Photodynamic Therapy: Disjointed Cell Cycle Phase- Related Activity Accounts for Synergistic Outcome in Metastatic Non–Small Cell Lung Cancer Cells (H1299)
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776 Low doses of cisplatin or gemcitabine plus Photofrin/ photodynamic therapy: Disjointed cell cycle phase- related activity accounts for synergistic outcome in metastatic non–small cell lung cancer cells (H1299) 1 1 Elvira Crescenzi, Angela Chiaviello, accumulation of G0-G1 cells (and increased p21 expres- Gianfranco Canti,2 Elena Reddi,3 sion). Like photodynamic therapy, low-dose gemcitabine 1 1 Bianca Maria Veneziani, and Giuseppe Palumbo targeted G0-G1 cells, which caused a massive accumula- tion of cells in S phase (and increased cyclin A expression). 1Dipartimento di Biologia e Patologia Cellulare e Molecolare Although we observed therapeutic reinforcement with ‘‘L. Califano’’ and Istituto di Endocrinologia ed Oncologia both drugs and photodynamic therapy, reinforcement was Sperimentale/Consiglio Nazionale delle Ricerche, Universita`di Napoli Federico II, Naples, Italy; 2Dipartimento di Farmacologia, more pronounced when the drug (CDDP) and photody- Universita`di Milano, Milan, Italy; and 3Dipartimento di namic therapy exert disjointed phase-related cytotoxic Biologia, Universita`di Padova, Padua, Italy activity. Thus, if photodynamic therapy is appropriately tuned, the dose of the cytostatic drug can be reduced without compromising the therapeutic response. [Mol Abstract Cancer Ther 2006;5(3):776–85] We compared the effects of monotherapy (photodynamic therapy or chemotherapy) versus combination therapy (photodynamic therapy plus a specific drug) on the non– Introduction small cell lung cancer cell line H1299. Our aim was to Although chemotherapy is the leading treatment for most evaluate whether the additive/synergistic effects of types and stages of cancers, photodynamic therapy is an combination treatment were such that the cytostatic dose effective anticancer procedure for selected tumors (1). This could be reduced without affecting treatment efficacy. procedure involves administration of a tumor-localizing Photodynamic therapy was done by irradiating Photofrin- photosensitizing agent that when activated by light of a preloaded H1299 p53/p16-null cells with a halogen lamp specific wavelength mediates cell destruction via the equipped with a bandpass filter. The cytotoxic drugs used production of singlet oxygen. Photodynamic therapy has were cis-diammine-dichloroplatinum [II] (CDDP or cisplatin) been shown to have few or no side effects in patients with and 2V,2V-difluoro-2V-deoxycytidine (gemcitabine). Vari- Barrett’s esophagus (high-grade dysplasia and early carci- ous treatment combinations yielded therapeutic effects noma) and in selected cases of early squamous cell (trypan blue dye exclusion test) ranging from additive to carcinoma (2). One of the most widely used photosensitiz- clearly synergistic, the most effective being a combination ing drugs is the hematoporphyrin-derivative Photofrin. of photodynamic therapy and CDDP. To gain insight into This nontoxic substance has been used for photodynamic the cellular response mechanisms underlying favorable therapy for f8 years and is the only one widely approved outcomes, we analyzed the H1299 cell cycle profiles and for human therapy. On exposure of Photofrin-loaded cells the expression patterns of several key proteins after to visible light, intracellular toxic oxygen species (singlet 1 monotherapy. In our conditions, we found that photody- oxygen, O2) are generated that can trigger cell apoptosis or namic therapy with Photofrin targeted G0-G1 cells, thereby necrosis (3, 4). causing cells to accumulate in S phase. In contrast, low- Anticancer drugs target several cellular components and dose CDDP killed cells in S phase, thereby causing an activate responses that go from cell repair to cell death. However, chemotherapy is effective only at high doses, because, at low doses, tumor cells may repair damage and resume their original high proliferation rate (5). Cis- Received 10/14/05; revised 12/23/05; accepted 1/12/06. diammine-dichloroplatinum [II] (CDDP or cisplatin) and Grant support: Ministero dell’Istruzione, dell’Universita`e della Ricerca- 2V,2V-difluoro-2V-deoxycytidine (gemcitabine) are widely COFIN program and Agenzia Spaziale Italiana (Rome, Italy). used in the management of various cancers. By forming The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked adducts in DNA, CDDP inhibits DNA replication and chain advertisement in accordance with 18 U.S.C. Section 1734 solely to elongation, which accounts for its antineoplastic activity. In indicate this fact. clinical practice, CDDP is often combined with other drugs. Note: E. Crescenzi and A. Chiaviello contributed equally to this work. Synergy between CDDP and other chemotherapeutic Requests for reprints: Giuseppe Palumbo, Dipartimento di Biologia e agents occurs by various pathways: increased intracellular Patologia Cellulare e Molecolare ‘‘L. Califano,’’ Universita`di Napoli Federico II, Via S. Pansini 5, 80131 Naples, Italy. Phone: 39-81-7463249; drug accumulation, enhanced binding to DNA, and Fax: 39-81-7701016. E-mail: [email protected] decreased DNA repair (6). Gemcitabine is a deoxycytidine Copyright C 2006 American Association for Cancer Research. analogue that is effective against solid tumors, including doi:10.1158/1535-7163.MCT-05-0425 non–small cell lung cancer. After transport into the cell, Mol Cancer Ther 2006;5(3). March 2006 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 777 gemcitabine requires phosphorylation by deoxycytidine stock solution was obtained by dissolving the powder in kinase to exert biological activity (7, 8). The enzyme water containing 5% glucose to obtain a final concentration responsible for this reaction is also the rate-limiting step of 2.5 mg/mL. This solution was stored in aliquots at in gemcitabine activation (8). It has been suggested that À20jC in the dark. Before measurements, appropriate the combination of photodynamic therapy and convention- aliquots of this solution were diluted to the desired al cancer treatments may become a workable anticancer concentration. Except for ‘‘dark toxicity assays,’’ Photofrin strategy (9). was always used at a concentration of 2.5 Ag/mL. All The aim of our study was to identify photodynamic treatments involving Photofrin were done on triplicate. therapy/chemotherapy combinations in which the dose of Cells were exposed to 2.5 Ag/mL Photofrin for 16 hours the most toxic compound could be decreased without a loss before irradiation. of efficacy. To this aim, we examined the response of H1299 Chemotherapy and Chemotherapy Schedule human lung metastatic non–small cell lung cancer cells Usually, 5 Â 104 H1299 cells were seeded in 35-mm tissue (cell viability, cell cycle, and protein expression) to culture dishes and exposed to the cytotoxic substances moderately toxic doses of CDDP or gemcitabine with (individual or combined with photodynamic therapy) 24 Photofrin/photodynamic therapy, administered separately hours later. This procedure was carried out in triplicate. and in appropriate combinations. Normally, 4 Â 105 cells were used for cell cycle measure- ments and protein extraction for Western blotting. To obtain CDDP/gemcitabine dose-response curves, culture Materials and Methods dishes were treated with CDDP (2.5–12 Amol/L) or Cell Cultures gemcitabine (2–12 nmol/L) and kept at 37jC for 24 hours. The NCI-H1299 human non–small cell lung cancer cell After incubation, cells were washed and released into fresh line was obtained from American Type Culture Collection medium. Cell viability was evaluated with the trypan blue (Rockville, MD). They were cultured in RPMI 1640, 2 assay (as detailed in ref. 10) 24 hours later, counting mmol/L L-glutamine, 10 mmol/L HEPES, 1 mmol/L between 15 and 30 cells per field. Two sets of data were sodium pyruvate, 4,500 mg/L glucose, 1,500 mg/L sodium collected for each drug. In combination experiments, after bicarbonate, 100 Ag/mL streptomycin, 100 units/mL peni- incubation with the drug (0–24 hours) and Photofrin (8–24 cillin, and 10% FCS. The medium was changed every 3 days. hours), cells were washed, placed in a colorless saline All media and cell culture reagents were purchased from solution (Hank’s), and immediately irradiated. After Life Technologies (San Giuliano Milanese, Italy). NCI-H1299 irradiation, cells were placed in fresh complete medium, cells are p53À/À and p16À/À. All treatments, individual and cells negative to trypan blue staining were counted 24 and in combination, were done in triplicate samples in tissue hours later (see Fig. 1). Residual viability was expressed as culture dishes (35 mm) in which 5 Â 104 cells were routinely percentage of trypan blue–negative cells versus untreated seeded. A larger number of cells (4 Â 105)wereseededfor controls. cell cycle measurements and for protein extraction for Photodynamic Therapy and Photodynamic Therapy Western blotting purposes. The effects of monotherapy with Schedule drugs or photodynamic therapy or combined therapy (drugs Cells were irradiated by a broadband light delivered with + photodynamic therapy) were evaluated (a)bytrypanblue a PTL-Penta apparatus (Teclas, Sorengo, Switzerland). This assays (to check residual cell viability), (b) by analyzing cell apparatus consists of a halogen lamp (Osram 250 W, 24 V cycle profiles (to determine the cell distribution in the Osram, Munich, Germany) equipped with a bandpass filter various conditions), and (c)