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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

Effect of Ko-143 on PPa treated colon cancer cell in PDT. a, Cells were irradiated by 670 nm light (4 J/cm2) after 16 h incubation with indicated concentration of PPa in the presence or absence of 1 μM Ko-143. At 24 h after PDT, the MTT assay was performed. b, Cells were incubated with 100 nM PPa for 16 h in the presence or absence of 1uM Ko-143 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 in the presence or absence of 1uM Ko-143 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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Fig3: Effect of Ko-143 on PPa treated colon cancer cell in PDT. a, Cells were irradiated by 670 nm light (4 J/cm2) after 16 h incubation with indicated concentration of PPa in the presence or absence of 1 μM Ko-143. At 24 h after PDT, the MTT assay was performed. b, Cells were incubated with 100 nM PPa for 16 h in the presence or absence of 1uM Ko-143 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 in the presence or absence of 1uM Ko-143 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 above findings proved that ABCG2 plays a major role in the resistance of PDT, which could be prevented by using Ko-143, an inhibitor of ABCG2 transporter [18]. To confirm whether the blockage of ABCG2 could increase the effect of PDT in colon cancer, we tested the cell survival rate and singlet oxygen production. Cells were pretreated with 1 μM of Ko-143 for 1 h and then incubated with PPa. There was no change in the SW480 cell survival rate after the inhibition of ABCG2 (Fig. 3a). Contrastingly, HT29 cells showed decreased cell survival rate derived from ABCG2 protection (Fig. 3a). Combined treatment of PPa with Ko-143 enhanced the sensitivity of HT29 cell to PDT. To further investigate the ABCG2 inhibition effect, we measured singlet oxygen production of ABCG2 treated cells and non-treated cells. Singlet oxygen production rate was increased afterABCG2 inhibition in both SW480 and HT29. SW480 cells showed a little increase in the singlet oxygen, with no effect on cell survival rate. On the other hand, HT29 cells treated with Ko-143 showed more singlet oxygen production compared to other cells (Fig. 3b). To clarify that ABCG2 was inversely related with PPa accumulation, we measured fluorescence of PPa in Ko-143treated cells using fluorescence microscope. The level of PPa fluorescence in HT29 cells was remarkably increased by Ko-143 (Fig. 3c). It seems that ABCG2 inhibition increased the sensitivity to PDT by blocking PPa efflux and thereby inducing high level of singlet oxygen.Fig. 3


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)

Effect of Ko-143 on PPa treated colon cancer cell in PDT. a, Cells were irradiated by 670 nm light (4 J/cm2) after 16 h incubation with indicated concentration of PPa in the presence or absence of 1 μM Ko-143. At 24 h after PDT, the MTT assay was performed. b, Cells were incubated with 100 nM PPa for 16 h in the presence or absence of 1uM Ko-143 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 in the presence or absence of 1uM Ko-143 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

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

Fig3: Effect of Ko-143 on PPa treated colon cancer cell in PDT. a, Cells were irradiated by 670 nm light (4 J/cm2) after 16 h incubation with indicated concentration of PPa in the presence or absence of 1 μM Ko-143. At 24 h after PDT, the MTT assay was performed. b, Cells were incubated with 100 nM PPa for 16 h in the presence or absence of 1uM Ko-143 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 in the presence or absence of 1uM Ko-143 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 above findings proved that ABCG2 plays a major role in the resistance of PDT, which could be prevented by using Ko-143, an inhibitor of ABCG2 transporter [18]. To confirm whether the blockage of ABCG2 could increase the effect of PDT in colon cancer, we tested the cell survival rate and singlet oxygen production. Cells were pretreated with 1 μM of Ko-143 for 1 h and then incubated with PPa. There was no change in the SW480 cell survival rate after the inhibition of ABCG2 (Fig. 3a). Contrastingly, HT29 cells showed decreased cell survival rate derived from ABCG2 protection (Fig. 3a). Combined treatment of PPa with Ko-143 enhanced the sensitivity of HT29 cell to PDT. To further investigate the ABCG2 inhibition effect, we measured singlet oxygen production of ABCG2 treated cells and non-treated cells. Singlet oxygen production rate was increased afterABCG2 inhibition in both SW480 and HT29. SW480 cells showed a little increase in the singlet oxygen, with no effect on cell survival rate. On the other hand, HT29 cells treated with Ko-143 showed more singlet oxygen production compared to other cells (Fig. 3b). To clarify that ABCG2 was inversely related with PPa accumulation, we measured fluorescence of PPa in Ko-143treated cells using fluorescence microscope. The level of PPa fluorescence in HT29 cells was remarkably increased by Ko-143 (Fig. 3c). It seems that ABCG2 inhibition increased the sensitivity to PDT by blocking PPa efflux and thereby inducing high level of singlet oxygen.Fig. 3

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