Invited Symposium: Photodynamic Therapy



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in Vitro Induction of Resistance in Human Tumour Cells to PDT by Various Photosensitizers

Contact Person: Joanne Taylor (joanne_taylor@hrcc.on.ca)


Interest in the phenomenon of the induced photodynamic therapy (PDT) resistance is recent and very few studies have been published. The use of PDT induced resistant cells in the elucidation of the mechanisms of PDT has shown that direct tumour cell kill is an important component of tumour responsiveness (1,2). However, the mechanisms responsible for a direct tumour cell kill have not been completely identified. Various cellular components have been identified as the primary targets for initiating a stress response in a cell that ultimately leads to apoptosis or necrosis of the tumour (3,4,5).

Selection of three photosensitizers namely Aluminum phthalocyanine (ALPcSu), Nile Blue A and Photofrin for comparison are based on the hypothesis that the mechanisms of action will be different for each of the different photosensitizers, since the intracellular localizations are unique and consequently the targets for direct tumour cell phototoxicity are also expected to be unique for each photosensitizer. In addition, the use of various human tumour cells with varying degree of inherent sensitivity to various photosensitizers will provide correlative evidence for particular subcellular targets based on the morphological differences in the cell types. This study shows that both inherent and induced resistance to various photosensitizers is indicative of subcellular targets.

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Materials and Methods

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.

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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.

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Discussion and Conclusion

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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1. Adams KE, Espiritu M, Wilson BC, Singh G. Photodynamic therapy-resistant cells maintain their resistance in vivo. Amer Soc. Photobiol, Atlanta (abstract). 1996.

2. Sharkey SM, Wilson BC, Moorehead R, Singh G. Mitochondrial alterations in photodynamic therapy resistant cells. Cancer Research: 5, 34994-4999, 1993.

3. Lin CW, Shoulok JR, et al. Photodynamic destruction of lysomes mediated by Nile Blue photosensitizers. Photochem Photobiol: 58(1), 81-91, 1993.

4. Singh G, Jeeves WP, Wilson BC, Jang D. Mitochondrial photosensitization by photofrin II. Photochem Photobiol: 46, 6450649, 1987.

5. Luna MC, Gomer CJ. Isolation and initial characterization of mouse tumour resistant to porphyrin-mediated photodynamic therapy. Cancer Res: 51, 4243-4249, 1991.

6. Singh G, Wilson BC, Sharkey SM, et al. Resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and chinese hamster ovary multidrug resistant cells in vitro. Photochem Photobiol: 54(2), 307-312, 1991.

7. Gomer CJ, Rucker N, Wong S. Porphyrin photosensitivity in cell lines expressing a hear resistant phenotype. Proc SPIE: 1203, 185-195, 1990.

8. Mithcell JB, Glatstein E, Cowan KH, Russo A. Photodynamic therapy of multidrug resistant cell lines. Proc AACR:29, 315, 1988.

9. Giannotti CA. Andriamanpandry A, Werner GH. Studies on resistance of human cancer cells to photosensitizing phthalocyamines. 3rd Biennial Meeting of International Photodynamic Association, Buffalo, NY. 1990 (Abstract No. I/4).

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Singh, G; Espiritu, M; Rainbow, A; (1998). in Vitro Induction of Resistance in Human Tumour Cells to PDT by Various Photosensitizers. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/rainbow/singh0761/index.html
© 1998 Author(s) Hold Copyright