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Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells.

Li T, Xu XH, Tang ZH, Wang YF, Leung CH, Ma DL, Chen XP, Wang YT, Chen Y, Lu JJ - Acta Pharmacol. Sin. (2015)

Bottom Line: Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis.In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight.Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT

Aim: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy.

Methods: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg(-1)·d(-1)) was intraperitoneally injected to BEL-7402-bearing mice for 21 days.

Results: Platycodin D (5-40 μmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 μmol/L at 24, 48 and 72 h, respectively. Platycodin D (5-20 μmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5-20 μmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 μmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight.

Conclusion: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.

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PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). bP<0.05, cP<0.01 vs control.
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fig3: PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). bP<0.05, cP<0.01 vs control.

Mentions: In our previous study, PD induced apoptosis in hepatocellular carcinoma HepG2 cells by inducing the protein expression of cleaved PARP and Bax as well as by inhibiting survivin protein expression15. PD also enhances the apoptotic effect of doxorubicin in breast cancer MCF-7 and MDA-MB-231 cells14. To investigate whether PD promotes BEL-7402 cell apoptosis, several apoptotic assays were employed. Firstly, cell death was measured in PD-treated BEL-7402 cells using an Annexin V/PI staining assay. As shown in Figures 3A and 3B, apoptotic cells increased in a concentration-dependent manner after PD treatment for 24 h. A significant increase (P<0.05) in the mean number of apoptotic cells was observed after treatment with 20 μmol/L PD with a fold change of 4.34±1.73. Secondly, morphological alteration of BEL-7402 cells after 24 h PD treatment was revealed by Hoechst 33342 staining. As shown in Figure 3C, no apoptotic nuclei were observed in the control group. In contrast, 5 μmol/L PD treatment for 24 h slightly altered BEL-7402 cell morphology, and cell nuclear condensation developed in a concentration-dependent manner (Figure 3C). The majority of the 20 μmol/L PD-exposed cells was shriveled and exhibited typical apoptotic morphology characterized by nuclear condensation and DNA fragmentation (Figure 3C). We further detected the levels of several proteins related to apoptosis. After PD treatment, the antiapoptotic protein Bcl-2 was significantly down-regulated, while the Bax/Bcl-2 ratio and the levels of cleaved PARP and caspase-3 were significantly up-regulated (Figures 3D and 3E), indicating that PD induces apoptosis in BEL-7402 cells.


Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells.

Li T, Xu XH, Tang ZH, Wang YF, Leung CH, Ma DL, Chen XP, Wang YT, Chen Y, Lu JJ - Acta Pharmacol. Sin. (2015)

PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). bP<0.05, cP<0.01 vs control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4816242&req=5

fig3: PD induces apoptosis in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) Apoptotic cells were detected by Annexin V/PI staining using flow cytometry. (B) Quantitation of the results obtained from (A). (C) The apoptotic cells were evaluated with Hoechst 33342 staining and imaged using the In Cell Analyzer 2000 System. (D) The expression of apoptosis-related proteins, including cleaved PARP, cleaved caspase-3, Bcl-2, and Bax, were analyzed by Western blot analysis. (E) Quantitation of the results obtained from (D). bP<0.05, cP<0.01 vs control.
Mentions: In our previous study, PD induced apoptosis in hepatocellular carcinoma HepG2 cells by inducing the protein expression of cleaved PARP and Bax as well as by inhibiting survivin protein expression15. PD also enhances the apoptotic effect of doxorubicin in breast cancer MCF-7 and MDA-MB-231 cells14. To investigate whether PD promotes BEL-7402 cell apoptosis, several apoptotic assays were employed. Firstly, cell death was measured in PD-treated BEL-7402 cells using an Annexin V/PI staining assay. As shown in Figures 3A and 3B, apoptotic cells increased in a concentration-dependent manner after PD treatment for 24 h. A significant increase (P<0.05) in the mean number of apoptotic cells was observed after treatment with 20 μmol/L PD with a fold change of 4.34±1.73. Secondly, morphological alteration of BEL-7402 cells after 24 h PD treatment was revealed by Hoechst 33342 staining. As shown in Figure 3C, no apoptotic nuclei were observed in the control group. In contrast, 5 μmol/L PD treatment for 24 h slightly altered BEL-7402 cell morphology, and cell nuclear condensation developed in a concentration-dependent manner (Figure 3C). The majority of the 20 μmol/L PD-exposed cells was shriveled and exhibited typical apoptotic morphology characterized by nuclear condensation and DNA fragmentation (Figure 3C). We further detected the levels of several proteins related to apoptosis. After PD treatment, the antiapoptotic protein Bcl-2 was significantly down-regulated, while the Bax/Bcl-2 ratio and the levels of cleaved PARP and caspase-3 were significantly up-regulated (Figures 3D and 3E), indicating that PD induces apoptosis in BEL-7402 cells.

Bottom Line: Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis.In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight.Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China.

ABSTRACT

Aim: Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy.

Methods: The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg(-1)·d(-1)) was intraperitoneally injected to BEL-7402-bearing mice for 21 days.

Results: Platycodin D (5-40 μmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 μmol/L at 24, 48 and 72 h, respectively. Platycodin D (5-20 μmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5-20 μmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 μmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg(-1)•d(-1)) significantly reduced relative tumor volume with decreased body weight.

Conclusion: Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors.

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Related in: MedlinePlus