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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 triggers autophagy in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) The cell morphology was directly observed under an Axiovert 200 inverted microscope. Bar: 20 μm. (B) The autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mmol/L MDC. (C) The protein levels of LC3 were determined by Western blot analysis. (D) The BEL-7402 cells were pretreated with 5 μmol/L CQ for 1 h followed by 20 μmol/L PD treatment for an additional 24 h, and the protein levels of LC3 were determined by Western blot analysis. cP<0.01 vs -CQ.
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fig4: PD triggers autophagy in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) The cell morphology was directly observed under an Axiovert 200 inverted microscope. Bar: 20 μm. (B) The autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mmol/L MDC. (C) The protein levels of LC3 were determined by Western blot analysis. (D) The BEL-7402 cells were pretreated with 5 μmol/L CQ for 1 h followed by 20 μmol/L PD treatment for an additional 24 h, and the protein levels of LC3 were determined by Western blot analysis. cP<0.01 vs -CQ.

Mentions: Morphological features of cytoplasmic vacuole accumulation are often observed in cells undergoing autophagy16,21,22. We have demonstrated that PD triggers autophagic characteristics, such as cytoplasmic vacuole accumulation, in many cell lines16. In this study, PD also induced the formation of cytoplasmic vacuoles in BEL-7402 cells as observed under an Axiovert 200 inverted microscope (Figure 4A). To confirm if autophagy is triggered in PD-treated BEL-7402 cells, we performed several experiments. MDC, which can accumulate in acidic vesicular organelles, was used to detect autophagic activity. The cells were treated with different concentrations of PD for 24 h and then labeled with 50 μmol/L MDC. The number of MDC-positive cells increased in a concentration-dependent manner in PD-treated cells after a 24 h incubation. The levels of LC3, a hallmark of autophagy, were also determined by Western blot analysis. As shown in Figure 4C, a concentration-dependent increase in LC3-II was detectable in BEL-7402 cells after a 24 h PD treatment. CQ has been widely used as late-stage autophagy inhibitor, and CQ leads to an increase of LC3-II levels due to the accumulation of undigested autophagosomes23,24,25. Western blot analysis revealed that LC3-II further accumulated in cells co-treated with CQ and PD compared to cells treated with CQ or PD alone (Figure 4D). Taken together, our results indicated that PD triggers autophagy 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 triggers autophagy in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) The cell morphology was directly observed under an Axiovert 200 inverted microscope. Bar: 20 μm. (B) The autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mmol/L MDC. (C) The protein levels of LC3 were determined by Western blot analysis. (D) The BEL-7402 cells were pretreated with 5 μmol/L CQ for 1 h followed by 20 μmol/L PD treatment for an additional 24 h, and the protein levels of LC3 were determined by Western blot analysis. cP<0.01 vs -CQ.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816242&req=5

fig4: PD triggers autophagy in BEL-7402 cells. BEL-7402 cells were treated with different concentrations of PD for 24 h. (A) The cell morphology was directly observed under an Axiovert 200 inverted microscope. Bar: 20 μm. (B) The autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mmol/L MDC. (C) The protein levels of LC3 were determined by Western blot analysis. (D) The BEL-7402 cells were pretreated with 5 μmol/L CQ for 1 h followed by 20 μmol/L PD treatment for an additional 24 h, and the protein levels of LC3 were determined by Western blot analysis. cP<0.01 vs -CQ.
Mentions: Morphological features of cytoplasmic vacuole accumulation are often observed in cells undergoing autophagy16,21,22. We have demonstrated that PD triggers autophagic characteristics, such as cytoplasmic vacuole accumulation, in many cell lines16. In this study, PD also induced the formation of cytoplasmic vacuoles in BEL-7402 cells as observed under an Axiovert 200 inverted microscope (Figure 4A). To confirm if autophagy is triggered in PD-treated BEL-7402 cells, we performed several experiments. MDC, which can accumulate in acidic vesicular organelles, was used to detect autophagic activity. The cells were treated with different concentrations of PD for 24 h and then labeled with 50 μmol/L MDC. The number of MDC-positive cells increased in a concentration-dependent manner in PD-treated cells after a 24 h incubation. The levels of LC3, a hallmark of autophagy, were also determined by Western blot analysis. As shown in Figure 4C, a concentration-dependent increase in LC3-II was detectable in BEL-7402 cells after a 24 h PD treatment. CQ has been widely used as late-stage autophagy inhibitor, and CQ leads to an increase of LC3-II levels due to the accumulation of undigested autophagosomes23,24,25. Western blot analysis revealed that LC3-II further accumulated in cells co-treated with CQ and PD compared to cells treated with CQ or PD alone (Figure 4D). Taken together, our results indicated that PD triggers autophagy 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.

Show MeSH
Related in: MedlinePlus