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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 singlet oxygen production rate & cell death between SW480 and SW480/ABCG2 cells. a, SW480, SW480/ABCG2, and HT29 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. b, Cells were harvested 4 h after photosensitization and stained with Annexin V/PI for FACS analysis. c, Cells were incubated with 100 nM PPa for 16 h followed by irradiation. Cell lysates were subjected to immunoblot with indicated antibodies. Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)
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Fig5: Differences of singlet oxygen production rate & cell death between SW480 and SW480/ABCG2 cells. a, SW480, SW480/ABCG2, and HT29 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. b, Cells were harvested 4 h after photosensitization and stained with Annexin V/PI for FACS analysis. c, Cells were incubated with 100 nM PPa for 16 h followed by irradiation. Cell lysates were subjected to immunoblot with indicated antibodies. Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)

Mentions: As mentioned above, ABCG2 plays a major role in inducing drug resistance and determining the efficacy of PDT. In order to show that ABCG2 is directly related with PDT efficacy, we established SW480 that stably overexpress ABCG2 using retrovirus. The expression level of ABCG2 in SW480 and SW480/ABCG2 was confirmed by western blot analysis and immunocytochemistry with anti-ABCG2 antibody (Fig. 4a). SW/ABCG2 cells with overexpression of ABCG2showed higher level of survival rate compared to SW480 and HT29, which indicates that overexpression of ABCG2 is directly related with the efficacy of PDT and drug resistance (Fig. 4b). SW480/ABCG2 cells showed low fluorescence of PPa, whereas SW480 cell showed strong signal of PPa (Fig. 4c). Also, singlet oxygen production rate was decreased in SW480/ABCG2 cells (Fig. 5a). To investigate the effect of PDT on cell viability, we checked for apoptosis or necrosis using flow cytometry analysis with Annexin V and PI. After irradiation, apoptosis and necrosis developed considerably in SW480. SW480 cells with overexpression of ABCG2 showed little necrosis while HT29 cells showed some areas of apoptosis and necrosis (Fig. 5b). In order to verify the effect of ABCG2 on PDT efficacy, we checked for the cleaved caspase-3 and LC3II level. Cleaved caspase-3 is known as a marker of apoptosis and LC3II suggests autophagic cell death [19, 20]. Cleaved caspase-3 level in SW480 was further increased at 24 h after PDT than SW480/ABCG2 (Fig. 5c) and SW480/ABCG2 cells showed low level of LC3II than SW480 at post-24 h PDT (Fig. 3c). The above data indicate that ABCG2 expression level in colon cancer cells is inversely related with PDT efficacy, and that ABCG2 inhibition could therefore increase cancer cell death.Fig. 4


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 singlet oxygen production rate & cell death between SW480 and SW480/ABCG2 cells. a, SW480, SW480/ABCG2, and HT29 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. b, Cells were harvested 4 h after photosensitization and stained with Annexin V/PI for FACS analysis. c, Cells were incubated with 100 nM PPa for 16 h followed by irradiation. Cell lysates were subjected to immunoblot with indicated antibodies. 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

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494642&req=5

Fig5: Differences of singlet oxygen production rate & cell death between SW480 and SW480/ABCG2 cells. a, SW480, SW480/ABCG2, and HT29 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. b, Cells were harvested 4 h after photosensitization and stained with Annexin V/PI for FACS analysis. c, Cells were incubated with 100 nM PPa for 16 h followed by irradiation. Cell lysates were subjected to immunoblot with indicated antibodies. Data are means ± SEM from three independent experiments (*P < 0.05, **P < 0.01, #P < 0.0001)
Mentions: As mentioned above, ABCG2 plays a major role in inducing drug resistance and determining the efficacy of PDT. In order to show that ABCG2 is directly related with PDT efficacy, we established SW480 that stably overexpress ABCG2 using retrovirus. The expression level of ABCG2 in SW480 and SW480/ABCG2 was confirmed by western blot analysis and immunocytochemistry with anti-ABCG2 antibody (Fig. 4a). SW/ABCG2 cells with overexpression of ABCG2showed higher level of survival rate compared to SW480 and HT29, which indicates that overexpression of ABCG2 is directly related with the efficacy of PDT and drug resistance (Fig. 4b). SW480/ABCG2 cells showed low fluorescence of PPa, whereas SW480 cell showed strong signal of PPa (Fig. 4c). Also, singlet oxygen production rate was decreased in SW480/ABCG2 cells (Fig. 5a). To investigate the effect of PDT on cell viability, we checked for apoptosis or necrosis using flow cytometry analysis with Annexin V and PI. After irradiation, apoptosis and necrosis developed considerably in SW480. SW480 cells with overexpression of ABCG2 showed little necrosis while HT29 cells showed some areas of apoptosis and necrosis (Fig. 5b). In order to verify the effect of ABCG2 on PDT efficacy, we checked for the cleaved caspase-3 and LC3II level. Cleaved caspase-3 is known as a marker of apoptosis and LC3II suggests autophagic cell death [19, 20]. Cleaved caspase-3 level in SW480 was further increased at 24 h after PDT than SW480/ABCG2 (Fig. 5c) and SW480/ABCG2 cells showed low level of LC3II than SW480 at post-24 h PDT (Fig. 3c). The above data indicate that ABCG2 expression level in colon cancer cells is inversely related with PDT efficacy, and that ABCG2 inhibition could therefore increase cancer cell death.Fig. 4

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