Endogenously generated protoporphyrin IX in drug-resistant and sensitive murine leukaemia cells

DIEZ, BERENICE; ERNTZ, G; CORDO RUSSO, R; TEIJO, JULIETA; HAJOS S; BATLLE, ALCIRA; FUKUDA, HAYDÉE

Cardiff

Congreso; International Porphyrins and Porphyrias Meeting; 2011

British Association of Dermatologists

Endogenous production of protoporphyrin IX (PpIX) is successfully exploited for photodynamic therapy (PDT) on malignant cells, following 5-aminolaevulinic acid (ALA) administration and light irradiation. We studied the synthesis of PpIX in three murine leukaemia cell lines: LBR- (sensitive to antineoplastic drugs), LBR-D160 (resistant to doxorubicin) and LBR-V160 (resistant to vincristine) after incubation with 1 mmol L)1 ALA for different lengths of time; LBR-D and LBR-V resistance is mediated by phospho-glycoprotein (Pgp). Porphyrin synthesis (pmol porphyrins/106 cells) increased with incubation time, and accumulated at highest levels in the LBR-V160 cell line. (LBR-: 3 h, 15Æ2 ± 0Æ6; 4 h, 22Æ5 ± 1Æ1; 5 h, 34Æ5 ± 0Æ9; 6 h, 35Æ4 ± 1Æ3. LBRD160: 3 h, 22Æ2 ± 0Æ8; 4 h, 27Æ5 ± 1Æ2; 5 h, 36Æ6 ± 1Æ3; 6 h, 35Æ4 ± 0Æ8. LBR-V160: 3 h, 25Æ2 ± 1Æ6; 4 h, 51Æ6 ± 0Æ7; 5 h, 66Æ1 ± 1Æ5; 6 h, 75Æ4 ± 1Æ1). We also evaluated intracellular and extracellular PpIX content. At short times (2–3 h) of incubation with ALA the proportion of extracellular PpIX was higher than the intracellular content. As the incubation time increased both values became similar, and after 20 h incubation again the extracellular porphyrin content increased (percentage of extracellular PpIX: LBR-: 3 h, 71Æ2 ± 2Æ1; 4 h, 51Æ2 ± 1; 5 h, 55Æ2 ± 1; 20 h, 78 ± 4Æ95. LBR-D160: 3 h, 63Æ4 ± 1Æ47; 4 h, 36Æ3 ± 1Æ62; 5 h, 43Æ4 ± 0Æ3; 20 h, 77Æ2 ± 8Æ05. LBR-V160: 3 h, 69Æ3 ± 2Æ1; 4 h, 50Æ8 ± 1Æ3; 5 h, 55Æ1 ± 2Æ4; 6 h, 66Æ7 ± 1Æ1; 20 h, 81Æ9 ± 0Æ6). Externalization of PpIX is probably not related to Pg-p as it also occurs in the sensitive cell line. To visualize the intracellular PpIX distribution with fluorescence and confocal microscopy, LysoTracker Green and MitoTracker Green were used for lysosome and mitochondria identification, respectively. The granular pattern and fluorescence distribution were similar in the three lines. At short times of incubation PpIX showed a high concentration in mitochondria and adjacent areas, and as the incubation time increased the fluorescence pattern diffused to all the cytoplasm. PpIX fluorescence was also observed in lysosomes but to a lesser extent than in mitochondria; however, it increased at long times of incubation. Illumination of cells after incubation with ALA and assessment of phototoxicity using the MMT assay showed that viability of cells decreases with the irradiation time. Although the LBR- cell line did not accumulate the highest level of PpIX, it was more sensitive to the PDT treatment than the resistant lines. The efficacy of ALA-PDT on LBR-D160 and LBR-V160 cell lines suggests the possible application of the therapy to overcome multidrug resistance to eradicate minimal residual disease in patients with leukaemia.