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. 2016 Sep 12;7(13):1877-1887.
doi: 10.7150/jca.15512. eCollection 2016.

Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10 Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway

Jinyu Li  1 , Jinyao Li  1 , Adila Aipire  1 , Li Gao  2 , Shixia Huo  2 , Jiaojiao Luo  1 , Fuchun Zhang  1
Affiliations

Phenylethanoid Glycosides from Cistanche tubulosa Inhibits the Growth of B16-F10 Cells both in Vitro and in Vivo by Induction of Apoptosis via Mitochondria-dependent Pathway

Jinyu Li et al. J Cancer. .

Abstract

Cistanche tubulosa phenylethanoid glycosides (CTPG) have been shown various biological activities including anti-allergy, hepatoprotective activity and bone regeneration. However, the anti-tumor activity of CTPG needs to be investigated. CTPG was used to treat B16-F10 cells both in vitro and in vivo. We found that CTPG dramatically changed the morphology of B16-F10 cells, and significantly reduced the viability of B16-F10 cells in a dose-dependent and time-dependent manner, which might be mediated by CTPG-induced apoptosis and cell cycle arrest. After CTPG treatment, the expressions of BAX and BCL-2 were up-regulated and down-regulated, respectively. Moreover, mitochondrial membrane potential was reduced and ROS generation was increased. Consequently, the levels of cytochrome c and cleaved-caspase-3 and -9 were up-regulated by CTPG treatment but not for cleaved-caspase-8. We further observed that CTPG significantly inhibited the tumor growth in vivo and improved the survival rate of tumor mice. We also observed that CTPG promoted the proliferation of splenocytes and increased the proportions of CD4+ and CD8+ T cells in spleens of tumor mice. The results showed that CTPG induced the apoptosis of B16-F10 cells through mitochondria-dependent pathway, suggesting that CTPG could be a potential candidate for treatment of cancer.

Keywords: B16-F10 cell apoptosis; Cistanche tubulosa phenylethanoid glycosides; ROS; cytochrome c; mitochondria-dependent pathway..

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Conflict of interest statement

All authors declare that they have no conflict of interests.

Figures

Figure 1
Figure 1
The qualify control of CTPG. The components of CTPG were qualitatively and quantitatively analyzed by HPLC and compared to the standards of echinacoside, acteoside and isoacteoside.
Figure 2
Figure 2
The inhibitory effect of CTPG on the growth of B16-F10 in vitro. (A) The morphological changes of B16-F10 cells after 24 h of CTPG treatment. The magnification folders for upper panel and lower panel are 10 and 20, respectively. (B) The cell viability and (C) inhibitory rate of B16-F10 cells. 24, 48 and 72 h after CTPG treatment, cell viability was analyzed by MTT assay and the inhibitory rate was calculated. Data are from 4 independent experiments and analyzed by one-way analysis of variance. ** p < 0.01; *** p < 0.001 compared to control.
Figure 3
Figure 3
The apoptosis of B16-F10 and 293T cells induced by CTPG treatment. Different concentrations of CTPG were used to treat B16-F10 and 293T cells for 24 h. (A) The apoptosis and necrosis of B16-F10 and 293T cells were analyzed by flow cytometry. The upper panel showed the individual dot plots and the lower panel showed the summary data. (B) After 24 h, total protein was isolated and the expressions of BAX and BCL-2 were detected by western blot. (C) The apoptosis and necrosis of 293T cells were analyzed by flow cytometry. Data are from 3 independent experiments and analyzed by one-way analysis of variance. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to control.
Figure 4
Figure 4
The chromosomal condensation and cell cycle arrest of B16-F10 and H22 cells induced by CTPG treatment. After 24 h, cells were stained with Hoechst 33342 or PI, and then the apoptotic nuclear morphology of B16-F10 cells was observed using fluorescent microscopy (A) and cell cycle distribution in B16-F10 (B) and H22 cells (C) was analyzed by flow cytometry. The arrows indicated the chromosomal condensation. Data are from 3 independent experiments and analyzed by one-way analysis of variance. *** p < 0.001 compared to control.
Figure 5
Figure 5
The reduction of Δψm and up-regulation of cytochrome c and ROS. (A) After 48 h of CTPG treatment, Δψm was detected by JC-1 staining and samples were analyzed by flow cytometry. The overlay (left panel) shows the fluorescent intensity of JC-1 in FL-1 channel. The frequency of FITC+ cells is shown in the right panel. (B) After 48 h of CTPG treatment, ROS generation was detected by fluorescent probe DCFH-DA and samples were analyzed by flow cytometry. The overlay (left panel) shows the level of ROS generation. The frequency of ROS+ cells is shown in the right panel. Data are from 3 independent experiments and analyzed by one-way analysis of variance. ** p < 0.01; *** p < 0.001 compared to control. (C) The level of cytochrome c was detected by western blot after CTPG treatment for 24 h.
Figure 6
Figure 6
The levels of cleaved-caspases upon CTPG treatment. After 24 h, proteins were isolated and the levels of cleaved-caspases were detected by western blot.
Figure 7
Figure 7
The inhibition of tumor growth in vivo. Tumor mouse model was induced by injection of B16-F10 cells. After 3 days, tumor mice (12 mice per group) were treated with or without CTPG. Tumor growth (A) and survival rate (B) were monitored at the indicated time points.
Figure 8
Figure 8
The proliferation of splenocytes and the proportions of CD4+ and CD8+ T cells in spleen. (A) Splenocytes were isolated from naïve C57BL/6 mice and stimulated with different concentrations of CTPG. After 24 and 48 h, the proliferation was detected by MTT assay. (B) Splenocytes were isolated from tumor mice and stained with FITC-anti-CD4 and PE-anti-CD8. Samples were analyzed by flow cytometry. Representative dot plots (upper panel) show the gate of CD4+ and CD8+ T cells. The numbers are the frequencies of CD4+ and CD8+ T cells in spleen. The summarized data are shown in lower panel. Data are analyzed by one-way analysis of variance. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to control.
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