in vitro study of cell death with 5-aminolevulinic acid based photodynamic therapy to improve the...

ISSN 1054�660X, Laser Physics, 2012, Vol. 22, No. 3, pp. 626–633.© Pleiades Publishing, Ltd., 2012.Original Text © Astro, Ltd., 2012.

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1 1. INTRODUCTION

Photodynamic therapy (PDT) has emerged as apromising therapeutic modality for the treatment ofoncological diseases [1, 2]. It has the advantage of dualselectivity, due to the preferential localization of thephotosensitizer by the malignant tissue and restrictionof photoactivation to the tumor site due to localizedlight irradiation [3, 4]. Direct tumor destruction,tumor vasculature shutdown and anti�tumor immuneresponse are three important cell death mechanisms inPDT. The combination of all three PDT mechanismsmay lead to long�term tumor control via anti�tumoraction against both the primary and metastatic tumors[5–8]. Photodynamic effects resulting either in apop�totic, mitotic, and necrotic cell death depend on thenature of the photosensitizer, cell type and the cellulartargets for photosensitization, concentration andintracellular localization of the sensitizer, the incuba�tion conditions and the light dose [9, 10]. Manipulat�ing light flounce rate, timing of illumination followingphotosensitizer administration or a combination ofthese it is possible to modulate the role played by eachof these factors in the final damage towards the tumor.Studies from in vitro culture systems and in vivo ani�mal models have indicated that both necrosis and apo�

1 The article is published in the original.

ptosis of the target cells or tissues represent the majortherapeutic effect of PDT [11–15].

In recent years, 5�aminolevulinic acid (ALA)�mediated photodynamic therapy (PDT) has becomeone of the most promising fields in PDT in vivo and invitro research. ALA is the pro�drug of the photosensi�tizer protoporphyrin IX (PPIX) [16, 17]. After ALAadministration, cells generate PPIX through the haembiosynthetic pathway. The main advantage of PPIXrelative to other photosensitizers is the short half�lifeof its photosensitizing effects, which do not last longerthan 48 [18, 19]. The light wavelengths used in PDTare in the red or infrared range of electromagneticwave. For a photobiological reaction to occur lightmust be absorbed by the photosensitizer. This is possi�ble when the wavelength of light matches the electronabsorption spectrum of the photosensitizer. For clini�cal use the activating light is usually between 600 and900 nm. Mostly low power 635 nm diode laser is usedto illuminate the ALA [20, 21].

In our previous work we have proved experimen�tally that basic features of PDT may contribute to thesolid establishment of dosimetry in PDT enhancing itsuse in clinical management of cancers and otherlesions [22–26]. One can imagine that the thresholddose for normal tissue is higher than for the tumor, wealso explained the effect of different photosensitizer invivo and in vitro experiments and explain that high

LASER METHODS IN CHEMISTRY,BIOLOGY, AND MEDICINE

In Vitro Study of Cell Death with 5�Aminolevulinic Acid Based Photodynamic Therapy to Improve the Efficiency

of Cancer Treatment1

S. Firdousa, *, M. Nawaza, M. Ikramb, and M. Ahmeda

a Biophotonics Lab., National Institute of Lasers and Optronics (NILOP), Islamabad, Pakistanb Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan

*e�mail: [email protected] October 11, 2011; in final form, October 25, 2011; published online February 6, 2012

Abstract—Photodynamic therapy (PDT) is a kind of photochemo therapeutic treatment that exerts its effectmainly through the induction of cell death. Distinct types of cell death may be elicited by different PDTregimes. In this study, efforts are underway to optimize PDT protocols for improved efficacy and combinationof all three PDT mechanisms involved in the different human carcinomas cell narcosis. Our in vitro cell cul�ture experiments with 5�aminolevulanic acid (ALA) a clinically approved photiosensitizer (PS) and 635 nmlaser light have yielded promising results, as follow: (1) (human cervical cancer (HeLa) cell line incubated,for 18 h, with 30 µg/ml of 5�ALA, treated with laser light dose of 50 J/cm2 can produce 85% of cell killing (2)human larynx carcinoma (Hep2c) cell line incubated, for 7 h, with 55 µg/ml of 5�ALA, treated with laser lightdose of 85 J/cm2 can produce 75% of cell killing (3) human liver cancer (HepG2) cell line incubated, for 22–48 h, with 262 µg/ml of 5�ALA, treated with laser light dose of 120 J/cm2 can produce 95% of cell killing (4)human muscle cancer (RD) cell line incubated, for 47 h, with 250 µg/ml of 5�ALA, treated with laser lightdose of 80 J/cm2 can produce 76% of cell killing (5) Human embryonic kidney (HEK293T) cell line incu�bated, for 18 h, with 400 µg/ml of 5�ALA, treated with laser light dose of 40 J/cm2 can produce 82% of cellkilling confirming the efficacy of photodynamic therapy.

DOI: 10.1134/S1054660X12030048

LASER PHYSICS Vol. 22 No. 3 2012

IN VITRO STUDY OF CELL DEATH 627

selectivity selection, ALA�PDT may have a greatpotential in the treatment of BCC and SCC tumors[27, 28]. Some researchers has quoted that differentcell line owing variation in PDT response due to dif�ferent immune response and photosensitivity, e.g.human hepatoblastoma (HUH6) and human neuro�blastoma (MHH�NB�11) cell lines did not surviveafter incubation with 5�ALA along with light dosesover 15 J/cm2, where as human fibroblastic cells suf�fered only about 20% reduction in viability at 20 J/cm2

under same conditions [29]. Some other researcherfounded that the immune response is initiated by thecellular components of osseous portion [30, 31]. Wehave concluded in our recent publish data by using dif�ferent cell lines that cell killing is light dose dependentassociated with photosensitizer concentration [32–36]. Current study is to optimize PDT parameters likePS concentration, incubation time and light dose onfive cancer cell lines and to study the cell viability.

2. MATERIALS AND METHODS

All the human cancer cell lines are specific cellsthat can be grown indefinitely given the appropriatemedium and conditions used in this study. The cellsare prepared in cell culturing facilities of NationalInstitute of Health (NIH), Islamabad and NCVI,National University of Science and Technology(NUST) Islamabad.

2.1. Cells and Culture Conditions

HeLa cell line (derived from human cervical ade�nocarcinoma) Hep2c cells (human larynx squamouscell carcinoma) HepG2 cells (human liver cancer cell)RD cells (human muscle cancer cell) and HEK293T(human embryonic kidney cell) was cultivated in Min�imum Essential Medium (MEM) (with Hank’s salts)containing 10% fetal bovine serum (FBS) and 2 mML�glutamine along with some non�essential aminoacids and antibiotics (penicillin, streptomycin andneomycin) and were incubated for 24–48 h for properattachment to the substratum. Cells were maintainedat 37°C in a moist environment as a sub confluentmonolayer and were routinely sub cultured in a moistenvironment as a sub confluent monolayer in 25 cm2

tissue culture flasks (Nunc, Wiesbaden, Germany)and were routinely subcultured twice or thrice weekly.The cell culture with 70–80% confluence was har�vested using 0.20–0.25% trypsin. Similar techniquewas adopted in our previous published data [32–36].

2.2. Photosensitizer (5�Aminolevulanic Acid) Administration

ALA stock solution (1000 μg/ml) was prepared inphosphate buffered saline (PBS, pH 7.4) and was keptin the dark. ALA were dissolved in normal saline toobtain different concentrations of ALA, 0–

1000 μg/ml. We count the cells using a haemocymeterbefore adding ALA. The cell viability was evaluatedusing 10 μL trypan blue solution in 90 μL of the cellculture in PBS. The best incubation time and concen�tration were observed.

2.3. Quantification of Cellular Uptake Timeto Photosensitizer

The cultured cell lines were incubated in a 96�wellflat�bottomed microtiter plate, approximately 1 ×105 cells/well with 0.05–1000 μg/ml of ALA at 37°Cfor 0–50 h. Exposure to light was avoided during incu�bation with ALA. After incubation cellular absorptionof ALA was quantified by measuring the optical den�sity of 405, 450 nm (but 405 nm was most suitable)light using microwell plate reader (AMP PLATOS R�496), after each hour for first 9 hour and then after 16,18, 24, and 48 hour. The time point corresponding tothe highest absorbance was considered as the optimalincubation time for PDT. All results are presented asmean absorbance ±σ of the experimental value mea�sured four times.

2.4. Cytotoxicity of ALA

A 96�well control plate containing only cells but noALA was also prepared in parallel. After incubation,cellular absorption of ALA was quantified by measur�ing the optical density using micro well plate reader ata wavelength of 405 nm corresponding to the absorp�tion wavelength of the aminolevulonic acid in cellsand the cytotoxicity was evaluated by means of neutralred assay as described in our previous work [32].

2.5. Photoxicity of Laser Light

Our experiments performed in two steps: in firststep the phototoxic effect of laser light on the cells wasstudied by culturing the cells in 96�well microplate ata density of 1 × 105 cells/well with different ALA con�centration ranging from 0–500 μg/ml. in each stepafter 48 h incubation with ALA. In second step cellswere incubated in separate 96�well plate for each ALAconcentration of 30, 55, 250, 262, and 400 μg/ml. Thecells were then irradiated through the clear bottom ofthe plate using a semiconductor diode laser (SAB 635�2, NILOP, Pakistan and LPhT�630/675�01�BIO�SPEC, Russia) at a wavelength of 635 nm. Light dosesin the range from 0 to 150 J/cm2 (4 wells/dose) wereused, and laser beams were transmitted to the cells in a6�mm diameter well by an optical fiber (NILOP SAB�II and BIOSPEC, TF�D). The exposure time wasadjusted to obtain a desired energy density. The cellsthat were not irradiated with light source served ascontrol. After irradiating the cells the medium wasremoved from each well and 100–300 μl fresh MEMmedium containing 10% FBS was added to each welland the plate was then returned to the incubator at

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37°C for 48 h. After 48 h the phototoxicity of cells wasmeasured using neutral red assay.

2.6. Cellular Viability Assay

Cellular viability was estimated by means of theneutral�red spectrophotometric assay. The mediumwas removed from the wells, containing the cells, andreplaced with 200–300 μl of fresh medium per wellcontaining 50–70 mg/ml of neutral red. The plateswere then returned to the incubator for 3–5 h. Themedium was subsequently removed, and the cultureswere washed rapidly with PBS. A mixture of 1% v/v

acetic acid�50% v/v ethanol (1 : 1) was then added toextract the neutral red. The plate was shaken for 60 sand left to stand at room temperature for 15 min. Theabsorbance of the solubilized dye was subsequentlyread at 490 nm. Quantification of the extracted dyewas correlated with the live cell number. Control wellswere prepared in parallel, and these cells were exposedto neutral red, but not to ALA. The percentage of via�ble cells in the cell population at each concentration ofthe test agent was calculated by means of cells viabilityformula:

3. RESULTS AND DISCUSSION

In the present study, the time dependent accumu�lation of ALA�PpIX, in the (HeLa, Hep2c, HepG2,RD, and HEK293T) cells were subjected to 0–1000 μg/ml of 5�ALA for up to 48 h. A significantincrease in the level of absorbance was found, after18 h of time of incubation in HeLa and HEK293T celllines, 7 h in Hep2c and 22–47 h in HepG2 and RDcell lines. The first criterion to evaluate the usefulnessof ALA�PpIX was the capacity of different malignantcells to absorb ALA (0–1000 μg/ml). It was noted thatin the first three h of incubation the ALA uptake in(HeLa and Hep2cand HepG2) cells was slow while atthe four hours of incubation it just shoot to maximum,becomes saturate till 18 h. It is suggested that in abovementioned (HeLa and HEK293T) cell line increasingbehavior in absorbance level was noted from four toalmost 20 h. Optimally the suitable incubation time forthe good outcome of PDT on the given cell line couldbe from four to eighteen hours of incubation. Butrather is the case in RD (human muscle cancer) andHEPG2 (Human liver cancer) cell lines. ALA absor�bance in RD cells is very low initially but after 22 h ofincubation time there is an accidentally increase inALA absorbance which shows behavior up till 47 h oftime of incubation. It is an excellent agreement withprevious reported results [32–36]. Our group hasalready investigated that two different selected con�centration of ALA present almost same uptake behav�ior. Currently, It has been investigated that ALA absor�bance in HepG2 cell line owing the almost sameuptake behavior as shown in RD cell line but startingfrom 22 h. No significant change was observed in thecurrent cell line. Ohgari et al. [27] explored in his pub�lished data that amount of PpIX in cancerous cells andsupernatant significantly increased 24 h after additionof ALA. A noticeable increase in ALA absorbance wasobserved after 30 h, because some authors has provedthat RD cell line resistant to actinomycin D has been

used as an in vitro model to investigate with light andelectron microscopy.

Many researcher quoted different incubations timedepending on Cell line type, a well known fact now[38]. Also for a photosensitizer other than ALA weneed different incubation time for different cellsshowing that the cellular uptake of a photosensitizermight be structure dependent. Cytotoxicity of photo�sensitizer was measured in given four malignant celllines. No prominent cytotoxic effect reported whileexposure to various PS concentrations in the absenceof light (hν) except higher concentrations shows athigher doses causing decrease in cellular viability, itmight be the toxicity of drug itself at higher concentra�tionsas shown in Figs. 1a, 1b, and 1e. In the currentstudy various concentration of drug 0–1000 μg/mlwere selected, the sharp absorbance peak lies at differ�ent concentrations (250 μg/ml for RD, 262 μg/ml forHepG2, 400 μg/ml for HEK293T, 55 μg/ml forHep2c, and 30 μg/ml for HeLa cell line), which issimply called the optimum concentration of PS,which shows that at this optimal dose of PS maximumcellular accumulation of PpIX in given cell lines, is asuitable drug dose. ALA itself does not play its role inPDT, but its precursor heme cycle into PpIX, an effec�tive photosensitizer agent. Huang [39] concluded intheir research paper that for fruitful PDT outcomes,optimal timing, light sources, doses and selection ofphotosensitizer for different cancer treatment are nec�essary factors. 5�ALA owing great importance espe�cially in skin cancer disease, our result are in consis�tence with the previous experimental studies made RDcells that light, a member of the TNF super family,induces morphological changes and delays prolifera�tion in current cell line Our experimental data showsthat influence of increasing concentration of ALA inthe absence of irradiation on the survival rates in thewells containing 1 × 105 viable HEK293T cells. Therewere remarkable cytotoxic effects observed due to drug

% Viability Mean absorbance of ALA treated cellsMean absorbance of control cells

�� 100.×=



injection into the cells without any light exposure. Forhigher ALA concentration some cytotoxicity wasobserved that might represent the toxicity of ALA itselfat higher concentrations with the cellular componentsof about 85% cell death occurred. Huang in [39]quoted in their work using HEK293T as experimentalmodel, that the mentioned cell line treated with ALAand light dose (visible region) are more sensitive ascompared with the ALA�treated control cells. Theyverified that cell death was observed when ALA�treated cells were exposed to visible light (Fig. 1), butno cell death was observed either in the absence ofALA or without exposure to light. They also examined

the photosensitivity of HeLa, Hep2c, HepG2, RD,and HEK293T cells, investigated that one fifth of thecells died treated with suitable ALA concentration and40–120 J/cm2. Ohgari et al. [27] reported that cancer�ous cells take up much more ALA than normal cells,and the potency of photodamage dependent on theaccumulation of protoporphyrin was also related tothe amount of ALA taken by the cells. They suggestedthat active neoplastic cells positively take up smallmolecules including ALA and Photofrin. Since theproduction of heme in tumor was higher than that inisolated tissue cells. Atif et al. [33] concluded that nei�

HeLa cells Hep2c cells

HepG2 cells RD cells

HEK 293T cells

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Fig. 1. Cellular viability images of different cell (a) HeLa, (b) Hep2c, (c) HepG2, (d) RD, and (e) HEK293T after suitable ALAconcentration and laser irradiation at 20× confocal microscope.

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ther sensitizer nor light doses have significant effect oncells viability when studied independently.

It was experimentally proved that 30–55 μg/ml,100 to 250 μg/ml, and 350 to 400 μg/ml concentrationof ALA, the viability for HeLa, Hep2c, HepG2, RD,and HEK293T cell lines becomes minimum of about18 and 28%, respectively, as shown in Fig. 2 and dis�cussed in our previous research papers [32–36]. Manyresearchers has worked with different photosensitizersin different cell line, e.g., Photofrin mediated PDT ingastric cancer cell line (MKN45) induces apoptosiswithin 60 min, and mitochondrial damage is likely asthe first event of apoptosis. Some authors quoted thatafter the administration of photosensitizer, it is local�ized at higher concentrations in cancer cells comparedwith normal cells.

In the present study various concentrations of drugranging from 0–1000 μg/ml were selected for differentmalignant cell line. It is investigated that very sharpabsorbance peak at 400, 250, 30, and 55 μg/ml wereobserved for HEK293T, RD, HeLa, HepG2, andHep2c cell line, the above mentioned drug dose forcell lines are called optimal dose. Our group exploredthe data for cytotoxicity and phototoxicity, PS uptake

for different malignant cell lines in our previous work[32–36]. It is worth mentioning that no protocol avail�able on PDT mediated aforementioned cell linesbefore our work. Our group has investigated that HeLacell line owing more light sensitivity as compare toother malignant cell lines.

Figure 3 shows the effect of different light doses inthe presence of ALA. Light dose of 80 J/cm2 seems tobe effective in combination with 250 μg/ml ALA con�centration. The appreciable decrease of cell viabilitymeasured with NR assay of 82% admits the role oflaser light in combination with ALA in cancer cells.Figure 4 shows the comparative results between ALAtreated non ALA treated HeLa and Hep2c cells withdifferent light doses, which has been published in ourprevious work [32]. Yow et al. explored the uptake ofALA on human hepatocellular carcinoma cell(HepG2) by spectrophotometer and proved that rateof cellular uptake increased slowly in early 18 h andsaturates after 30 h of time of incubation. Manyresearchers explored that penetration of light into tis�sue is a complex mechanism dependent on many fac�tors including tissue density, organ pigmentation,blood flow, surface geometry and tissue interfaces.

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Figure 5 shows the % viability vs. incubation timegraph with optimum ALA concentration of 262 μg/mland 250 μg/ml for HepG2 and RD cell lines, respec�tively. From Fig. 4, it is clear that % viability of cells atselected ALA concentration along with suitable635 nm light dose is at minimum level. But in case ofHep2c cell line % viability reaches to 22% with PDT

parameters of 85 J/cm2 along with 55 μg/ml, the resulthas been already discussed in our published data [32–36]. Many researchers explored that 5�ALA (PpIX)have good therapeutic results due to high uptake, invitro as well as in vivo study. Moreover, the currentphotosensitizer claims fruitful results for the treatmentin clinical side, which agrees to our results.

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Fig. 4. Comparison of ALA treated HeLa and Hep2c cells viability for irradiation dose of 0, 50 J/cm2 for HeLa and 85 J/cm2 forHep2c. Each data point corresponds to mean ±σ (n = 4).

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4. CONCLUSIONS

The present study demonstrates that PDT usingALA�PpIX at 635 nm laser indices cell death in(HeLa, Hep2c, HepG2, RD, and HEK293T) cells invitro. It is concluded that all the cell lines owing goodtherapeutic results by PDT point of view at differentphotosensitizer uptakes, concentration and light dose.It has been extensively studied that ALA�PpIX is anefficient photosensitizer that can produce cytotoxicresults during photodynamic therapy. Results providethat neither ALA nor laser light alone can produce anydamage to tumor cells, the proper combination of drugand light doses and perhaps the proper time of irradi�ation may cause to maximal damage to cancerous cellswhile carrying out PDT. We are hopeful from theexperimental result and the elucidation of the mecha�nisms of PDT action will provide us with more effec�tive treatment regimens and clinical improvements forpeople suffering from cancer.

ACKNOWLEDGMENTS

We acknowledge higher education commission(HEC) for financial support through laser tissue, Bio�photonics, University linkage program of PIEAS andHarvard university, USA, and UNESCO chair atPIEAS. We are thankful to peoples from Biophotonicsgroup, National Institute of Lasers and Optronics(NILOP), Physics Department, PIEAS, WellmanCenter for Photo medicine, Harvard Medical School,USA, University of São Paulo, Brazil, S. S. Z. Zaidi,and R. Suleman, National Institute of Health, Islam�abad, NCVI, National University of Science andTechnology (NUST) Islamabad., PAEC hospital,Islamabad persons and all our students (M. Phil andPhD at PIEAS especially, A. Khurshid, F. Alam,H. Ullah, R. Mehmood, and Tanzeel) and members

of our group (Atif, Aziz, Shahzad) and others whosenames are not mentioned that deserves acknowledge�ment.

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in vitro study of cell death with 5-aminolevulinic acid based photodynamic therapy to improve the...

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