Methotrexate Enhances 5-Aminolevulinic Acid-Mediated

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provided by Elsevier - Publisher Connector Journal of the Formosan Medical Association (2014) 113,88e93

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ORIGINAL ARTICLE Methotrexate enhances 5-aminolevulinic acid-mediated photodynamic therapy- induced killing of human SCC4 cells by upregulation of coproporphyrinogen oxidase Deng-Fu Yang a, Jeng-Woei Lee b, Hsin-Ming Chen c,d, Zheng Huang e, Yih-Chih Hsu f,g,h,*

a Graduate Program, Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan, Taiwan b Department of Life Science, Tzu Chi University, Hualien, Taiwan c Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan d Department of Dentistry, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan e Cancer Center, University of Colorado Denver Cancer Center, CO, USA f Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan, Taiwan g Center for Nanotechnology, Chung Yuan Christian University, Taoyuan, Taiwan h Center of Biomedical Technology, Chung Yuan Christian University, Taoyuan, Taiwan

Received 9 December 2013; accepted 24 December 2013

KEYWORDS Background/Purpose: Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA- 5-aminolevulinic PDT) is effective for treatment of oral precancerous and cancerous lesions. This in vitro study acid; tried to examine whether the SCC4 cell killing by ALA-PDT was enhanced by pretreatment of coproporphyrinogen methotrexate (MTX). oxidase; Methods: To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTXeALA- ferrochelatase; PDT), the SCC4 cells were pretreated with 0 mg/L, 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or human squamous cell 1 mg/L of MTX for 72 hours, then incubated with 0 mM, 0.0625 mM, 0.125 mM, 0.187 mM, carcinoma 4 cell; 0.25 mM, or 0.375 mM ALA for 4 hours, and subsequently illuminated with a 640-nm light- 2 methotrexate; emitting diode array at a light dose of 10 J/cm . The 3-(4,5-dimethylthiazol-2-yl)- photodynamic 2,5-diphenyltetrazolium bromide assay was conducted at 24 hours to quantify SCC4 cell sur- therapy vival rates after MTXeALA-PDT treatment. Western blot analyses were used to examine the MTX-mediated enhancement in the expressions of the heme production-related enzymes,

Conﬂicts of interest: The authors have no conﬂicts of interest relevant to this article. * Corresponding author. Department of Bioscience Technology, Chung Yuan Christian University, 200, Chung-Pei Road, Chung-Li, 32023 Taoyuan, Taiwan. E-mail address: [email protected] (Y.-C. Hsu).

coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX), and ferrochelatase, in the MTX-preconditioned SCC4 cells. Results: Pretreatment of SCC4 cells by 0.001 mg/L MTX for 72 hours resulted in a significant augmentation in MTXeALA-PDT-induced killing of SCC4 cells (p < 0.05). The SCC4 cells treated with 0.001 mg/L MTX for 72 hours showed a significant and 1.65-fold increase in CPOX expression compared with the control SCC4 cells without MTX treatment (p < 0.05). However, no significant changes in the expressions of PPOX and ferrochelatase were observed in the SCC4 cells pretreated with different concentrations of MTX. Conclusion: MTX enhances ALA-PDT-induced SCC4 cell killing through upregulation of CPOX expression and subsequent increase in intracellular protoporphyrin IX production in SCC4 cells. Copyright ª 2014, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

e Introduction epithelial or cancer cells after exposure to the light.23 28 Therefore, we hypothesize that when oral cancer cells Oral squamous cell carcinoma is the sixth most common are pretreated with a nontoxic and low dose of MTX and cancer in the world.1 Oral cancer is also the sixth most then treated with ALA-PDT, the PDT efficacy can be frequent cancer in Taiwan.2 Because of the continuously augmented through upregulation of CPOX expression and increased prevalence of oral cancer and poor survival for subsequent increase in PpIX production in oral cancer cells. patients with advanced oral cancers, it is very important to In this study, we used human SCC4 cell line to measure the find an effective treatment modality for oral cancer.3 SCC4 cell killing abilities by MTX-pretreated ALA-PDT e Traditional treatment for oral precancers and cancers is (MTX ALA-PDT). The SCC4 cells were pretreated with e total surgical excision that always leads to scar formation for 0 1 mg/L of MTX for 72 hours, then incubated with e e a large precancerous or cancerous lesion.4 6 Photodynamic 0 0.375 mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode (LED) array at a light therapy (PDT) is another effective treatment option for 2 human precancerous and cancerous lesions because it is dose of 10 J/cm . The 3-(4,5-dimethylthiazol-2-yl)-2,5- noninvasive, is well tolerated by patients, can be used diphenyltetrazolium bromide (MTT) assay was performed repeatedly without cumulative side effects, and results in at 24 hours to quantify SCC4 cell survival rates after e little scar formation.7 5-Aminolevulinic acid (ALA) itself is MTX ALA-PDT treatment. Western blot analyses were used not a photosensitizer but serves as the biological precursor of to examine the MTX-mediated enhancement in the ex- the photosensitizer, protoporphyrin IX (PpIX), in the heme pressions of CPOX, protoporphyrinogen oxidase (PPOX), and biosynthesis pathway. When ALA is topically applied on the ferrochelatase in the MTX-pretreated SCC4 cells. oral lesions or systemically administrated by the patients, it will be absorbed by the lesional epithelial cells and results in Materials and methods accumulation of a relatively high concentration of PpIX in 8e12 these cells. ALA is superior to other photosensitizers In vitro survival assays because it can be rapidly cleared from the tissues and the body within 48 hours and patients after ALA-PDT treatment e An in vitro study was carried out to examine the roles of MTX have no problem of prolonged skin photosensitivity.7 12 in the MTXeALA-PDT-induced killing of human SCC4 cells. PDT involves two individual nontoxic components, The SCC4 cells were purchased from Bioresource Collection namely, light and photosensitizer. When a photosensitizer and Research Center (Hsinchu, Taiwan) and cultured at 37 C in tissues is activated by a light of specific wavelength, it in a humidified CO incubator in Dulbecco’s Modified Eagle transfers energy from light to molecular oxygen, resulting 2 Medium/F12 medium (Invitrogen, Grand Island, NY, USA) in the generation of reactive oxygen species (ROS).7 There supplemented with 10% fetal bovine serum (Invitrogen). The are three main mechanisms by which PDT mediates tumor SCC4 cells were plated in 24-well plates at a cell number of destruction. First, the ROS can kill tumor cells directly. 1 104 (the control group) or 4 104 (the MTXeALA-PDT Second, PDT can damage the tumor-associated vasculature, group) and pretreated with the MTX (SigmaeAldrich Corp., leading to thrombus formation and subsequent tumor St. Louis, MO, USA) at the concentration of 0 mg/L, infarction. Third, PDT can also activate an immune 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or 1 mg/L for 72 hours. The response against tumor cells.7 SCC4 cells were then incubated with 0 mM, 0.0625 mM, Previous studies have shown that ALA-PDT is effective e 0.125 mM, 0.187 mM, 0.25 mM, or 0.375 mM ALA for 4 hours, for the treatment of both oral cancer and precancers.9 22 and susequently exposed to a self-made 640-nm LED array at However, for relatively large oral precancerous lesions, a light dose of 10 J/cm2. The MTT assay was conducted at 24 four to six topical ALA-PDT treatments are needed to ach- e hours to quantify the cell survival rate. ieve a complete regression of the lesion.17 22 Previous studies found that pretreatment of prostate cancer cells and skin keratinocytes or carcinoma cells with a low-dose Western blot analyses for key enzymes for PpIX methotrexate (MTX) can have a two- to fourfold upregula- synthesis in SCC4 cells tion of expression of coproporphyrinogen oxidase (CPOX) proteins, resulting in a two- to fourfold increase in intra- The SCC4 cells were treated with 0 mg/L, 0.001 mg/L, cellular PpIX production and subsequent killing of these 0.01 mg/L, 0.1 mg/L, or 1 mg/L MTX for 72 hours and then 90 D.-F. Yang et al. lysed in the lysis buffer (PRO-PREP protein extraction so- experimental groups pretreated with 0.01 mg/L, 0.1 mg/L, lution; Intron Biotechnology Inc., Seoul, Korea). Samples or 1 mg/L MTX for 72 hours. containing an equal quantity of protein, as determined by bicinchoninic acid protein assay (Thermo Fisher Scientific MTX-induced CPOX protein expression in SCC4 cells Inc., Rockford, IL, USA), were denatured in sample buffer for e 10 minutes at 100 C and resolved on a 4 12% Bis-Tris To investigate the biochemical mechanisms underlying the acrylamide gel along with molecular-size markers. Electro- MTX-mediated enhancement of ALA-PDT-induced killing of phoresis was carried out at a constant voltage (200 V) at room SCC4 cells, we evaluated changes in the expression of en- temperature. Proteins were electrophoretically transferred zymes involved in the PpIX synthesis in SCC4 cells pre- to a nitrocellulose membrane (Whatman GmbH, Dassel, treated with different concentrations of MTX for 72 hours. Germany) at a constant voltage (100 V) for 5 hours at 4 C. Western blotting was performed to evaluate the expression e The blot was then blocked with 10% casein phosphate- levels of the CPOX (40 kDa), PPOX (26 kDa), ferrochelatase buffered saline, incubated with rabbit primary antibodies (48 kDa), and GAPDH (36 kDa, control) proteins. The against CPOX (GenTex, Taipei, Taiwan) at the dilution of semiquantitative Western blotting analyses showed a sig- 1:1000, PPOX (Abnova, Taipei, Taiwan) at the dilution of nificant increase (1.65-fold higher than the control level) in 1:500, ferrochelatase (GenTex) at the dilution of 1:1000, and the level of CPOX expression in the SCC4 cells pretreated glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Gen- with 0.001 mg/L MTX, compared with corresponding levels Tex) at the dilution of 1:1000, followed by peroxidase- in SCC4 cells pretreated with 0 mg/L (control), 0.01 mg/L, conjugated goat antirabbit immunoglobulin G (GenTex) at 0.1 mg/L, or 1 mg/L MTX, respectively (p < 0.05; Fig. 2). the dilution of 1:5000, and then developed in enhanced However, no significant changes in the expression of PPOX, chemiluminescence substrate (PerkinElmer, Boston, MA, ferrochelatase, and GAPDH proteins were observed in the USA). Intensities of hybridized protein bands on Western SCC4 cells pretreated with five different concentrations of blots were semiquantified by VisionWorkers LS software MTX for 72 hours. (UVP, LLC, Upland, CA, USA). Blots were scanned using the BioDoc-It 220 imaging system (UVP, Upland, CA, USA). Discussion

LED light source This study found that the SCC4 cells pretreated with 0.001 mg/L MTX for 72 hours showed a significant and 1.65- An LED array light was used for the in vitro cell study. The fold augmentation in the CPOX expression, compared with peak of LED light source is 640 nm, and full width and half the CPOX expression in the control SCC4 cells without MTX maximum is 20 nm. The power density of the LED light treatment. The significant increase in CPOX protein 2 system was 64 mW/cm and the period of light treatment expression can lead to an increase in intracellular PpIX 2 was set at 159 seconds to deliver a light dose of 10 J/cm . production in SCC4 cells that in turn augments MTXeALA- PDT-induced killing of SCC4 cells after PDT treatment. However, no significant changes in the expressions of PPOX Statistical analysis and ferrochelatase were observed in the SCC4 cells pretreated with different concentrations of MTX. These find- Duncan test was used to compare the SCC4 cell survival ings indicate that MTX-induced increase in the CPOX e rates after MTX ALA-PDT treatments between any two of expression in SCC4 cells is the main mechanism responsible the five groups treated with different concentrations of for an augmented MTXeALA-PDT-induced killing of SCC4 MTX at a fixed ALA concentration. Student t test was used cells. to compare the mean CPOX, PPOX, and ferochelatase The efficacy of ALA-PDT depends on the amount of PpIX enzyme expression levels in SCC4 cells between any two of accumulated in the lesional epithelial or cancer cells. PpIX the five groups pretreated with five different concentra- is the seventh product in the heme biosynthesis pathway. tions of MTX. The increase in the expression of CPOX (the enzyme that is responsible for the sixth step in heme production, con- verting coproporphyrinogen III into protoporphyrinogen IX) Results and the decrease in the expression of ferrochelatase (the enzyme that catalyzes the terminal or eighth step in the MTX enhances ALA-PDT-induced killing of human biosynthesis of heme, catalyzing the insertion of Fe2þ into SCC4 cells PpIX to form heme) are responsible for the increased accumulation of PpIX in lesional epithelial or cancer The cell killing experiments showed that, in SCC4 cells cells.29,30 Previous studies have shown that the agents pretreated with 0 mg/L, 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, capable of inducing cellular differentiation can also cause or 1 mg/L MTX for 72 hours, the ALA-PDT-induced killing of an elevation in the level of PpIX in prostate cancer cells and SCC4 cells increased in an ALA dose-dependent manner skin epithelial cells.23e28 Because MTX can promote dif- (Table 1 and Fig. 1). The cell killing abilities by MTXeALA- ferentiation and PpIX accumulation in carcinoma cells, MTX PDT when using 0.001 mg/L MTX and 0.0625 mM, 0.125 mM, may serve as a drug to augment the efficacy of ALA-PDT for or 0.187 mM ALA were significantly greater (p < 0.05 based tumor cell killing. Sinha et al25 studied the MTX-pretreated on the Duncan test) than the corresponding data of the ALA-PDT for the killing of prostate cancer cells. They found control group without MTX pretreatment and of other that prostate cancer cells pretreated by MTX and followed MTXeALA-PDT for SCC4 cells 91

Table 1 Cell survival rates of SCC4 cells pretreated with methotrexate (MTX) at five different concentrations for 72 hours, then treated with ALA at five different concentrations for 4 hours, and subsequently illuminated with an LED array at a light dose of 10 J/cm2. MTX level (mg/L) ALA concentration (mM) 0.0625* 0.125* 0.187* 0.25* 0.375 0 98.2 1.5** 83.6 1.3** 75.7 3.3** 61.7 2.7** 49.7 7.9** 0.001 84.6 6.2*** 74.5 1.2*** 65.1 1.0*** 54.0 7.2** 40.3 1.6** 0.01 98.9 0.6** 95.5 5.3** 89.0 3.0** 78.9 10.4** 66.8 15.0** 0.1 95.8 0.5** 96.9 1.3** 90.2 3.7** 78.2 08.1** 64.5 17.1** 1 101.5 3.3** 96.5 3.7** 81.3 7.4** 66.7 14.9** 50.5 22.0** *Duncan test was used to compare the cell survival rates of SCC4 cells after MTXeALA-PDT treatments between any two of the five groups pretreated with five different concentrations of MTX at a fixed ALA concentration. Groups with different asterisks (**,***) indicate a significant difference between the two groups with p < 0.05. Moreover, groups with ‘**’ indicate no significant difference between the two groups. ALA Z 5-aminolevulinic acid; LED Z light-emitting diode; PDT Z photodynamic therapy.

by ALA incubation can result in a threefold increase in preferentially augment intracellular PpIX levels two- to intracellular PpIX level. The threefold increase in PpIX level fourfold in skin carcinoma cells versus normal keratino- in prostate cancer cells is due to the threefold augmenta- cytes. Photodynamic killing is synergistically enhanced by tion in expression of CPOX induced by MTX. Transfection of the MTX-combined therapy compared with PDT alone. MTX prostate cancer cells with a CPOX-expressing vector also enhancement of PpIX levels is achieved over a broad MTX stimulates the accumulation of PpIX in cells. In addition, concentration range (0.0003e1.0 mg/L). PpIX enhancement after exposure to 512-nm light, killing is significantly correlates with a fourfold increase in expression of CPOX enhanced in MTX-preconditioned cells. These findings sug- and no change or a slight decrease in expression of ferro- gest that MTX, when used to modulate intracellular pro- chelatase. MTX preconditioning also enhances PpIX accu- duction of endogenous PpIX, may provide a new mulation in organotypic cultures of immortalized combination PDT approach for certain cancers.25 Previous keratinocytes, chemically induced skin tumors in mice, and studies also demonstrated that low-dose MTX can enhance human A431 squamous cell tumors implanted subcutane- ALA-PDT in skin carcinoma cells in vitro and in vivo.26e28 ously in mice.26 The findings of the aforementioned studies MTX preconditioning of monolayer cultures can and the present study indicate that different carcinoma cells may have different responses of the expression of CPOX and ferrochelatase after pretreatment with the same concentration of MTX for the same period.23e28 Neverthe- less, the nontoxic and low dose of MTX pretreatment for 3 days is an effective method to increase intracellular PpIX production in carcinoma cells; this in turn may augment the efficacy of ALA-PDT on different carcinoma cells. In conclusion, MTX enhances ALA-PDT-induced SCC4 cell killing through the upregulation of CPOX expression and subsequent increase in intracellular PpIX production in SCC4 cells. In addition to MTX, vitamin D is also a useful biological enhancer of ALA-PDT.31,32 Sato et al31 found that vitamin D enhances ALA-mediated PpIX production and photodynamic cell death in three-dimensional organotypic cultures of keratinocytes. Anand et al32 also showed that vitamin D3 can raise tumoral accumulation of the PpIX up to 10-fold, mainly due to the augmentation of CPOX expres- Figure 1 Cell survival rates of SCC4 cells after MTXeALA- sion and the reduction of ferrochelatase expression in PDT. The SCC4 cells were treated with 0 mg/L (control), tumor cells. Moreover, very low doses of vitamin D3 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or 1 mg/L MTX for 72 hours, (0.1e1 mg/kg body weight) are sufficient to elicit tumor- then incubated with 0 mM, 0.0625 mM, 0.125 mM, 0.187 mM, selective enhancement to ALA-PDT efficacy. Cryotherapy 0.25 mM, or 0.375 mM ALA for 4 hours, and subsequently illu- can also be used prior to topical ALA-PDT at the same visit minated with a self-made 640-nm LED array at a light dose of to facilitate the diffusion of ALA into the precancerous and 10 J/cm2. * The mean cell survival rates of SCC4 cells after cancerous epithelial cells to improve the clinical outcome MTXeALA-PDT treatments were compared by Duncan test be- of topical ALA-PDT.33 Mi et al34 used the combination tween any two of the five groups pretreated with five different therapy of cryotherapy plus topical ALA-PDT and cryo- concentrations of MTX at a fixed ALA concentration with therapy alone to treat 80 patients with multiple condylo- p < 0.05. ALA Z 5-aminolevulinic acid; LED Z light-emitting mata acuminata. They found that the combination therapy diode; MTX Z methotrexate; PDT Z photodynamic therapy. of cryotherapy followed by topical ALA-PDT can achieve a 92 D.-F. Yang et al.

administration and local application or injection of steroid.36e38 Further human clinical trials are needed to evaluate the efﬁcacy of the combination therapy of drug pretreatment or cryotherapy plus ALA-PDT on oral precancers or cancers.

Acknowledgments

The work was supported by grants NSC 100-2221-E-033-021 and NSC 101-2221-E-033-001-MY2 from the National Science Council, Taipei, Taiwan, R.O.C.

References

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