Paclitaxel Augments Cytotoxic Effect of Photodynamic Therapy Using Verteporﬁn in Gastric and Bile Duct Cancer Cells

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PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences Paclitaxel augments cytotoxic effect of photodynamic therapy using verteporﬁn in gastric and bile duct cancer cells

Seungwoo Park,†a Sung Pil Hong,†a Tae Yoon Oh,b Seungmin Bang,a Jae Bock Chunga and Si Young Song*a,b

Received 11th December 2007, Accepted 25th April 2008 First published as an Advance Article on the web 15th May 2008 DOI: 10.1039/b719072g

Photodynamic therapy (PDT) shows a limited antitumor effect in treating gastrointestinal tumors because of improper light penetration or insufficient photosensitizer uptake. The aim of this study was to evaluate the cytotoxic effect of PDT combined with paclitaxel on in vitro cancer cells. In vitro photodynamic therapy was performed in gastric cancer cells (NCI-N87) and bile duct cancer cells (YGIC-6B) using verteporfin (2 ug mL−1) and a PTH light source (1 000 W, Oriel Co.) with 665–675 nm narrow band pass filter. Cytotoxicity was compared using the MTT assay between cancer cells treated with PDT alone or pretreated with paclitaxel (IC25). Apoptotic changes were evaluated using DAPI staining, DNA fragmentation analysis, Annexin V-FITC apoptosis assay, cell cycle analysis, and western blots for cytochrome c, Bax, and Bid. The PDT-induced cytotoxicity was potentiated by pretreating with low dose paclitaxel (P < 0.001). The enhanced cytotoxicity was due to an augmented apoptotic response mediated by exaggerated cytochrome c released from mitochondria, without Bax or Bid activation. These results show that paclitaxel pretreatment enhances PDT-mediated cancer therapy.

Introduction effect by improving tumor oxygenation and increasing red light penetration in tumors.16 New photosensitizers that can be strongly Photodynamic therapy (PDT) has been used to treat various activated at longer wavelengths have been developed to increase gastrointestinal tumors, including esophageal cancer, dysplasia the ablating zones.13 PDT combined with other drugs, such as in Barrett’s esophagus, stomach cancer, cholangiocarcinoma, inhibitor of vascular endothelial growth factor, cyclooxygenase-2, 1–7 metastatic cancer of the liver, and pancreatic cancer. PDT or matrix metalloproteinase, and anticancer agents also enhanced was performed by administering a photosensitizer followed by the apoptotic response.17–20 Among them, anticancer agents applying a specific wavelength of light. This treatment activates could be easily applied to the cancer patients combined with photosensitizer,which transfers photo-energy to molecular oxygen PDT. 8,9 to generate cytotoxic single oxygen within the irradiated tissues. Paclitaxel, a well-known radiosensitizer, was isolated from the ROS generated by PDT have a direct necrotic and apoptotic effect bark of the Pacific yew, Taxus brevifolia, and binds specifically 10 on tumor cells. PDT also induces vascular injury, tumor hypoxia, to microtubules, alters their dynamics, promotes reorganization and immune response with concomitant expression of several of the microtubule network into bundles or asters, and stabilizes 11,12 growth factors and cytokines. microtubules against disruption by various agents. Microtubule A pitfall of PDT is that the induction of cell death is highly stabilization induced by paclitaxel leads to cell cycle arrest dependent on the penetration depth of the laser and the cellular at the G2/M phase, the most vulnerable phase, resulting in 13 uptake of photosensitizers. Optimal cytotoxic effect can be tumor cell sensitization to ionizing radiation.21,22 The paclitaxel obtained when sufficient photosensitizers are activated by enough radiosensitizing effect in the setting of external radiation might number of photons. Due to improper penetration of light, the be beneficial in PDT treatment. Previous report showed that maximum effect of PDT is less than 1 cm in depth, which limits paclitaxel also enhanced the antitumor effect of photodynamic 14 PDT of curative intent only to early cancers. PDT in advanced therapy in vivo mouse model, however, the action mechanism 5,15 cancers, at most, attains palliation of obstructive symptoms. of paclitaxel combined with phototherapy has not been well Suboptimal delivery of light may cause sublethal damage to elucidated.23 Because there was no difference of antitumor effect cancer cells, leading to recurrence of tumor during follow up. between paclitaxel combined with photodynamic therapy without Many efforts to overcome the sublethal tumor cell damage have photosensitizer and paclitaxel alone, which means that pacli- been tested. A hyperbaric condition enhanced the PDT antitumor taxel itself have no direct phototoxicity, there might be another mechanism to enhance the antitumor effect of phototherapy by aDivision of Gastroenterology, Department of Internal Medicine, Yonsei paclitaxel. University College of Medicine, 134 Shinchon-Dong, Seodaemun-Gu, 120- In the present study, we evaluated the cytotoxic effect and 752, Seoul, South Korea. E-mail: [email protected]; Fax: 82-2- mechanism of PDT using verteporfin combined with paclitaxel 2227-7900; Tel: 82-2-2228-1981 bBrain Korea 21 Project for Medical Science, Yonsei Institute of Gastroen- in gastric and bile duct cancer cells. We treated cells with low terology, Yonsei University College of Medicine, South Korea dose paclitaxel before PDT to minimize the cytotoxicity of † Seungwoo Park and Sung Pil Hong contributed equally to this article. paclitaxel.

This journal is © The Royal Society of Chemistry and Owner Societies 2008 Photochem. Photobiol. Sci., 2008, 7, 769–774 | 769 Results and discussion Table 1 Dose enhancement ratio after PDT combined with low dose paclitaxel. DER was calculated as the dose for PDT alone divided by the Photodynamic therapy using verteporﬁn induced cell death in dose of PDT plus paclitaxel (normalized for drug toxicity) for a surviving fraction of 0.25, 0.5, and 0.75 NCI-N87 and YGIC-6B cells

Cells DER (IC25) DER (IC50) DER (IC75) The MTT assay was performed to determine the IC25 and IC50 light doses at 24 h after PDT using verteporﬁn (2.78 uM) in NCI-N87 NCI-N87 1.27 1.25 1.24 and YGIC-6B cells. Cancer cell survival decreased according to YGIC-6B 1.28 1.19 1.16 −2 light dose intensity.The IC25 and IC50 light doses were 310 mJ cm and 565 mJ cm−2 in NCI-N87, and 240 mJ cm−2 and 450 mJ cm−2 of paclitaxel. Fig. 1 shows dose-response survival curves of the in YGIC-6B cells, respectively (Fig. 1). NCI-N87 and YGIC-6B cells when treated with PDT with or

without paclitaxel. The IC50 value was signiﬁcantly decreased in paclitaxel pretreated NCI-N87 cells compared with PDT alone cells (565 mJ cm−2 versus 453 mJ cm−2, P < 0.001). In YGIC-

6B cells, IC50 value showed signiﬁcant fall after PDT combined with paclitaxel (451 mJ cm−2 versus 379 mJ cm−2, P < 0.001). To evaluate the enhancing cytotoxicity of paclitaxel pretreatment, we borrowed the concept of dose enhancement ratio which has been used to measure the radiosensitizing effect of an anticancer drug.24,25 A value greater than 1.0 denotes an enhancer and greater than 1.1 denotes a strong enhancer. the dose enhancement ratio

(DER) was calculated at IC25,IC50,andIC75 dose of photosensi-

tization in NCI-N87 and YGIC-6B cells (Table 1). DER of IC25,

IC50,andIC75 were 1.27, 1.25, and 1.24 in NCI-N87 cells and 1.28, 1.19, and 1.16 in YGIC-6B cells, respectively. These results that DERs were greater than 1.1 for both cells demonstrated that paclitaxel pretreatment augmented PDT-induced cytotoxicity. Paclitaxel, a microtubule stabilizer, showed promising antitumor activity and a radiosentitizing effect when combined with external radiation for gastric and bile duct cancer.26–29 Paclitaxel inhibits chromosomal segregation by stabilizing microtubules and leads to a cell cycle arrest at the G2-M phase. This arrest results in apoptosis and tumor cell sensitization to ionizing radiation.21,22 Our results suggested that paclitaxel could be a potent radiosensitizer to overcome limited antitumor effect of PDT.

Pretreatment with low dose paclitaxel potentiated PDT-induced apoptosis

To elucidate the mechanism of PDT-induced cytotoxicity, we ﬁrst investigated cellular morphologic changes by 4-6-diamidino- 2-phenylindole (DAPI) staining at 4 h and 24 h after PDT

(IC50) in NCI-N87 and YGIC-6B cells. As shown in Fig. 2, Fig. 1 Survival curve of NCI-N87 and YGIC-6B cells treated with PDT pycknotic nuclear changes and chromatin condensation were alone versus PDT combined with paclitaxel pretreatment. Cytotoxicity seen 4 h after PDT, which is suggestive apoptotic change. The was measured using the MTT assay 24 h after PDT using verteporﬁn (2 ug mL−1) with or without paclitaxel (NCI-N87, 4.8 nM; YGIC-6B, 5.1 nM). morphological changes were exaggerated 24 h after PDT. As Survival curves were corrected for the cytotoxic effect of paclitaxel. Bars, showninFig.3,DNAfragmentation,whichisatypicalsignof mean ± standard deviation. apoptosis, appeared as early as 1 h after PDT in NCI-N87 cells and the DNA fragmentation was augmented as time dependent manner. Apoptotic fractions measured by ﬂow cytometry with Pretreatment with low dose paclitaxel enhanced PDT-induced Annexin V-FITC staining at 0 h, 1 h, and 3 h after PDT in ± ± ± cytotoxicity NCI-N87 cells were 11.1 1.7%, 13.6 2.2%, and 30.9 4.1%, respectively (data was not shown). These results showed that PDT-

Initially, the paclitaxel IC25 and IC50 doses were measured using induced cytotoxicity with verteporﬁn was mediated by apoptotic the MTT assay (4.8 nM and 11.3 nM in NCI-N87, and 5.1 nM mechanism, which occurred shortly after photosensitization. and 13.1 nM in YGIC-6B cells, respectively; data not shown). To evaluate the mechanism of enhanced cytotoxicity of PDT The MTT assay was done after PDT in cancer cells with or after paclitaxel pretreatment, DAPI staining, DNA fragmentation without paclitaxel pretreatment to evaluate enhancing cytotoxicity analysis, Annexin V-FITC apoptosis assay, and cell cycle analysis

770 | Photochem. Photobiol. Sci., 2008, 7, 769–774 This journal is © The Royal Society of Chemistry and Owner Societies 2008 Fig. 3 DNA fragment analysis after PDT. DNA fragment analysis was performed 0 h, 1 h, and 3 h after PDT in paclitaxel pretreated NCI-N87 cells (4.8 nM) compared with PDT alone (565 mJ cm−2). Pct, paclitaxel.

after PDT by DAPI staining (Fig. 2). DNA fragmentation was Fig. 2 Evaluation of morphological changes following PDT with pacli- increased as early as 1 h after PDT in paclitaxel pretreated NCI- taxel pretreatment. Morphological changes were evaluated 4 h and 24 h N87 cells compared to PDT alone (Fig. 3) and the difference of after PDT in paclitaxel pretreated cells (NCI-N87, 4.8 nM; YGIC-6B, DNA fragmentation between two groups were more prominent − 5.1 nM) compared with PDT alone (NCI-N87, 565 mJ cm 2;YGIC-6B, at 3 h after PDT. The apoptotic fraction, measured by ﬂow −2 450 mJ cm ) by DAPI staining. Pct, paclitaxel. cytometry with Annexin V-FITC staining, was increased in paclitaxel pretreated NCI-N87 cells compared to PDT alone were performed. When paclitaxel was pretreated, apoptotic cells (24.6 ± 2.1% versus 13.6 ± 1.7%) at 1 h after PDT, which were prominent 4 h after PDT compared to PDT alone treated was consistent to DNA fragmentation results (Fig. 4(A)). Our cells and almost all cells were undergoing apoptotic process 24 h data demonstrated that PDT-induced apoptotic process was

Fig. 4 Annexin V-FITC apoptosis assay and cell cycle analysis using ﬂow cytometry. (A) Apoptosis assay using Annexin V-FITC was performed 1 h after PDT with or without paclitaxel pretreatment in NCI-N87 cells. (B) Cell cycle analysis was performed using ﬂow cytometry 4 h and 24 h after PDT with or without paclitaxel pretreatment in NCI-N87 cells. The values of each cell cycle phase were included in DNA histograms. Pct, paclitaxel.

This journal is © The Royal Society of Chemistry and Owner Societies 2008 Photochem. Photobiol. Sci., 2008, 7, 769–774 | 771 augmented by paclitaxel pretreatment, which resulted in enhanc- Paclitaxel pretreatment enhanced the PDT-induced apoptotic ing PDT-induced cytotoxicity. process by augmenting cytochrome c release from mitochondria Previously, adriamycin was introduced in a combination treatment with PDT in an in vivo mouse model. Although adriamycin Different photosensitizers, however, differ in their intracellular is taken up by mitochondria and increases ROS in tumor cells, distribution and mechanism of cytotoxicity. While mitochondria- it has failed to demonstrate an enhancing effect by combination uptaken photosensitizers induce cytotoxicity by mitochondrially- with PDT.19 Paclitaxel absorbs 227 nm wavelength of light and the mediated apoptosis, photosensitizers uptaken in membranous extinction coefficient is 3.13 × 104 M−1 cm−1.30 Ma et al. reported structures induce cell necrosis via release of cytoplasmic content 32 that the antitumor effect of paclitaxel combined phototherapy by disruption of membranes. Verteporfin, a benzoporphyrin without photosensitizer did not differ from that of paclitaxel alone derivative monoacid ring A, is a potent second generation photo- in an in vivo mouse model.23 This means that the augmenting sensitiser and is taken up by mitochondria and induces apoptosis 33 effect induced by pretreatment with paclitaxel is not attributed via direct cytochrome c release after photosensitization. to the intrinsic sensitizing effect of paclitaxel. It is assumed that To investigate the potential mechanism of augmented apoptosis paclitaxel which has a different mechanism of action compared to by paclitaxel pretreatment, we measured the cytosolic cytochrome adriamycin, puts the cancer cells in vulnerable state by inducing c level, which is released from mitochondria after photosensitiza- G2/M phase arrest and might increase sensitivity to apoptotic tion. Cytochrome c was promptly released from mitochondria and stimuli induced by PDT,31 just like that cells in G2/M phase increased in a time-dependent manner after photosensitization are more sensitive to radiation therapy.21,22 The rapid and intense (Fig. 5(A)). The cytosolic fraction of cytochrome c was increased induction of DNA fragmentation by PDT in paclitaxel-pretreated after photosensitization and exaggerated by paclitaxel pretreat- cells supports this notion. It will be necessary to investigate ment (Fig. 5(B)). We performed western immunoblot for Bax and the differences in sensitivity to PDT according to the cell Bid, which were known to be involved in cytochrome c release cycle. in mitochondria-mediated apoptosis. The Bax and Bid levels did Cell cycle analysis demonstrated that paclitaxel induced G2/M not change after PDT with or without paclitaxel (data not shown). arrest, which phase is sensitive to radiation (Fig. 4(B)). In NCI- These results demonstrated that cytochrome c was directly released N87 cells, the percentage of cells in G2/M phase was increased from photochemically damaged mitochondria without activation from 9.9 ± 1.2% in the untreated cells, to 34.4 ± 2.1% after 4 h of Bax and Bid system. The paclitaxel pretreatment increased the incubation with 4.8 nM paclitaxel and 24 h with fresh medium. cytochrome c release from mitochondria, which in turn might After another 24 h incubation with fresh medium, the paclitaxel induced G2/M arrest was reversed to 11.1 ± 1.5%. After paclitaxel pretreatment, the subG0/G1 fraction was increased compared to PDT alone groups (3.9 ± 0.8% versus 13.9 ± 1.7% at 4 h after PDT, and 37.5 ± 2.1% versus 53.5 ± 2.3% at 24 h after PDT,respectively). YGIC-6B cells showed similar results to NCI-N87 cells (data not shown). Our data showed that enhanced cytotoxicity of PDT combined with paclitaxel might be related to paclitaxel induced G2/M arrest in cancer cells. A previous report demonstrated that vincristine and paclitaxel enhanced antitumor effect of PDT in vivo mouse model and interestingly, the cytotoxicity was dependent on the injection time of microtubule inhibitor prior photosensitization.23 Previous results were consistent to our cell cycle analysis data, that paclitaxel-induced G2/M arrest was reversed after 24 h incubation with fresh medium. When applying to clinical setting, it will be important to validate the exact time of paclitaxel administration prior PDT to maximize cytotoxicity. Several suggestions can be made from our study. First, more rapid and complete cell death can be induced by pretreatment of cancer cells with paclitaxel before PDT. Second, pretreatment with paclitaxel is advantageous in preventing local recurrence after photodynamic therapy which would be caused by sublethal damage due to insufficient light penetration or photosensitizer uptake and resultant insufficient production of ROS to induce irreversible apoptosis. Third, combination of paclitaxel is particularly useful Fig. 5 Western blot analysis of cytochrome c after photosensitization. (A) The cytochrome c levels were measured by western blot 0 min, because it can be conveniently applied to cancer patients in the 30 min, and 120 min after PDT with or without paclitaxel pretreatment clinical field. It is plausible that the cytotoxic mechanisms leading in cytosolic fraction and whole cell lysate of NCI-N87 and YGIC-6B to apoptosis are not very different between ionizing radiation cells. (B) Individual band intensity of cytosolic fraction of cytochrome c and PDT. As is the case, it is assumed that paclitaxel enhances was quantified by densitometry and normalized by the intensity of actin the cytotoxic effect of PDT by a similar mechanism as inducing in each samples. The data were expressed in relative ratio divided with sensitization to radiotherapy. control. Pct, paclitaxel.

772 | Photochem. Photobiol. Sci., 2008, 7, 769–774 This journal is © The Royal Society of Chemistry and Owner Societies 2008 enhance PDT-induced cytotoxicity. In physiological conditions, Heidelberg, Germany). Each experiment with triplicate was per- the release of cytochrome c is mediated by mitochodrially- formed three times. The dose response curves were analyzed and translocated proapoptotic proteins such as Bax or Bid, which, the IC values were calculated using Prism software (GraphPad in turn, binds to a permeability transition pore complex (PTPC) Software, San Diego, CA, USA). Radiosensitization was expressed resulting in increased permeability.28 Therefore full activation of by the dose enhancement ratio (DER).24,25 DER was calculated as mitochondrial apoptosis takes 1 or 2 days and is accompanied by the dose for PDT alone divided by the dose for PDT plus paclitaxel a reduction of the cytoplamic level of Bax or Bid. Meanwhile, (normalized for paclitaxel toxicity) for a surviving fraction of 0.25, cytochrome c release mediated by activated photosensitizers, 0.5, and 0.75. however, does not require translocation of proapoptotic proteins and is mediated by direct damage of mitochondria by the ROS DAPI staining generated by photosensitizers.34 Therefore there is no lag phase between PDT and cytochrome c release. In the present study, we DAPI staining was done in control and paclitaxel pretreated 36 demonstrated a prompt release of cytochrome c asearlyas30min cells 4 and 24 h after PDT, as described previously. Briefly, after PDT without change in cytosolic Bax or Bid level. cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min. Fixed cells were permeabilized with TBST [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, and 0.1% Triton X-100] for Experimental 3min.CellswerewashedwithPBSandtreatedwith1%bovine Cell cultures serum albumin for 20 min at room temperature. A DAPI solution (1 : 4 000) was added for 5 min. After washing with PBS, the cells Human gastric cancer cells (NCI-N87) were purchased from were mounted with glycerol plastine and observed by fluorescence American Tissue Culture Collection (Manassas, VA). A human microscopy (Olympus BX51, Japan) using Olympus WU filter bile duct cancer cells (YGIC-6B) has been established in the (excitation wavelength 372 nm and emission wavelength 456 nm). author’s laboratory. NCI-N87 and YGIC-6B cells were grown as monolayer cultures in RPMI 1640 and DMEM with 10% FBS, DNA fragment analysis penicillin (100 units mL−1), amphotericin (2.5 units mL−1), and streptomycin (100 g mL−1)at37◦C in a humidified atmosphere Prepared cell pellets were lysed in 10 mM Tris (pH 7.6), 10 mM EDTA, 50 mM NaCl, 0.2% SDS, and 200 lgmL−1 proteinase K with 5% CO2, respectively. overnightat42◦C. After centrifugation at 16 000 g for 20 min at ◦ Drugs 4 C, low molecular DNA was extracted from the supernatant using phenol–chloroform–isoamyl alcohol (25 : 24 : 1). The extracted Verteporfin, a second-generation photosensitizer, was purchased DNA was precipitated in ethanol containing 0.3 M sodium acetate, from CIBA Vision (Duluth, GA, USA) and used as a PDT pho- and the precipitant was melt in 10 mM Tris (pH 8.0), 1 mM ◦ tosensitizer. Verteporfin was dissolved in saline and stored at 4 C EDTA, and 100 lgmL−1 RNase A for 30 min at 37 ◦C. The DNA in the dark. Paclitaxel was purchased from Sigma (St. Louis, MO, electrophoresis was performed in 2% agarose gel. The DNA was ◦ USA), dissolved in DMSO and stored at −20 C. The paclitaxel stained with ethiodium bromide and visualized under ultraviolet stock solution was diluted in culture medium immediately before light. use. Cell cycle analysis using flow cytometry In vitro treatment protocol and MTT assay Flow cytometry was performed to determine DNA ploidy and For in vitro PDT, a QTH light source was used (1 000 W, Oriel, apoptotic fraction. Briefly, trypsinized cells were collected, cen- Stratford, CT, USA). A narrow band pass filter was attached trifuged and washed with PBS. Cells were fixed in cold 70% ethanol to the light source to allow for selective illumination with 665– and kept at −20 ◦C overnight. Cells were washed twice in PBS 35 675 nm light. Light was delivered at a fixed dose rate of and resuspended in 400 lL PBS, added with 100 propidium iodide −2 4 0.35 mW cm .10 cells were incubated for 24 h in complete −1 −1 ◦ solution (propidium iodide 5 mg mL , RNase 20 mg mL ,and medium at 37 C. Cells were then incubated with verteporfin 0.1% TritonX-100),incubated at room temperature for 30 min. The −1 ◦ (2 ug mL ) in complete medium for 1 h in the dark at 37 C. Cells number of cells at each cell cycle was evaluated with FACSCalibur were rinsed with PBS twice, placed in fresh medium, and irradiated (Becton Dickinson, San Jose, CA). The ModFit (Verity Software −2 for varying durations, making the exposure range from 0 to 2 J m . House, Topsham, ME) software was used to generate DNA To evaluate the effect of paclitaxel combined with PDT, cells were ◦ content frequency histograms and quantify the number of cells treated with the IC25 dose of paclitaxel for 4 h at 37 C. Cells in the individual cell cycle phase including subG0/G1 (apoptotic were rinsed twice in PBS and incubated in fresh medium for 20 h ◦ cells), G0/G1, S, and G2/M. Each experiment was performed in in the dark at 37 C. The cells were then irradiated with various triplicate. doses of light. NCI-N87 and YGIC-6B cell survival were measured 24 h after photosensitization using the 3-(4,5-dimethylthiazol-2yl)- Apoptosis assay using Annexin V-FITC staining 2,5-diphenyltetrazolium bromide (MTT) assay. Treated cells were rinsed twice with PBS, incubated in 50 ul MTT solution (Sigma Apoptosis was evaluated using Annexin V-FITC staining. Briefly, St. Louis, MO, USA) for 4 h at 37 ◦C, and 100 uL DMSO was after PDT with or without paclitaxel, 105 cells were incubated with added. The absorbance of each well was measured at 570 nm Annexin V-FITC and propidium iodide for 15 min in the dark. using Tilter-Tech 96-well multiscanner (Beckton and Dickenson, Samples were analyzed by flow cytometry using FACSCalibur

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774 | Photochem. Photobiol. Sci., 2008, 7, 769–774 This journal is © The Royal Society of Chemistry and Owner Societies 2008