .

Abstract

Limited success has been achieved in extending the survival of patients with metastatic and hormone-refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. We have shown that flavokawain B (FKB), a kava chalcone, is about 4- to 12-fold more effective in reducing the cell viabilities of androgen receptor (AR)-negative, HRPC cell lines DU145 and PC-3 than AR-positive, hormone-sensitive prostate cancer cell lines LAPC4 and LNCaP, with minimal effect on normal prostatic epithelial and stromal cells. FKB induces apoptosis with an associated increased expression of proapoptotic proteins: death receptor-5, Bim and Puma and a decreased expression of inhibitors of apoptosis protein: XIAP and survivin. Among them, Bim expression was significantly induced by FKB as early as 4 hr of the treatment. Knockdown of Bim expression by short-hairpin RNAs attenuates the inhibitory effect on anchorage-dependent and -independent growth and caspase cleavages induced by FKB. These findings suggest that the effect of FKB, at least in part, requires Bim expression. In addition, FKB synergizes with TRAIL for markedly enhanced induction of apoptosis. Furthermore, FKB treatment of mice bearing DU145 xenograft tumors results in tumor growth inhibition and increases Bim expression in tumor tissues. Together, these results suggest robust mechanisms for FKB induction of apoptosis preferentially for HRPC and the potential usefulness of FKB for prevention and treatment of HRPC in an adjuvant setting.

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Figures

Figure 1
Figure 1
FKB is the most potent agent among flavokawains and their combinations against the growth of PCa cell lines and exhibits a differential effect on prostatic cells. Cells were treated with indicated treatment in the figure for 48 hours and cell densities were measured by MTT assay. The combination indexes (CIs) were calculated as described in Materials and Methods. Points, mean of four independent plates; bars, SE. (A) Growth inhibitory effects of flavokawain A, B, C and the 1:1/31:1/38 mixture of flavokawain A, B, and C on DU145 cells. (B) Graphical presentation of the CI with respect to the fraction of cell growth inhibition by mixtures of flavokawain A, B and C in DU145 cells. (C) Growth inhibitory effects of FKB on AR-positive and –negative PCa cell lines, as well as on normal prostatic cells. (D) & (E) time-dependent inhibitory effect of FKB on the growth of DU145 and PC-3 cells.
Figure 2
Figure 2
FKB induces apoptosis and activates caspase 3/8/9. (A) Live cell morphology under phase-contrast light microscope (Magnification: X100). A representative picture was shown from a random field. (B) DAPI staining of nuclear morphology under fluorescence microscope (Magnification: X400). A representative picture was shown from a random field. (C) Cells were stained by Annexin V and PI and analyzed by flowcytometry. Data represent the means from three independent experiments. Standard errors are less than 5%. (D) Caspase activation was determined with a caspase-3/7, caspase-9 or caspase-8 activity assay. Bars are means ± SE of three independent quantitative measures.
Figure 3
Figure 3
FKB induces mRNA and protein expression of DR5 and synergizes with TRAIL for enhanced cell growth inhibitory and apoptotic effect. (A) The expression of DR5 after indicated treatments for 24 hours was analyzed by Western blot. β-Actin was detected as a loading control. A representative blot was shown from three independent experiments. (B) Real-time RT-PCR analysis of DR5 mRNA expression. Bars are means ± SE of three independent quantitative measures. (C) The combined effect of FKB and TRAIL on PC-3 cell viability. Columns, mean for percentage of cell viability relative to control (n = 4); bars, SE. (D) The combined effect of FKB and TRAIL on apoptosis of PC3 cells. Cells were stained by Annexin V and PI and analyzed by flowcytometry. Data represent the means from three independent experiments. SEs are less than 5%.
Figure 4
Figure 4
FKB increases the expression of Bim, Puma and active Bax, decreases the expression of XIAP and survivin and induces a formation of Bim/Bax immunocomplex. (A) The protein levels of Puma, Bim, Active Bax, XIAP, and survivin after indicated treatments for 24 hours were analyzed by Western blotting. β-Actin was detected as a loading control. A representative blot was shown from three independent experiments. (B) Immunoprecipitation assay of the Bim/Bax complex. DU145 cell lysates after indicated treatments for 24 hours were incubated with conformation-specific anti-Bax 6A7 antibody and protein A-agarose beads and probed with anti-Bim antibody. A representative blot was shown from three independent experiments. (C) The protein levels of DR5, Puma, Bim, active Bax, XIAP, and survivin after 17.6 μM for indicated times were analyzed by Western blotting. β-Actin was detected as a loading control. A representative blot was shown from three independent experiments.
Figure 5
Figure 5
Knock-down of Bim expression by ShRNAs attenuates the anchorage-dependent and -independent growth inhibitory effects and caspase cleavage induced by FKB. PC-3 and DU145 cells were transfected with ShLacZ, ShBIM117, and ShBIM 459. Stable clones were obtained via G418 selection, and pooled clones for each line were used. (A) Western blotting verified knockdown of Bim expression. (B). PC3 or DU145/ShLacZ, PC3 or DU145/ShBim117, and PC3 or DU145/ShBim459 cells were treated with 0.1% DMSO or indicated concentrations of FKB for 48 hours. Cell viabilities were measured by MTT assays. Points: mean of four independent plates; bars: SE. Each sample was counted in duplicate. (C) PC3 or DU145/ShLacZ, PC3 or DU145/ShBim117, and PC3 or DU145/ShBim459 cells were grown in soft agar in six-well plates and treated with 0.1% DMSO or FKB at the indicated concentrations for 30 days. The number of colonies was determined by counting them under an inverted phase-contrast microscope at ×100 magnification; a group of >10 cells was counted as a colony. Columns: mean of four independent wells at 30 days after the start of cell seeding; bars: SE. (D) PC3/ShLacZ, PC3/ShBim117, and PC3/ShBim459 cells were treated with 0.1% DMSO or indicated concentrations of FKB for 24 hours. Cell deaths were measured by cell death ELISA kit. Caspase activation was determined with a caspase-3/7, caspase-9 or caspase-8 activity assay. Each result is expressed as a percentage relative to control. Bars are means ± SE of three independent quantitative measures.
Figure 6
Figure 6
FKB reduces tumor growth of DU145 cells in a xenograft model and decreases Bim expression in tumor tissues. (A) The mice bearing DU145 tumors were randomly divided, pair-matched into treatment and control groups of ten mice each, and daily dosing was begun with vehicle or FKB at 50 mg/kg. Tumor volumes were recorded, and presented as mean ± SE. (B) At the termination of the study, tumors were excised from each mouse in different groups and weighed. Wet weight of tumors is represented as mean of 10 tumors from individual mouse in each group. Bars, ± SE. (C) Immunostainings of Bim protein in DU145 xenograft tumors. Control immunostaining was performed with IgG isotype alone; Slides were counterstained with hematoxylin and photographed using a light microscope. Original magnification: X200 and X40 (insert). (D) Western blotting analysis of Bim expression in control (lanes, 1, 2, 3, 4, and 5) – or FKB (lanes 6, 7, 8, 9, and 10) - treated DU145 xenograft tumors.
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