Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy and occurs in less than 5% of thyroid carcinoma with an annual incidence of 1-2 cases/million.1 Although the pathogenesis of ATC is unclear, it is considered that ATCs might have multiple genetic abnormalities.1 Although genetic mutation might have an important role in initiating or promoting carcinogenesis and its progression, the understanding of which genetic mutations was affected, and how, is unknown.1 ATC has poor prognosis because it does not respond well to conventional treatments such as surgery, radiation therapy, and chemotherapy. The median survival time has been reported to be about 3-9 months and only 10%-15% alive at 2 years.2
Photodynamic therapy (PDT) is a minimally invasive treatment involving light and a chemical substance a photosensitizing agent.3 Photosensitized cell is activated by a specific wavelength. The photosensitizer absorbs light and transforms from ground state to the excited singlet state. At this point, oxygen free radicals are released and act as cell damage.4 The extent of photodamage and cytotoxicity after PDT in vitro is multifactorial and can depend on the photosensitizing molecule, its localization at the time of irradiation the total administered dose, the total light exposure dose, light fluence rate, the time between administration of the photosensitizer and irradiation, the type of tumor and its level of oxygenation.5 Three independent processes were contributed to efficient PDT-induced tumor destruction: direct cancer cell death (apoptosis), destruction of tumor vasculature and activation of an immune response.5 Among three independent processes, it is considered that direct cancer cell death (apoptosis) is main mechanism of PDT induced cell death.
PDT is recognized as alternative modality of cancer treatment, besides PDT is used in ophthalmology as anti-angiogenesis treatment and dermatology.6,7 The advantage of PDT is that it is non-invasive, lack of pain, and can be combined with conventional treatments such as surgery, radiation therapy, and chemotherapy. In addition, there is no limit to the number of times photosensitizer injection of laser therapy can be performed.4 Photofrin is one of the widely used photosensitizer. Photofrin-mediated PDT was first given approval in 1993 by the Canadian health agency for use against bladder cancer and later in Japan, USA and parts of Europe for use against cancers of the esophagus and non-small cell lung cancer.3 Photofrin has a maximum absorption wavelength at 625 nm. Therefore, the skin penetration depth is shallow. Despite this drawback, photofrin is currently the most widely used photosensitizer.8
Currently, the treatment of ATC centered not on its cure, but on its salvage. PDT is usually not applied to the treatment of ATC, but its efficacy is being proven by various other studies.9,10 Therefore, this study will focus on PDT’s validity and efficacy on the anaplastic thyroid cancer cell line.
The photosensitizer, porfimer sodium, sold as Photofrin (QTL Photo Therapeutic Inc., Vancouver, BC, Canada) has maximum absorption peak at 630 nm. Photofrin was diluted in the culture medium just before used. For cell culture, all medium supplements (RPMI-1640 [Roswell Park Memorial Institute] medium, fetal bovine serum [FBS], and antibiotics) were supplied by Welgene (Daegu, Republic of Korea). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenlyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Hoechst 33342 dye, propidium iodide (PI) dye were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bradford dye reagent was supplied by Bio-rad (Hercules, CA, USA). Caspase-3, caspase-8 and poly (ADP-ribose) polymerase (PARP) were from Calbiochem (San Diego, CA, USA); caspase-9 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Abcam (Cambridge, UK). MitoTracker® and ER-TrackerTM were supplied by Molecular Probes (Eugene, OR, USA).
As the cell line, SNU-80 derived from human anaplastic thyroid carcinoma was used. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% streptomycin/penicillin (Gibco, BRL, Waltham, MA, USA) in an atmosphere of 5% CO2 and 95% air at 37°C in a humidified incubator.
SNU-80 cells were diluted at a concentration 5 × 104 cells/ml and inoculated into a 96-well microplate at a volume of 100 μl. Cells were incubated for 24 hours in an incubator (5% CO2, 37°C) for attachment to well. The culture medium was changed before laser irradiation at room temperature. Various concentrations of photofrin (0-50 μg/ml) were treated to the cells. After incubation for 6 hours in dark conditions, the cells were irradiated with 630 nm diode laser at an intensity of 2.0 J/cm2 for 15 minutes. After irradiation, cells were incubated for 4, 8, or 24 hours.
Cell viability was measured by MTT assay. The cells were diluted at a concentration 5 × 104 cells/ml and inoculated into a 96-well microplate and were treated by protocol of PDT. Twenty four hours after PDT, 50 μl of MTT solution (2 mg/ml) was added to the cells and the cells were incubated for 4 hours. The culture medium and MTT solution were replaced by 100 μl of DMSO. After 30 seconds of shaking, absorbance at 540 nm measured by microplate reader (Bio-rad 550; Bio-rad).
Cell viability (%) = mean absorbance in PDT group/mean absorbance in control group
To evaluate apoptosis and necrosis induced by Photofrin-PDT, morphologic and color change of nuclei were detected by double-staining. Cells were seeded on 6-well plate (1 × 105 cells/ml) and were treated by protocol of PDT. After different time points (4, 8, 24 hours) after PDT, cells were incubated with Hoechst 33342 (2 μg/ml) for 30 minutes. After culture medium was changed, cells were incubated with PI (2 μg/ml) for 10 minutes before observation by confocal microscope (Zeiss LSM510 META; Carl Zeiss, Jena, Germany). In Hoechst 33342 and PI double staining, nuclear condensation and fragmentation stained bright blue by Hoechst are considered as apoptosis. Cells whose nuclei are stained red by PI is considered as necrotic cells because PI cannot penetrate the cell membrane.
To identify the localization of photofrin, cells were incubated with 3.2 μg/ml photofrin in culture medium for 6 hours. During the last 30 minutes, Cells were co-loaded with MitoTracker®, ER-TrackerTM. Signal from organic probes and photofrin was observed with confocal microscope. MitoTracker® was excited with a 488 nm argon laser and ER-TrackerTM excited with 405 nm diode laser. Photofrin was excited a 543 nm helium/neon laser.
The treated cells were washed twice with cold Dulbecco’s phosphate-buffered saline (DPBS) and total proteins were extracted in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 0.1% deoxy sulfate) with protease and phosphatase inhibitor. The protein concentration was evaluated with a Bradford assay. Equivalent amounts of protein from each sample were loaded onto 12% polyacrylamide gels, subjected to electrophoresis, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% skin milk for 1 hour, polyvinylidene difluoride (PVDF) membranes were incubated with the primary antibodies at 4°C overnight. After treated with primary antibodies (caspase-3, -8, -9 and PARP) for 1 hour at room temperature, the membrane was washed (×3) with phosphate buffered saline with tween (PBST) for 30 minutes. Each membrane was probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody for 1 hour. The bounded protein bands were detected by a Kodak image analyzer (Eastern Kodak, Rochester, NY, USA).
The experiments were carried out according to the guidelines set by the Institutional Animal Care and Use Committee at Dankook University.
Statistical analysis was performed using IBM SPSS statistics ver. 20 (IBM Co., Armonk, NY, USA). Analysis was performed using the Mann-Whitney U-test, comparing cell viability as it related to photofrin concentration.
To evaluation cytotoxiciy of photofrin-PDT, MTT assay was performed for variable concentration of photofrin. The extent of cytotoxicity was dependent on concentration of photofrin. Cell viability was 46.7 ± 6.3% and 10.4 ± 8.3% at the concentration of photofrin 1.6 μg/ml and 3.2 μg/ml respectively (Fig. 1). We selected these two concentrations for experimental groups.
The fluorescence signal revealed red, green, and blue-white for photofrin, mitochondria, and endoplasmic reticulum (ER) respectively. Most of the fluorescence signal from photofrin overlapped with the location of the mitochondria in merge image, while there was only partial overlap with ER (Fig. 2). It suggested that photofrin, which was photosensitizer, was accumulated mainly mitochondria in the cytoplasm of SNU-80 anaplastic thyroid cancer cell line.
Hoechst 33342/PI double staining was used to evaluated apoptotic and necrotic cell death. As shown in Fig. 3, control group showed intact homogeneous blue nuclei as time was progressed. At 4 hours after PDT, PDT group using photofrin concentration of 1.6 μg/ml (PDT 1.6 group) and photofrin 3.2 μg/ml (PDT 3.2 group) showed similar morphology of nuclei with control group. On the other hand, condensed or fragmented bright blue nuclei were observed in PDT 1.6 group at 8 hours after PDT, which proved that apoptosis was initiated. At 24 hours after PDT, condensed/fragmented bright blue and condensed pink nuclei were observed in PDT 1.6 group, which meant apoptosis and necrosis. Condensed/fragmentated bright blue and pink nuclei were observed in PDT 3.2 group at 8 hours after PDT. At 24 hours after PDT, there were more fragmented and condensed pink nuclei in PDT 3.2 group than PDT 1.6 group (Fig. 3).
Apoptotic ratio was measured using confocal microscope. The apoptotic cells were observed as nuclear condensation or fragmentation in Hoechst staining. Apoptotic cells were counted at 4, 8, and 24 hours after PDT. We counted the number of apoptotic cells with confocal microscope at five random fields from ×200 magnification power and calculated the ratio of apoptotic and total cell number. PDT 3.2 group had apoptosis ratio of 25.4 ± 4.8% at 8 hours after PDT, which turned to 4.6 ± 2.5% after 24 hours. In the PDT 1.6 group, the apoptosis ratio after PDT was 15.1 ± 3.1% at 8 hours, and 6.8 ± 3.2% at 24 hours (Fig. 4). Necrotic ratio was also measured using confocal microscope. The nucleus of necrotic cell was stained red in PI stating. Necrotic cells were counted at 4, 8, and 24 hours after PDT. We counted the number of necrotic cells with confocal microscope at five random fields from ×200 magnification power and calculated the ratio of necrotic and total cell number. In the PDT 3.2 group, the necrosis ratio after PDT was 1.4 ± 1.5% at 8 hours, and 17.4 ± 3.8% at 24 hours. In the photofrin 1.6 group, there was no necrosis at 8 hours after PDT, while the necrosis ratio at 24 hours was 12.6 ± 2.3% (Fig. 5).
In comparison with control group, the expression of caspase-3, -9, and PARP was increased in PDT group, whereas caspase-8 expression in PDT group was similar pattern in control group. The expression of caspase-3 increased according to increasing concentrations of photofrin. The expression of caspase-9 and PARP, the initiator caspase of mitochondrial death pathway and the native substrate of caspase-3, respectively, proved similar expression patterns with caspase-3. Enhanced apoptotic cell death, due to increase of caspase-3, -9 and PARP cleavage, was increased (Fig. 6).
PDT has been used in a wide variety of settings since its first introduction in the 1980’s. PDT was applied for the treatment of oral, laryngeal, esophageal, gynecologic, and lung cancers. Recently, it is being used in an ever-expanding variety of settings, for example by ophthalmologists for its anti-angiogenesis effects to treat new vessel growth, as well as by dermatologists on non-neoplastic skin lesions.6
PDT is not being used in the clinical setting to treat ATC, but several studies have confirmed its efficacy. Al-Watban and Zhang9 reported that PDT using topical ALA (5-aminolevulinic acid) cream is effective in delaying the growth of ATC-bearing nude mice. ALA is a photosensitizer and frequently used as topical type like cream. In this study, the percentage of tumor growth delay was –48.13 ± 13.76 at 7 days after PDT, –54.9 ± 8.83 at 14 days after PDT, and –51.11 ± 8.95 at 21 days after PDT.9 Catalano et al.10 reported that PDT, high energy shock waves, and paclitaxel were used as multimodal therapy to increase their cytotoxic effects, and again this showed enhancement in the induction of apoptosis in vitro.
In several other study, photofrin-PDT has cytotoxicity of various cancer cells such as malignant melanoma, esophageal cancer, and lung cancer.7,11 PDT has been applied for early stage oral or laryngeal cancer and overall rate of complete remission was 85%. It is also indicated for symptom relief of end-stage obstructive esophageal cancer and advanced airway obstructive lung cancer. Photodynamic therapy for skin cancer was tried in basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, and malignant melanoma, and showed a therapeutic effect of about 40% in squamous cell carcinoma or Kaposi's sarcoma. However, the therapeutic effect is low in basal cell carcinoma or malignant melanoma with high pigmentation, and treatment is attempted in malignant melanoma with low pigmentation. PDT is delivered differently according to the tumor sites. In larynx cancer, tracheobronchial and lung cancer or esophageal cancer, Laser irradiation is delivered through direct laryngoscope, bronchoscope or esophagoscope. In melanoma or thyroid cancer, laser irradiation is done through skin.
In our study, we demonstrated that photofrin-PDT has cytotoxicity for anaplastic thyroid carcinoma cell line. Cell viability of 24 hours after PDT was 46.7 ± 6.3% and 10.4 ± 8.3% at the concentration of photofrin 1.6 μg/ml and 3.2 μg/ml respectively. And these results showed significantly decreased cell viability than that of photofrin only group (Fig. 1). There was a significantly increased number of condensed/fragmented blue nuclei and condensed/fragmented pink nuclei in PDT group than those in control group (Fig. 3). Also, the apoptosis of SNU-80 anaplastic cancel cells peaks at 8 hours, although not measured every hour. Furthermore, we proved that apoptosis and necrosis were progressed with increasing concentration of photofrin and delaying the time after PDT.
It is generally accepted that the subcellular localization of the photosensitizer coincides with the primary site of photodamage.5 Meanwhile, the molecular nature of the photo-oxidized targets has a profound influence on the signaling pathways and mode of cell death initiated following PDT.12 Therefore, the lipophilic sensitizers, such as photofrin, preferentially accumulate in the lipophilic compartments of the tumor cells, including the plasma, mitochondria, endoplasmic reticulum, nuclear and lysosomal membranes.13 Our data revealed that mitochondria was mainly localized site in SNU-80 anaplastic thyroid cancer cell line (Fig. 3). Chang et al.14 suggested the similar subcellular localization in malignant melanoma cells as our study, whereas the other study suggested that photofrin was localized in Golgi apparatus, plasma membrane.15 Because photofrin was localized in the mitochondria of SNU-80 cells in our study, we expected that main cell death mechanism of photofrin-PDT is mitochondrial apoptosis.
The apoptotic caspase pathways have two main converging pathways, called extrinsic and intrinsic, in which initiator caspase-8/-10 and -9. directly activate the effector caspase-3, -7.5 Caspase-3 plays an important role in the execution of apoptosis and is primarily responsible for the cleavage of PARP during cell death.16 The activation of PARP by DNA strand breaks contributes to the consumption of nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) that occurs in cells undergoing apoptosis.16 In our study, there was enhanced expression of cleaved form of caspase-3, -9 and PARP in PDT group compared to control group (Fig. 6). Meanwhile, the expression of casapase-8 in PDT group was similar to that in control group (Fig. 6). We analyzed it, thinking that showing a dose dependent pattern would be more evidence that PDT has cytotoxicity in anaplastic thyroid cancer cells. In Hoechst/PI double staining, cytotoxicity was dose-dependent, and in western blot analysis, there was no strict difference between PDT 1.6 and PDT 3.2 group. However, compared to control group (number 1), it can be seen that apoptosis increases through caspase pathway clearly in PDT groups (number 2 and 3) (Fig. 6). The increased expression of cleaved caspase-3, -9 and PARP coincide with an enhanced cytotoxic and apoptotic effect for PDT. Therefore, we suggested that the mechanism of photofrin-PDT may relate to mitochondria-mediated apoptosis.
The limitation of this study is that the dose-dependent relationship is not clearly confirmed in MTT because photofrin could be related to its inherent cytotoxicity at higher concentrations. Also, this study is limited within the context of killing cancer cells and did not cover the appropriate dose of photofrin which has minimal effect on normal cells under PDT. In conclusion, our study demonstrated that PDT using photofrin as photosensitizer has cytotoxic effect on SNU-80 anaplastic thyroid carcinoma cell line. Furthermore, photofrin-PDT led to in vitro cell death through mitochondrial mediated apoptosis.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1I1A3072797).
No potential conflict of interest relevant to this article was reported.
Concept and design: SJL. Analysis and interpretation: SHW. Data collection: SHW. Writing the article: KWK. Critical revision of the article: SJL. Final approval of the article: SJL. Statistical analysis: PSC. Obtained funding: PSC. Overall responsibility: SJL.