Chemicals. Photofrin II was supplied by QLT Photo Therapeutics Inc., Vancouver, BC, Canada. Nile Blue-A was obtained from Sigma-Aldrich Canada, Ltd. And Aluminum Phthalocyanine Tetrasulphonate was obtained from Porphyrin Products, Logan, Utah, USA.

Cells. Human cell lines HT29, HT1376 and SK-N-MC were obtained from The American Type Culture Connection, Rockville, Maryland, USA. PDT-induced resistant variants were derived from these cells. All cell lines were maintained as monolayer cultures in Alpha-Minimum Essential Medium containing L-glutamine, deoxyribonucleoside and ribonucleoside, supplemented with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 mg/ml) amphotericin-B as fungizone (0.25 mg/ml), that were obtained from GIBCO/BRL, Canada. The cells were incubated at 37o C in a humidified 5% CO2 atmosphere.

To isolate PDT-resistant variants, we used LD90 which was first determined by dose response of the wild type cell lines to the three photosensitizers (Photofrin II, Aluminum Phthalocyanine Tetrasulphonate, Nile Blue-A). Cells were incubated with photosensitizers (LD90) for 24 hours (PH II AIPcS4) or one hour for Nile Blue-A. Then light irradiated for five minutes with PH II and AIPcS4 or irradiated for fifteen minutes with Nile Blue-A. Single surviving colonies were harvested, grown individually and subjected to further courses of PDT. Eight to eleven cycles were necessary to generate resistant variants.

We have successfully generated resistant variants to AIPCS4 , Nile Blue A and Photofrin in HT29 cells and to Nile Blue A in HT1376. However, we have been unable to induce resistance to any photosensitizer in SK-N-MC cells. Table 1 below illustrates the D10 ratio of resistant versus parental cells.

Table 1

        Nile Blue-A     Photofrin II      AIPcS4
HT29    2.62 ± 0.265    1.50 ± 0.117    2.16 ± 0.42
HT1376  2.81 ± 0.36     1.15 ± 0.04     0.99 ± 0.02
SK-N-MC 1.08 ± 0.07     0.99 ± 0.032    1.11 ± 0.041

Our data presented in Figures 1 to 3 shows intrinsic sensitivity to the three photosensitizers, namely Photofrin, Aluminum phthalocynaine tetrasulphonate and Nile Blue A in four human cell lines namely human ovarian carcinoma (2008), human neuroblastoma (SK-N-Mc), human colon adenocarcinoma (HT29) and human bladder carcinoma cells (HT1376). The data clearly demonstrates that the inherent sensitivity of various tumour cells is cell type dependent and furthermore, the sensitivity is also dependent on the type of photosensitizer.

The primary goals of this study were to derive PDT-resistant variants of human tumour cells and to examine the inherent sensitivity of different tumour cells to various photosensitizers. The various early results are intriguing and suggest two ways in which such induced resistance may be important. First, the existence of PDT-induced resistance in vitro may also imply that a degree of resistance could also be induced in vivo, depending on the role played by direct tumour cell killing. This may have to be considered in clinical PDT when multiple or fractionated treatments are given. Secondly, and of special interest is the potential to use induced resistance as a powerful tool for understanding the mechanisms of inherent sensitivity of cells and pathways that are important in PDT cellular sensitivity.

Cells do not appear to be cross-resistant to hyperthermia and photofrin-mediated PDT, suggesting different cytotoxic mechanisms of cellular resistance by dark exposure to photosensitizers suggests mechanisms akin to those of classical p-glycoprotein mediated multi-drug resistance, whereas, PDT (photosensitizer + light)-induction does not appear to be drug-transport related (6,7). Studies of PDT cross-resistance in MDR cells are conflicting with both positive (6,8,9) and negative (5) findings. We will examine mechanisms of resistance to various photosensitizers in the unique resistant variants that we have generated.

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