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Enhanced efficacy of photodynamic therapy by inhibiting ABCG2 in colon cancers.

Kim JH, Park JM, Roh YJ, Kim IW, Hasan T, Choi MG - BMC Cancer (2015)

Bottom Line: Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells.Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells.The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT.

View Article: PubMed Central - PubMed

Affiliation: Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea. jhsubzero@naver.com.

ABSTRACT

Background: Photodynamic therapy (PDT) contains a photosensitizing process, which includes cellular uptake of photosensitizer and delivery of light to the target. ATP-binding cassette subfamily G2 (ABCG2) regulates endogenous protoporphyrin levels. In human colon cancers, it is not fully examined the role of ABCG2 in porphyrin-based photodynamic therapy.

Methods: SW480 and HT29 cells were selected because they showed low and high ABCG2 expression levels, respectively. Pyropheophorbid-a (PPa) was used as a photosensitizer. Cells were exposed to a 670 nm diod laser. Cell viability and necrosi apoptosis was examined. Production level of singlet oxygen was detected with the photomultiplier-tube s/ -based singlet oxygen detection system.

Results: SW480 cells, which expressed lower level of ABCG2, showed the higher uptake of PPa than HT-29 cells. The uptake level of PPa was significantly correlated with the decreased cell viability after PDT. Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells. To confirm the ABCG2 effect on PDT, we established ABCG2 over-expressing stable cells in SW480 cells (SW480/ABCG2). Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells. The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT.

Conclusion: ABCG2 plays an important role in determining the PDT efficacy by controlling the photosensitizer efflux rate. This implies the control of ABCG2 expression may be a potential solution to enhance photosensitivity.

No MeSH data available.


Related in: MedlinePlus

Differences of PDT effect between SW480 and HT29 cells depending on ABCG2 expression level. a, SW480 and HT29 cells were irradiated with a PDT laser (4 J/cm2) after a 16 h pretreatment of PPa at indicated concentrations. The MTT assay was performed in triplicate 24 h following the irradiation. b, Cells were incubated with 100 nM PPa for 16 h and then lysed with methanol, followed by application of PMT-based singlet oxygen monitoring system. c, Cells were incubated with 200 nM PPa for 16 h and then subjected to immunocytochemical staining for anti-abcg2 (Green), PPa (Red), and DAPI (blue). Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)
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Fig2: Differences of PDT effect between SW480 and HT29 cells depending on ABCG2 expression level. a, SW480 and HT29 cells were irradiated with a PDT laser (4 J/cm2) after a 16 h pretreatment of PPa at indicated concentrations. The MTT assay was performed in triplicate 24 h following the irradiation. b, Cells were incubated with 100 nM PPa for 16 h and then lysed with methanol, followed by application of PMT-based singlet oxygen monitoring system. c, Cells were incubated with 200 nM PPa for 16 h and then subjected to immunocytochemical staining for anti-abcg2 (Green), PPa (Red), and DAPI (blue). Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)

Mentions: The aim of this study was to see whether ABCG2 is a target protein in enhancing colon cancer PDT efficacy. To confirm ABCG2 effect in PDT, we tested ABCG2 expression level in colon cancer cell lines (Fig. 1a, b). In colon cancer cells, HT29 cell showed the highest expression of ABCG2 mRNA and protein. SW480, DLD1, LOVO, and HCT116 cells showed low expression of ABCG2. Among them, SW480 cells showed the lowest ABCG2 mRNA level. SW480 and HT29 cells were selected, which showed the lowest and highest ABCG2 expression level, respectively, among the tested colon cancer cells (Fig. 1c). Cells were incubated with PPa for 16 h and then irradiated with 4 J/cm2 red right. There were differences in the cell survival rate and singlet oxygen production between SW480 and HT29. Cell survival rate was measured using MTT assay. After PDT, higher treatment efficacy was obtained in SW480 cells compared to HT29 (Fig. 2a). After treating with 100 nM PPa, there was differences of three times in phototoxicity between SW480 and HT29. Singlet oxygen played a main role in killing cancer cells in PDT. We checked the singlet oxygen production using PMT-based singlet oxygen monitoring system. SW480 cell showed lower production rate of singlet oxygen than HT29 (Fig. 2b). These results indicate that high expression of ABCG2 induced PPa release out of the cell by the efflux function. To further explain the effect of ABCG2 in PDT, we verified localization of ABCG2 and accumulation of PPa using light fluorescence microscopy (Fig. 2c). SW480 cells showed no fluorescence of ABCG2, but strong red fluorescence of PPa. In contrast, HT29 cells showed ABCG2 fluorescence without accumulation of PPa. These results indicate that ABCG2 is related with the resistance to PDT derived from the efflux of photosensitizer in colon cancer.Fig. 1


Enhanced efficacy of photodynamic therapy by inhibiting ABCG2 in colon cancers.

Kim JH, Park JM, Roh YJ, Kim IW, Hasan T, Choi MG - BMC Cancer (2015)

Differences of PDT effect between SW480 and HT29 cells depending on ABCG2 expression level. a, SW480 and HT29 cells were irradiated with a PDT laser (4 J/cm2) after a 16 h pretreatment of PPa at indicated concentrations. The MTT assay was performed in triplicate 24 h following the irradiation. b, Cells were incubated with 100 nM PPa for 16 h and then lysed with methanol, followed by application of PMT-based singlet oxygen monitoring system. c, Cells were incubated with 200 nM PPa for 16 h and then subjected to immunocytochemical staining for anti-abcg2 (Green), PPa (Red), and DAPI (blue). Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Differences of PDT effect between SW480 and HT29 cells depending on ABCG2 expression level. a, SW480 and HT29 cells were irradiated with a PDT laser (4 J/cm2) after a 16 h pretreatment of PPa at indicated concentrations. The MTT assay was performed in triplicate 24 h following the irradiation. b, Cells were incubated with 100 nM PPa for 16 h and then lysed with methanol, followed by application of PMT-based singlet oxygen monitoring system. c, Cells were incubated with 200 nM PPa for 16 h and then subjected to immunocytochemical staining for anti-abcg2 (Green), PPa (Red), and DAPI (blue). Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)
Mentions: The aim of this study was to see whether ABCG2 is a target protein in enhancing colon cancer PDT efficacy. To confirm ABCG2 effect in PDT, we tested ABCG2 expression level in colon cancer cell lines (Fig. 1a, b). In colon cancer cells, HT29 cell showed the highest expression of ABCG2 mRNA and protein. SW480, DLD1, LOVO, and HCT116 cells showed low expression of ABCG2. Among them, SW480 cells showed the lowest ABCG2 mRNA level. SW480 and HT29 cells were selected, which showed the lowest and highest ABCG2 expression level, respectively, among the tested colon cancer cells (Fig. 1c). Cells were incubated with PPa for 16 h and then irradiated with 4 J/cm2 red right. There were differences in the cell survival rate and singlet oxygen production between SW480 and HT29. Cell survival rate was measured using MTT assay. After PDT, higher treatment efficacy was obtained in SW480 cells compared to HT29 (Fig. 2a). After treating with 100 nM PPa, there was differences of three times in phototoxicity between SW480 and HT29. Singlet oxygen played a main role in killing cancer cells in PDT. We checked the singlet oxygen production using PMT-based singlet oxygen monitoring system. SW480 cell showed lower production rate of singlet oxygen than HT29 (Fig. 2b). These results indicate that high expression of ABCG2 induced PPa release out of the cell by the efflux function. To further explain the effect of ABCG2 in PDT, we verified localization of ABCG2 and accumulation of PPa using light fluorescence microscopy (Fig. 2c). SW480 cells showed no fluorescence of ABCG2, but strong red fluorescence of PPa. In contrast, HT29 cells showed ABCG2 fluorescence without accumulation of PPa. These results indicate that ABCG2 is related with the resistance to PDT derived from the efflux of photosensitizer in colon cancer.Fig. 1

Bottom Line: Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells.Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells.The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT.

View Article: PubMed Central - PubMed

Affiliation: Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, South Korea. jhsubzero@naver.com.

ABSTRACT

Background: Photodynamic therapy (PDT) contains a photosensitizing process, which includes cellular uptake of photosensitizer and delivery of light to the target. ATP-binding cassette subfamily G2 (ABCG2) regulates endogenous protoporphyrin levels. In human colon cancers, it is not fully examined the role of ABCG2 in porphyrin-based photodynamic therapy.

Methods: SW480 and HT29 cells were selected because they showed low and high ABCG2 expression levels, respectively. Pyropheophorbid-a (PPa) was used as a photosensitizer. Cells were exposed to a 670 nm diod laser. Cell viability and necrosi apoptosis was examined. Production level of singlet oxygen was detected with the photomultiplier-tube s/ -based singlet oxygen detection system.

Results: SW480 cells, which expressed lower level of ABCG2, showed the higher uptake of PPa than HT-29 cells. The uptake level of PPa was significantly correlated with the decreased cell viability after PDT. Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells. To confirm the ABCG2 effect on PDT, we established ABCG2 over-expressing stable cells in SW480 cells (SW480/ABCG2). Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells. The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT.

Conclusion: ABCG2 plays an important role in determining the PDT efficacy by controlling the photosensitizer efflux rate. This implies the control of ABCG2 expression may be a potential solution to enhance photosensitivity.

No MeSH data available.


Related in: MedlinePlus