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Sanguisorba officinalis L synergistically enhanced 5-fluorouracil cytotoxicity in colorectal cancer cells by promoting a reactive oxygen species-mediated, mitochondria-caspase-dependent apoptotic pathway

View Article: PubMed Central - PubMed

ABSTRACT

Sanguisorba officinalis L. radix is a widely used herb called DiYu (DY) in China and has an extensive range of bioactivities, including anti-cancer, anti-inflammatory, and anti-oxidative activities. However, there is little evidence to support its anti-cancer effects against colorectal cancer (CRC). The first-line chemotherapeutic agent 5-fluorouracil (5-FU) is used to treat CRC, but its efficiency is hampered by acquired drug resistance. This study found that a water extract of DY exerted anti-proliferative effects against two CRC cell lines (HCT-116 and RKO), and it sensitized CRC cells to 5-FU therapy by activating a reactive oxygen species (ROS)-mediated, mitochondria-caspase-dependent apoptotic pathway. Co-treatment of DY and 5-FU significantly elevated ROS levels, up-regulated Bax/Bcl-2 ratio and triggered mitochondrial dysfunction, followed by a release of cytochrome c and up-regulation of proteins such as cleaved-caspase-9/3 and cleaved-PARP. Additionally, the induction of autophagy may be involved in mediating synergism of DY in HCT-116 cells. Gallic acid (GA), catechinic acid (CA) and ellagic acid (EA) were identified as the potential chief constituents responsible for the synergistic effects of DY. In conclusion, co-treatment of DY, specifically GA, CA and EA, with 5-FU may be a potential alternative therapeutic strategy for CRC by enhancing an intrinsic apoptotic pathway.

No MeSH data available.


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DY and 5-FU synergistically triggered the generation of ROS and disruption of MMP in CRC cells.(a) Cells were pre-treated with or without NAC, followed by DY and/or 5-FU. ROS levels of CRC cells loaded with DCFH-DA probes were evaluated by FCMS. (b) MMP levels were detected by JC-1 dye. JC-1 emits red fluorescence at high MMP levels by forming aggregate, while it emits green fluorescence as monomers at low MMP condition. The ratio of red/green fluorescence is used to indicate MMP levels. The distribution rates of J-aggregate and J-monomer in cells with DY/5-FU treatments are showed in the dot plots and cellular MMP levels are calculated in histograms. (c) After the indicated treatments, the cytosolic and mitochondrial fractions were isolated based on the manufacturer’s introduction of Cytochrome c Apoptosis Assay Kit. The expressions of cytosolic cytochrome c in two CRC cell lines were measured by western blotting analysis and normalized by β-actin, respectively. The levels of cytosolic cytochrome c were quantified in right histogram. The results are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001, vs control group. While *P < 0.05, **P < 0.01, ***P < 0.001, vs (5-FU + DY) group. αP < 0.05, βP < 0.05 and γP < 0.05, vs the corresponding group without NAC pre-treatment.
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f3: DY and 5-FU synergistically triggered the generation of ROS and disruption of MMP in CRC cells.(a) Cells were pre-treated with or without NAC, followed by DY and/or 5-FU. ROS levels of CRC cells loaded with DCFH-DA probes were evaluated by FCMS. (b) MMP levels were detected by JC-1 dye. JC-1 emits red fluorescence at high MMP levels by forming aggregate, while it emits green fluorescence as monomers at low MMP condition. The ratio of red/green fluorescence is used to indicate MMP levels. The distribution rates of J-aggregate and J-monomer in cells with DY/5-FU treatments are showed in the dot plots and cellular MMP levels are calculated in histograms. (c) After the indicated treatments, the cytosolic and mitochondrial fractions were isolated based on the manufacturer’s introduction of Cytochrome c Apoptosis Assay Kit. The expressions of cytosolic cytochrome c in two CRC cell lines were measured by western blotting analysis and normalized by β-actin, respectively. The levels of cytosolic cytochrome c were quantified in right histogram. The results are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001, vs control group. While *P < 0.05, **P < 0.01, ***P < 0.001, vs (5-FU + DY) group. αP < 0.05, βP < 0.05 and γP < 0.05, vs the corresponding group without NAC pre-treatment.

Mentions: Treatment with DY or 5-FU alone elevated ROS levels in both cell lines, and higher ROS levels were detected in the RKO cells. The combined treatment significantly increased the generation of ROS in both cell lines (Fig. 3a). When cells were pre-treated with NAC, the ROS levels of the cells following the single or combined treatments showed a significant decrease, except for a slight decline in the levels of RKO cells treated with DY. Annexin V-FITC/PI assays were conducted to evaluate the involvement of ROS in the synergistic apoptosis induced by DY and 5-FU. The results indicate that the induction of apoptosis was alleviated in cells pre-treated with NAC (see Supplementary Fig. S2a). Additionally, when ROS generation was suppressed by NAC, cell growth inhibition was partially abrogated in the DY and/or 5-FU group (Supplementary Fig. S2c). The results indicate that DY can synergistically trigger the generation of ROS in CRC cells when co-administered with 5-FU, which may be involved in the apoptosis induced by DY/5-FU.


Sanguisorba officinalis L synergistically enhanced 5-fluorouracil cytotoxicity in colorectal cancer cells by promoting a reactive oxygen species-mediated, mitochondria-caspase-dependent apoptotic pathway
DY and 5-FU synergistically triggered the generation of ROS and disruption of MMP in CRC cells.(a) Cells were pre-treated with or without NAC, followed by DY and/or 5-FU. ROS levels of CRC cells loaded with DCFH-DA probes were evaluated by FCMS. (b) MMP levels were detected by JC-1 dye. JC-1 emits red fluorescence at high MMP levels by forming aggregate, while it emits green fluorescence as monomers at low MMP condition. The ratio of red/green fluorescence is used to indicate MMP levels. The distribution rates of J-aggregate and J-monomer in cells with DY/5-FU treatments are showed in the dot plots and cellular MMP levels are calculated in histograms. (c) After the indicated treatments, the cytosolic and mitochondrial fractions were isolated based on the manufacturer’s introduction of Cytochrome c Apoptosis Assay Kit. The expressions of cytosolic cytochrome c in two CRC cell lines were measured by western blotting analysis and normalized by β-actin, respectively. The levels of cytosolic cytochrome c were quantified in right histogram. The results are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001, vs control group. While *P < 0.05, **P < 0.01, ***P < 0.001, vs (5-FU + DY) group. αP < 0.05, βP < 0.05 and γP < 0.05, vs the corresponding group without NAC pre-treatment.
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f3: DY and 5-FU synergistically triggered the generation of ROS and disruption of MMP in CRC cells.(a) Cells were pre-treated with or without NAC, followed by DY and/or 5-FU. ROS levels of CRC cells loaded with DCFH-DA probes were evaluated by FCMS. (b) MMP levels were detected by JC-1 dye. JC-1 emits red fluorescence at high MMP levels by forming aggregate, while it emits green fluorescence as monomers at low MMP condition. The ratio of red/green fluorescence is used to indicate MMP levels. The distribution rates of J-aggregate and J-monomer in cells with DY/5-FU treatments are showed in the dot plots and cellular MMP levels are calculated in histograms. (c) After the indicated treatments, the cytosolic and mitochondrial fractions were isolated based on the manufacturer’s introduction of Cytochrome c Apoptosis Assay Kit. The expressions of cytosolic cytochrome c in two CRC cell lines were measured by western blotting analysis and normalized by β-actin, respectively. The levels of cytosolic cytochrome c were quantified in right histogram. The results are presented as mean ± SD (n = 3). #P < 0.05, ##P < 0.01, ###P < 0.001, vs control group. While *P < 0.05, **P < 0.01, ***P < 0.001, vs (5-FU + DY) group. αP < 0.05, βP < 0.05 and γP < 0.05, vs the corresponding group without NAC pre-treatment.
Mentions: Treatment with DY or 5-FU alone elevated ROS levels in both cell lines, and higher ROS levels were detected in the RKO cells. The combined treatment significantly increased the generation of ROS in both cell lines (Fig. 3a). When cells were pre-treated with NAC, the ROS levels of the cells following the single or combined treatments showed a significant decrease, except for a slight decline in the levels of RKO cells treated with DY. Annexin V-FITC/PI assays were conducted to evaluate the involvement of ROS in the synergistic apoptosis induced by DY and 5-FU. The results indicate that the induction of apoptosis was alleviated in cells pre-treated with NAC (see Supplementary Fig. S2a). Additionally, when ROS generation was suppressed by NAC, cell growth inhibition was partially abrogated in the DY and/or 5-FU group (Supplementary Fig. S2c). The results indicate that DY can synergistically trigger the generation of ROS in CRC cells when co-administered with 5-FU, which may be involved in the apoptosis induced by DY/5-FU.

View Article: PubMed Central - PubMed

ABSTRACT

Sanguisorba officinalis L. radix is a widely used herb called DiYu (DY) in China and has an extensive range of bioactivities, including anti-cancer, anti-inflammatory, and anti-oxidative activities. However, there is little evidence to support its anti-cancer effects against colorectal cancer (CRC). The first-line chemotherapeutic agent 5-fluorouracil (5-FU) is used to treat CRC, but its efficiency is hampered by acquired drug resistance. This study found that a water extract of DY exerted anti-proliferative effects against two CRC cell lines (HCT-116 and RKO), and it sensitized CRC cells to 5-FU therapy by activating a reactive oxygen species (ROS)-mediated, mitochondria-caspase-dependent apoptotic pathway. Co-treatment of DY and 5-FU significantly elevated ROS levels, up-regulated Bax/Bcl-2 ratio and triggered mitochondrial dysfunction, followed by a release of cytochrome c and up-regulation of proteins such as cleaved-caspase-9/3 and cleaved-PARP. Additionally, the induction of autophagy may be involved in mediating synergism of DY in HCT-116 cells. Gallic acid (GA), catechinic acid (CA) and ellagic acid (EA) were identified as the potential chief constituents responsible for the synergistic effects of DY. In conclusion, co-treatment of DY, specifically GA, CA and EA, with 5-FU may be a potential alternative therapeutic strategy for CRC by enhancing an intrinsic apoptotic pathway.

No MeSH data available.


Related in: MedlinePlus