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Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

Zhu X, Wang H, Zheng L, Zhong Z, Li X, Zhao J, Kou J, Jiang Y, Zheng X, Liu Z, Li H, Cao W, Tian Y, Wang Y, Yang L - Int J Nanomedicine (2015)

Bottom Line: Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis.The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release.This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin, People's Republic of China.

ABSTRACT
Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

No MeSH data available.


Related in: MedlinePlus

Relative reactive oxygen species (ROS) generation induced by UCNPs-Ce6-mediated PDT.Notes: (A) Fluorescent photomicrographs of THP-1 macrophages stained using DCFH-DA, indicating intracellular ROS generation. (B) The alteration of ROS following PDT. (C) The effect of active oxygen scavengers on cell viability following PDT. To determine the primary inducer of apoptosis following PDT, several free radical scavengers, including 200 U/mL catalase (CAT, a scavenger of hydrogen peroxide), 100 mg/mL superoxide dismutase (SOD, a scavenger of superoxide anion radicals), 100 mM mannitol (a scavenger of the hydroxyl radical), and 10 mM sodium azide (NaN3, a scavenger of singlet oxygen molecules), were employed prior to treatment. Cell viability was examined via a CCK-8 assay. (D) MDA content following UCNPs-Ce6-mediated PDT. **P<0.001 vs control group. ***P<0.001 vs control group. #P<0.05 vs PDT group. Scale bar =0.1 mm.Abbreviations: UCNPs, upconversion nanoparticles; Ce6, chlorin e6; PDT, photodynamic therapy; DCFH-DA, 2′-7′-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; MDA, malondialdehyde; h, hours.
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f4-ijn-10-3719: Relative reactive oxygen species (ROS) generation induced by UCNPs-Ce6-mediated PDT.Notes: (A) Fluorescent photomicrographs of THP-1 macrophages stained using DCFH-DA, indicating intracellular ROS generation. (B) The alteration of ROS following PDT. (C) The effect of active oxygen scavengers on cell viability following PDT. To determine the primary inducer of apoptosis following PDT, several free radical scavengers, including 200 U/mL catalase (CAT, a scavenger of hydrogen peroxide), 100 mg/mL superoxide dismutase (SOD, a scavenger of superoxide anion radicals), 100 mM mannitol (a scavenger of the hydroxyl radical), and 10 mM sodium azide (NaN3, a scavenger of singlet oxygen molecules), were employed prior to treatment. Cell viability was examined via a CCK-8 assay. (D) MDA content following UCNPs-Ce6-mediated PDT. **P<0.001 vs control group. ***P<0.001 vs control group. #P<0.05 vs PDT group. Scale bar =0.1 mm.Abbreviations: UCNPs, upconversion nanoparticles; Ce6, chlorin e6; PDT, photodynamic therapy; DCFH-DA, 2′-7′-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; MDA, malondialdehyde; h, hours.

Mentions: Results (Figure 1B) have confirmed that UCNPs-Ce6 is capable of generating 1O2 after receiving 980 nm laser irradiation. Therefore, we examined the production of ROS in live cells. As compared with the control group, the green fluorescence increased significantly in the PDT group at 6 hours following treatment, whereas slight increases in the green fluorescence were observed in both the UCNPs-Ce6 group and the laser group (Figure 4A). This fluorescence was inhibited by the addition of the ROS scavenger, NAC, and the singlet scavenger, NaN3. We investigated the cause of the ROS alteration within 6 hours following PDT treatment, which was important in the study of apoptosis following PDT. We also evaluated the time course of ROS generation following PDT. Interestingly, we observed that ROS generation increased within the first 3 hours before decreasing with time (Figure 4B). The time course of ROS generation indicated that PDT increased the level of ROS, which peaked at 3 hours. The initial increase in ROS generation was most likely due to the reactivity of the 1O2 formed early following PDT and its ability to induce the formation of other ROS species that were detected via DCFH-DA (DCFH-DA does not detect 1O2 but reacts with hydroperoxides). To determine which chemical was more efficient in altering PDT-induced apoptosis, several other free radical scavengers, including mannitol, SOD, CAT, and NaN3, were tested prior to treatment. As shown in Figure 4C, both mannitol and NaN3 exerted strong effects in preventing the decline in cell viability. These results indicated that the hydroxyl radical may be the primary ROS that decreases cell viability, whereas 1O2 may act as a trigger for the induction of an imbalance in ROS generation in THP-1 macrophages (Figure 4C). A previous study demonstrated that the accumulation of ROS in tumor cells may cause oxidizing reactions in biomolecules that disrupt the integrity of both lipid membranes and proteins.30 We measured MDA levels and observed that in both the UCNPs-Ce6 and the laser groups, the MDA levels were slightly increased compared with the control group (Figure 4D). The MDA level in the PDT group was significantly increased as it was almost three times that in the control group. However, the MDA level in the PDT group decreased following pretreatment with NAC.


Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

Zhu X, Wang H, Zheng L, Zhong Z, Li X, Zhao J, Kou J, Jiang Y, Zheng X, Liu Z, Li H, Cao W, Tian Y, Wang Y, Yang L - Int J Nanomedicine (2015)

Relative reactive oxygen species (ROS) generation induced by UCNPs-Ce6-mediated PDT.Notes: (A) Fluorescent photomicrographs of THP-1 macrophages stained using DCFH-DA, indicating intracellular ROS generation. (B) The alteration of ROS following PDT. (C) The effect of active oxygen scavengers on cell viability following PDT. To determine the primary inducer of apoptosis following PDT, several free radical scavengers, including 200 U/mL catalase (CAT, a scavenger of hydrogen peroxide), 100 mg/mL superoxide dismutase (SOD, a scavenger of superoxide anion radicals), 100 mM mannitol (a scavenger of the hydroxyl radical), and 10 mM sodium azide (NaN3, a scavenger of singlet oxygen molecules), were employed prior to treatment. Cell viability was examined via a CCK-8 assay. (D) MDA content following UCNPs-Ce6-mediated PDT. **P<0.001 vs control group. ***P<0.001 vs control group. #P<0.05 vs PDT group. Scale bar =0.1 mm.Abbreviations: UCNPs, upconversion nanoparticles; Ce6, chlorin e6; PDT, photodynamic therapy; DCFH-DA, 2′-7′-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; MDA, malondialdehyde; h, hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4447170&req=5

f4-ijn-10-3719: Relative reactive oxygen species (ROS) generation induced by UCNPs-Ce6-mediated PDT.Notes: (A) Fluorescent photomicrographs of THP-1 macrophages stained using DCFH-DA, indicating intracellular ROS generation. (B) The alteration of ROS following PDT. (C) The effect of active oxygen scavengers on cell viability following PDT. To determine the primary inducer of apoptosis following PDT, several free radical scavengers, including 200 U/mL catalase (CAT, a scavenger of hydrogen peroxide), 100 mg/mL superoxide dismutase (SOD, a scavenger of superoxide anion radicals), 100 mM mannitol (a scavenger of the hydroxyl radical), and 10 mM sodium azide (NaN3, a scavenger of singlet oxygen molecules), were employed prior to treatment. Cell viability was examined via a CCK-8 assay. (D) MDA content following UCNPs-Ce6-mediated PDT. **P<0.001 vs control group. ***P<0.001 vs control group. #P<0.05 vs PDT group. Scale bar =0.1 mm.Abbreviations: UCNPs, upconversion nanoparticles; Ce6, chlorin e6; PDT, photodynamic therapy; DCFH-DA, 2′-7′-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; MDA, malondialdehyde; h, hours.
Mentions: Results (Figure 1B) have confirmed that UCNPs-Ce6 is capable of generating 1O2 after receiving 980 nm laser irradiation. Therefore, we examined the production of ROS in live cells. As compared with the control group, the green fluorescence increased significantly in the PDT group at 6 hours following treatment, whereas slight increases in the green fluorescence were observed in both the UCNPs-Ce6 group and the laser group (Figure 4A). This fluorescence was inhibited by the addition of the ROS scavenger, NAC, and the singlet scavenger, NaN3. We investigated the cause of the ROS alteration within 6 hours following PDT treatment, which was important in the study of apoptosis following PDT. We also evaluated the time course of ROS generation following PDT. Interestingly, we observed that ROS generation increased within the first 3 hours before decreasing with time (Figure 4B). The time course of ROS generation indicated that PDT increased the level of ROS, which peaked at 3 hours. The initial increase in ROS generation was most likely due to the reactivity of the 1O2 formed early following PDT and its ability to induce the formation of other ROS species that were detected via DCFH-DA (DCFH-DA does not detect 1O2 but reacts with hydroperoxides). To determine which chemical was more efficient in altering PDT-induced apoptosis, several other free radical scavengers, including mannitol, SOD, CAT, and NaN3, were tested prior to treatment. As shown in Figure 4C, both mannitol and NaN3 exerted strong effects in preventing the decline in cell viability. These results indicated that the hydroxyl radical may be the primary ROS that decreases cell viability, whereas 1O2 may act as a trigger for the induction of an imbalance in ROS generation in THP-1 macrophages (Figure 4C). A previous study demonstrated that the accumulation of ROS in tumor cells may cause oxidizing reactions in biomolecules that disrupt the integrity of both lipid membranes and proteins.30 We measured MDA levels and observed that in both the UCNPs-Ce6 and the laser groups, the MDA levels were slightly increased compared with the control group (Figure 4D). The MDA level in the PDT group was significantly increased as it was almost three times that in the control group. However, the MDA level in the PDT group decreased following pretreatment with NAC.

Bottom Line: Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis.The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release.This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin, People's Republic of China.

ABSTRACT
Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

No MeSH data available.


Related in: MedlinePlus