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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 synergistically induced apoptosis in CRC cells when treated with 5-FU.(a) Cells treated with DY and/or 5-FU for 48 h were loaded with Hoechst 33258 probes, and cells with condensed or fragmentary nucleus (arrow) were observed under a fluorescence microscope (200×). The mean fluorescence intensity of Hoechst 33258 in single cell and apoptotic rates were quantified (see right histograms) as well. (b) Apoptotic CRC cells stained with Annexin V-FITC/PI were detected by flow cytometry system (FCMS), and apoptotic rates are showed in right bars. (c) Cells were stained by PI & RNase and DNA contents were then measured by FCMS. Cells arrested in the sub-G1 phase are considered to undergo late apoptosis. All data are expressed 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 (DY + 5-FU) group.
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f2: DY synergistically induced apoptosis in CRC cells when treated with 5-FU.(a) Cells treated with DY and/or 5-FU for 48 h were loaded with Hoechst 33258 probes, and cells with condensed or fragmentary nucleus (arrow) were observed under a fluorescence microscope (200×). The mean fluorescence intensity of Hoechst 33258 in single cell and apoptotic rates were quantified (see right histograms) as well. (b) Apoptotic CRC cells stained with Annexin V-FITC/PI were detected by flow cytometry system (FCMS), and apoptotic rates are showed in right bars. (c) Cells were stained by PI & RNase and DNA contents were then measured by FCMS. Cells arrested in the sub-G1 phase are considered to undergo late apoptosis. All data are expressed 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 (DY + 5-FU) group.

Mentions: The chemotherapeutic drug 5-FU has been widely used on clinic for CRC therapy. Agents that enhance apoptosis are likely to suppress drug resistance to 5-FU18. Therefore, we determined whether the synergism between DY and 5-FU is resulted from increased apoptosis. Data of Hoechst 33258 assay shows that condensed and bright chromatin, as well as some cell debris were observed in the DY-treated HCT-116 cells (Fig. 2a), indicating that apoptosis was induced by DY in HCT-116 cells. However, DY only slightly induced apoptosis in RKO cells at the given dose. When cells were treated with a combination of 5-FU and DY, the mean fluorescence intensity in single cell remarkably increased, which were observed in both cell lines (Fig. 2a). To confirm the above results, Annexin V-FITC/PI and PI & RNase staining assays were then conducted. Following treatment with the DY and 5-FU combination, the percentages of apoptotic cells were 45.59 ± 1.27% and 11.61 ± 0.78% in HCT-116 and RKO cells, respectively, which were dramatically higher than those in the single treatment groups (Fig. 2b). Additionally, cell cycle analysis revealed that co-treatment of DY and 5-FU resulted in a significant accumulation of cells in sub-G1 phase (Fig. 2c). In contrast to the HCT-116 cells, the RKO cells showed more necrosis but less apoptosis following DY treatment (Fig. 2b), and late apoptosis was predominantly responsible for the apoptosis observed in the RKO cells (Fig. 2c). Taken together, the apoptosis induced by DY may relieve defective apoptosis induced by 5-FU in both CRC cells.


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 synergistically induced apoptosis in CRC cells when treated with 5-FU.(a) Cells treated with DY and/or 5-FU for 48 h were loaded with Hoechst 33258 probes, and cells with condensed or fragmentary nucleus (arrow) were observed under a fluorescence microscope (200×). The mean fluorescence intensity of Hoechst 33258 in single cell and apoptotic rates were quantified (see right histograms) as well. (b) Apoptotic CRC cells stained with Annexin V-FITC/PI were detected by flow cytometry system (FCMS), and apoptotic rates are showed in right bars. (c) Cells were stained by PI & RNase and DNA contents were then measured by FCMS. Cells arrested in the sub-G1 phase are considered to undergo late apoptosis. All data are expressed 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 (DY + 5-FU) group.
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Related In: Results  -  Collection

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f2: DY synergistically induced apoptosis in CRC cells when treated with 5-FU.(a) Cells treated with DY and/or 5-FU for 48 h were loaded with Hoechst 33258 probes, and cells with condensed or fragmentary nucleus (arrow) were observed under a fluorescence microscope (200×). The mean fluorescence intensity of Hoechst 33258 in single cell and apoptotic rates were quantified (see right histograms) as well. (b) Apoptotic CRC cells stained with Annexin V-FITC/PI were detected by flow cytometry system (FCMS), and apoptotic rates are showed in right bars. (c) Cells were stained by PI & RNase and DNA contents were then measured by FCMS. Cells arrested in the sub-G1 phase are considered to undergo late apoptosis. All data are expressed 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 (DY + 5-FU) group.
Mentions: The chemotherapeutic drug 5-FU has been widely used on clinic for CRC therapy. Agents that enhance apoptosis are likely to suppress drug resistance to 5-FU18. Therefore, we determined whether the synergism between DY and 5-FU is resulted from increased apoptosis. Data of Hoechst 33258 assay shows that condensed and bright chromatin, as well as some cell debris were observed in the DY-treated HCT-116 cells (Fig. 2a), indicating that apoptosis was induced by DY in HCT-116 cells. However, DY only slightly induced apoptosis in RKO cells at the given dose. When cells were treated with a combination of 5-FU and DY, the mean fluorescence intensity in single cell remarkably increased, which were observed in both cell lines (Fig. 2a). To confirm the above results, Annexin V-FITC/PI and PI & RNase staining assays were then conducted. Following treatment with the DY and 5-FU combination, the percentages of apoptotic cells were 45.59 ± 1.27% and 11.61 ± 0.78% in HCT-116 and RKO cells, respectively, which were dramatically higher than those in the single treatment groups (Fig. 2b). Additionally, cell cycle analysis revealed that co-treatment of DY and 5-FU resulted in a significant accumulation of cells in sub-G1 phase (Fig. 2c). In contrast to the HCT-116 cells, the RKO cells showed more necrosis but less apoptosis following DY treatment (Fig. 2b), and late apoptosis was predominantly responsible for the apoptosis observed in the RKO cells (Fig. 2c). Taken together, the apoptosis induced by DY may relieve defective apoptosis induced by 5-FU in both CRC cells.

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