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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 19, Iss. 22 — Oct. 24, 2011
  • pp: 21145–21154
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NADH fluorescence as a photobiological metric in 5-aminolevlinic acid (ALA)-photodynamic therapy

Guan-Chin Su, Yau-Huei Wei, and Hsing-Wen Wang  »View Author Affiliations


Optics Express, Vol. 19, Issue 22, pp. 21145-21154 (2011)
http://dx.doi.org/10.1364/OE.19.021145


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Abstract

Photodynamic therapy (PDT) dosimetry is complex as many factors are involved and varied interdependently. Monitoring the biological consequence of PDT such as cell death is the most direct approach to assess treatment efficacy. In this study, we performed 5-aminolevlinic acid (ALA)-PDT in vitro to induce different cell death modes (i.e., slight cell cytotoxicity, apoptosis, and necrosis) by a fixed fluence rate of 10 mW/cm2 and varied fluences (1, 2, and 6 J/cm2). Time course measurements of cell viability, caspase-3 activity, and DNA fragmentation were conducted to determine the mode of cell death. We demonstrated that NADH fluorescence lifetime together with NADH fluorescence intensity permit us to detect apoptosis and differentiate it from necrosis. This feature will be unique in the use of optimizing apoptosis-favored treatments such as metronomic PDT.

© 2011 OSA

1. Introduction

Photodynamic therapy (PDT) is a cancer therapy that involves photosensitizer interacting with light and oxygen to generate reactive oxygen species (ROS) or singlet oxygen that cause cell death and tumor destruction. It has been approved for treatments of head and neck cancer and basal-cell carcinoma in the European Union, esophageal and endobronchial cancer in the United States, and cervical and gastric cancers in Japan [1

1. D. E. Dolmans, D. Fukumura, and R. K. Jain, “Photodynamic therapy for cancer,” Nat. Rev. Cancer 3(5), 380–387 (2003). [CrossRef] [PubMed]

]. Clinical PDT dosimetry is complex because not only several treatment factors are involved (i.e., light dose, drug dose, light-drug duration, oxygen) but also these factors varied dynamically and interdependently. As a result, the biological consequence of PDT is not necessarily proportional to a single factor such as the irradiance dose rate in ionizing radiation therapy. Generally, four PDT dosimetry strategies are pursued: explicit, implicit, and direct dosimetry, and biological tissue response monitoring [2

2. B. C. Wilson, M. S. Patterson, and L. Lilge, “Implicit and explicit dosimetry in photodynamic therapy: a New paradigm,” Lasers Med. Sci. 12(3), 182–199 (1997). [CrossRef] [PubMed]

]. Explicit dosimetry directly measures the three PDT components (light, drug, and oxygen) and sometimes incorporates a dose model to simulate the outcome [3

3. K. K.-H. Wang, S. Mitra, and T. H. Foster, “A comprehensive mathematical model of microscopic dose deposition in photodynamic therapy,” Med. Phys. 34(1), 282–293 (2007). [CrossRef] [PubMed]

]. Implicit dosimetry intended to measure a single metric such as photosensitizer photobleaching that is predictive of the biological damage or outcome under certain circumstances. Direct dosimetry measures the ROS, particularly singlet oxygen, that is widely believed to dominate the causes of biological damage for most current photosensitizers and treatment doses used [4

4. M. T. Jarvi, M. J. Niedre, M. S. Patterson, and B. C. Wilson, “Singlet oxygen luminescence dosimetry (SOLD) for photodynamic therapy: current status, challenges and future prospects,” Photochem. Photobiol. 82(5), 1198–1210 (2006). [CrossRef] [PubMed]

]. With the availability of the new technology, direct dosimetry shows advantages over explicit and implicit dosimetry for its simplicity to measure single metric only without any dose model [5

5. M. J. Niedre, A. J. Secord, M. S. Patterson, and B. C. Wilson, “In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy,” Cancer Res. 63(22), 7986–7994 (2003). [PubMed]

]. However, studies have shown that singlet oxygen monitoring approach could fail to predict the tumor response [6

6. K. K. Wang, S. Mitra, and T. H. Foster, “Photodynamic dose does not correlate with long-term tumor response to mTHPC-PDT performed at several drug-light intervals,” Med. Phys. 35(8), 3518–3526 (2008). [CrossRef] [PubMed]

]. In situations like this, reporters of biological response to therapy would be necessary. At present, the biological response monitoring is limited to measure the tumor volume, tumor perfusion, and treatment-induced necrosis [2

2. B. C. Wilson, M. S. Patterson, and L. Lilge, “Implicit and explicit dosimetry in photodynamic therapy: a New paradigm,” Lasers Med. Sci. 12(3), 182–199 (1997). [CrossRef] [PubMed]

,6

6. K. K. Wang, S. Mitra, and T. H. Foster, “Photodynamic dose does not correlate with long-term tumor response to mTHPC-PDT performed at several drug-light intervals,” Med. Phys. 35(8), 3518–3526 (2008). [CrossRef] [PubMed]

]. Advanced technologies such as MRI and optical spectroscopy allow in vivo monitoring tumor perfusion such as blood flow and hemoglobin oxygenation that are undergoing active evaluation by several groups for the prediction of long-term response to PDT treatment [7

7. S. Gross, A. Gilead, A. Scherz, M. Neeman, and Y. Salomon, “Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI,” Nat. Med. 9(10), 1327–1331 (2003). [CrossRef] [PubMed]

11

11. B. Chen, B. W. Pogue, I. A. Goodwin, J. A. O’Hara, C. M. Wilmot, J. E. Hutchins, P. J. Hoopes, and T. Hasan, “Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor,” Radiat. Res. 160(4), 452–459 (2003). [CrossRef] [PubMed]

].

2. Materials and methods

2.1 Cell line and photodynamic therapy treatment

H1299 non-small cell lung carcinoma were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Invitrogrn Corp., Carlsbad, California, USA) supplemented with 5% fetal bovine serum, 100 units/ml penicillin and 50 mg/ml streptomycin at 37°C in 5% CO2 humidified atmosphere. Cells were seeded on 24 mm diameter glass coverslips (Paul Marienfeld GmbH & Co., Lauda- Konigshofen, Germany) at 2x105 cells 24 hours before ALA-PDT treatment. Before fluorescence lifetime imaging microscopy (FLIM), 5-ALA (A7793; Sigma, Buchs, Switzerland) was prepared by resuspending in the culture medium to a concentration of 1 mM. Cells were washed with the serum-free medium and incubated in serum-free medium [24

24. F. Giuntini, L. Bourré, A. J. MacRobert, M. Wilson, and I. M. Eggleston, “Improved peptide prodrugs of 5-ALA for PDT: rationalization of cellular accumulation and protoporphyrin IX production by direct determination of cellular prodrug uptake and prodrug metabolization,” J. Med. Chem. 52(13), 4026–4037 (2009). [CrossRef] [PubMed]

] containing 1 mM 5-ALA for four hours. The drug dose (1 mM) used here was based on the cell viability study that there was no significant change over a range of drug dose from 0.5 to 2 mM. At the dose of 1 mM, PPIX fluorescence was detected maximal at 4 hr incubation time (data not shown). The cells were then washed once by phosphate buffer solution (PBS) and incubated in PBS. Subsequently the cells were irradiated by an 8 x 12 cm2 LED array with a wavelength peak at 633 nm. The light fluence rate at 10 mW/cm2 and three different light fluences at 1, 2, or 6 J/cm2 were used to treat the cells. Immediately after PDT treatment, the cells were either imaged for NADH fluorescence lifetime or incubated with fresh DMEM and 5% FBS at 37°C in 5% CO2 incubator for use in the analysis of cell viability, caspase-3 activity, and DNA fragmentation. Control cells were performed with light treatment only, drug treatment only, and no drug and light treatment. For fluorescence lifetime image, the cell-seeded coverslip was washed twice using PBS and then transferred into an imaging chamber filled with 1 ml DMEM with 5% FBS. All images were taken at 256 x 256 pixels resolution with the field of view (FOV) of 100 x 100 μm and the acquisition time of 20 minutes to obtain enough photons for reliable analysis of the NADH fluorescence lifetime. Time-lapsed NADH fluorescence lifetime images were obtained at the same site (same FOV) before, immediately after (0-20 minutes), and up to 2 hr after ALA-PDT.

2.2 Cell viability assay

Cell viability was determined by using the CellTiter-Blue® cell viability assay (Promega Corp., Madison, Wisconsin, USA). Cells at a concentration of 2x105 cells were seeded per 35 mm plate. After ALA-PDT treatment, we added the medium containing CellTiter-Blue® reagent into the cells, which were then incubated for 1 hour at 37°C in 5% CO2 humidified atmosphere. The viable cells were detected by a Victor2 1420 Multilabel counter (PerkinElmer, Foster, Massachusetts, USA) with an excitation wavelength of 560 nm and emission wavelength of 590 nm.

2.3 Analysis of sub-G1 contents

An aliquot of 1x106 cells were seeded on a 100-mm plate for 24 hr. After ALA-PDT treatment, the adherent and floating cells were collected and then centrifuged at 1500 rpm. The pellet was resuspended in 500 μl PBS to a concentration of 1x106 cells/ml. These resuspended cells were then fixed with 1 ml ice-cold methanol overnight at −20°C. The fixed cells were washed twice in 2 ml cold PBS. The cells were resuspended in 1 ml staining buffer containing RNase (50 g/ml) and propidium iodide (PI, 60 mg/ml) (Sigma, Buchs, Switzerland) in PBS. After incubation at 20°C for 30 minutes in the dark, the stained cells were analyzed in a fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson, Franklin Lakes, New Jersey, USA). The percentage of cells in sub-G1 phase was analyzed using the WinMDI software (Becton Dickinson, Franklin Lakes, New Jersey, USA).

2.4 Caspase-3 activity assay

Caspase-3 activity was determined by fluorescent measurement of 7-amino-4 trifluomethylcoumarin (AFC) released from fluorogenic substrate Ac-DEVD-AFC (caspase-3 substrate) following ALA-PDT of H1299 cells. The cells were lysed in 50 μl lysis buffer (12.5 mM Tris-HCl, 1 mM dithiothreitol, 0.125 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, and an aliquot of complete protease inhibitor mixture (Roche Applied Sciences, Mannheim, Germany) pH 7.0, and centrifuged at 12000 rpm and4 °C for 15 minutes. A 100 μg aliquot of protein was incubated with 20 μM fluorogenic substrate of caspase-3 (Calbiochem, San Diego, California, USA) in 500 μl assay buffer (50 mM Tris-HCl, 1 mM EDTA, and 10 mM EGTA (ethyleneglycol-bis- (β-aminoethylether)-N, N, N’, N’-tetraacetic acid), pH 7.0) at 37 °C for 30 min in the dark as described previously [19

19. H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, “Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells,” J. Biomed. Opt. 13(5), 054011 (2008). [CrossRef] [PubMed]

]. The fluorescence intensity was determined by a spectrofluorometer (Hitachi F-3000, Tokyo, Japan) at an excitation wavelength of 408 nm and an emission wavelength of 505 nm.

2.5 NADH fluorescence lifetime imaging microscopy (FLIM)

Time-domain FLIM was performed using a 60X 1.45NA PlanApochromat oil objective lens (Olympus Corp., Tokyo, Japan) on a modified two-photon laser scanning microscope (FV300 with the IX71 inverted microscope, Olympus Corp., Japan) as described previously [19

19. H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, “Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells,” J. Biomed. Opt. 13(5), 054011 (2008). [CrossRef] [PubMed]

]. In brief, NADH fluorescence was two-photon excited at 740 nm by a mode locked Ti:sapphire laser (Mira F-900, Coherent Inc., Santa Clara, California, USA) pumped by a solid-state continuous wave 532 nm Verdi laser (Coherent Inc., Santa Clara, California, USA). The emitted NADH fluorescent light was detected using a band pass filter of 450 ± 40 nm (Edmund Optics, Inc., Barrington, New Jersey, USA). Time-resolved detection was conducted by the single photon counting SPC-830 printed circuit board (Becker & Hickl GmbH, Berlin, Germany). Data were analyzed with the commercially available SPCImage v2.8 software (Becker & Hickl GmbH, Berlin, Germany) via a convolution of a double-exponential model function, F(t)=a1et/τ2+a2et/τ2 and the instrument response function (IRF). The convoluted results were fitted to the experiment data to extract lifetime parameters τ1, τ2, a1, a2 and τm. τm is the mean lifetime defined as (a1τ1 + a2τ2)/(a1 + a2). IRF was measured using a second harmonic generated signal from a periodically poled lithium niobate crystal.

NADH fluorescence lifetime has recently been investigated as the biomarker of cell metabolic state [16

16. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]

,17

17. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007). [CrossRef] [PubMed]

] and cell death [19

19. H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, “Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells,” J. Biomed. Opt. 13(5), 054011 (2008). [CrossRef] [PubMed]

,20

20. J. S. Yu, H. W. Guo, C. H. Wang, Y. H. Wei, and H. W. Wang, “Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells,” J. Biomed. Opt. 16(3), 036008 (2011). [CrossRef] [PubMed]

]. This “fluorescence lifetime” usually refers to parameters like free NADH fluorescence lifetime (τ1~400 to 500 ps), bound NADH fluorescence lifetime (τ2 ~2000 to 3000 ps), the corresponding amplitudes a1 and a2, mean lifetime (τm = a1τ1 + a2τ2), and/or the ratio of relative amplitudes of two lifetime components (a1/a2). In this study, we only report τm as the fluorescence lifetime.

2.6 NADH fluorescence intensity measurement

NADH fluorescence intensity is measured by using a Victor2 1420 multilabel counter (PerkinElmer, Foster, Massachusetts, USA) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Treated cells were resuspended in 200 μl PBS and transferred into a black OptiPlate-96F 96-well plate (Packed Bioscience, Perkin-Elmer, Foster, Massachusetts, USA) to measure the NADH fluorescence and NADH fluorescence intensity was normalized to cell numbers.

3. Results

3.1 Characterization of cell death pathways induced by three different light doses of ALA-PDT

Slight cell death, primary apoptosis, and necrosis or non-apoptotic death was induced by treating cells at 1, 2, or 6 J/cm2, respectively, with a fixed fluence rate of 10 mW/cm2. In Fig. 1A
Fig. 1 Effect of different light fluences in response to controls and ALA-PDT treated H1299 cells on (A) cell viability, (B) caspase-3 activity and (C) sub-G1 contents. Sub-G1 contents were determined by FACS analysis of propidium iodide-stained cells for control cells (a), 5-ALA only treated cells (b), light only treated cells (6 J/cm2) (c), 5-ALA treated cells irradiated with light dose of 1 J/cm2 (d), 2 J/cm2 (e), and 6 J/cm2 (f). A total of 3 samples were used for each condition for average ± standard deviation. Symbol * indicates a statistic difference from controls (a) with a p-value less than 0.05 by student t-test.
, the cell viability of control cells was measured for cells without light and drug, with light only (6 J/cm2,10 mW/cm2), and with drug only treatments. No cell death was observed in all controls. The cell viability of PDT treated cells decreased slowly under the lowest fluence at 1 J/cm2 to remain 62% at 4 hr after PDT. It decreased rapidly within the first hour to be less than 30% with the highest dose at 6 J/cm2. At 2 J/cm2, cell viability decreased monotonically to 28% at 4 hr after PDT treatment. We then measured caspase-3 activity and DNA fragmentation to confirm the pathway of cell death as published reports [23

23. N. L. Oleinick, R. L. Morris, and I. Belichenko, “The role of apoptosis in response to photodynamic therapy: what, where, why, and how,” Photochem. Photobiol. Sci. 1(1), 1–21 (2002). [CrossRef] [PubMed]

]. Figure 1B shows the time course caspase-3 activity immediately after PDT (0 hr), and at 1, 2, and 3 hr after PDT treatment. The maximal caspase-3 activity appeared at 2 and 1 hr after PDT for light fluence of 1 and 2 J/cm2, respectively. No caspase-3 activity was shown at 6 J/cm2. Figure 1C shows DNA fragmentation for three different controls and for PDT treated cells at 4 hr after PDT. The light fluence of 2 J/cm2 induced 35.89 ± 11.17% sub-G1 contents (Fig. 1C (e)). No obvious sub G1 content was observed for controls (Figs. 1C(a)-(c)) and PDT treated cells at 1 J/cm2 (Fig. 1C(d)). Cells treated with 6 J/cm2 did not show any cell cycle distribution (Fig. 1C(f)). Table 1

Table 1. Statistic Summary for Experiments Statistically Different from Controls

table-icon
View This Table
summarizes the statistic results for those experiments with a p-value less than 0.05 using student t-test by comparing with controls without drug and light. Taken together, we conclude that the light fluence of 2 J/cm2 induced cell death primarily by apoptosis. Cells treated with 6 J/cm2 underwent non-apoptotic pathway that neither their caspase-3 activity nor DNA fragmentation showed any sign of apoptosis. The expression of caspase-3 activity for cells treated with 1 J/cm2 suggested that partial cells died by apoptosis.

3.2 The increase of NADH fluorescent lifetime was only detectable in cells that mainly died by apoptosis

To determine whether PDT induced cell death is detectable by NADH fluorescence lifetime changes, we acquired time course micrographs of NADH fluorescent lifetime of H1299 cells within the same field of view (FOV) for three different controls (Fig. 2
Fig. 2 Time course of NADH fluorescence lifetime micrographs of H1299 cells at the condition of 3 controls (treatment with 5-ALA only, light irradiation (6 J/cm2) only, and without 5-ALA and light). The light fluence rate was fixed at 10 mW/cm2 for the light control. The time course images at all conditions were taken at time points of 0 to 20 min (a), 20 to 40 min (b), 60 to 80 min (c), 100 to 120 min (d) after 4 hr serum-free medium treatment for drug controls or after the light irradiation for light controls. Each micrograph has a field of view 100 x 100 μm. The white scale bar indicates 20 μm.
) and for PDT treated cells immediately after PDT for up to two hours under three different fluence conditions (Fig. 3
Fig. 3 Time course of NADH fluorescence lifetime micrographs of H1299 cells at the condition of 5-ALA PDT treatment where cells with the light fluence of 1, 2, 6 J/cm2 in response to PDT were recorded from the same field of view. The light fluence rate was fixed at 10 mW/cm2 for all ALA-PDT treatment conditions. The time course images at all conditions were taken at time points of 0 to 20 min (a), 20 to 40 min (b), 60 to 80 min (c), 100 to 120 min (d) after 4 hour serum-free medium treatment and after the light irradiation. Each micrograph has a field of view 100 x 100 μm. The white scale bar indicates 20 μm.
). Controls without light and drug treatments were imaged at 0 to 20, 20 to 40, 60 to 80, and 100 to 120 minutes after the same preparation time for drug controls. Light controls (6 J/cm2, 10 mW/cm2) were imaged after light irradiation. Drug controls were imaged after 4 hr ALA incubation. PDT treated cells were imaged after 4 hr ALA incubation and light irradiation. Thus, time point at 0-20 minutes in Fig. 3 represents the time point immediately after PDT treatment. All micrographs are displayed using the same color bar of the time scale (200 to 3000 ps). We could examine cell morphology as well through these micrographs.

In Fig. 2, controls without drug and light treatments and light controls show no change on cell morphology and NADH fluorescence lifetime. Drug controls show shrinkage in cell morphology but no change in NADH fluorescence lifetime. In Fig. 3 (top row), cells treated with 1 J/cm2 did not show NADH fluorescence lifetime change. These cells tended to shrink immediately after PDT like drug controls shown in Fig. 2 (middle row). However, different from drug controls, these PDT treated cells returned back to their original size before PDT (as controls in Fig. 2, the top row) although the distribution of mitochondria (i.e., where NADH fluorescent) tended to be more punctuated and aggregated toward to nuclei. Cells treated with 2 J/cm2 (Fig. 3, middle row) became round shapes at 20-40 minutes and after. Their membranes showed blebbing structures (white arrow head 1) at 20-40 minute time point. At 1 hour after PDT, cells show ring-like signatures (white arrowhead 2), nuclei shrinkage, and mitochondria relocation toward to nuclei, which became more significant at 2 hr time point. The NADH fluorescence lifetime increased as early as immediately after PDT (blue color as indicated by white arrow head 3 at 0-20 minute micrograph), but the cell morphology at this time point remained the same as controls (Fig. 2, top row). Higher NADH fluoresce lifetime (blue dots as indicated by yellow arrow heads) appeared in those enlarged round cells. Finally under light treatment of 6 J/cm2 (Fig. 3, bottom row), cell morphology indicates that cells were not intact anymore, cell nucleus became small, and the nuclear structure was impaired. The NADH fluorescence lifetime shows no change that all cells appeared yellowish.

3.3 NADH fluorescent lifetime and intensity together differentiated between apoptosis, necrosis, and slight cytotoxicity and controls

Figure 4A
Fig. 4 The average ± standard deviation of NADH fluorescence lifetime (τm) (A) and NADH fluorescence intensity (B) respond to ALA-PDT with different light fluences. All PDT-treated cells were incubated with 5-ALA for 4 hours, and then irradiated with different light doses. A total of 6 and 3 samples for lifetime and intensity measurements, respectively, were repeated at each condition. Symbols ** and * indicates a statistic difference from controls with a p-value less than 0.005 and 0.05, respectively, by student t-test. P-values are summarized in Table 1. NADH fluorescence intensity was normalized to cell numbers.
shows the average results of NADH fluorescence lifetime on 6 samples (i.e., 6 independent replicated experiments) for each condition in control cells (without drug and light, light only, and drug only) and PDT treated cells at the fluence of 1, 2, or 6 J/cm2. Same as we observed in Figs. 2 and 3, NADH fluorescence lifetime only increased under light fluence of 2 J/cm2 in which cells underwent apoptotic pathway (Fig. 1B). Cells treated with 1 and 6 J/cm2 did not show NADH fluorescence lifetime change where cells underwent slight cell death and necrosis, respectively. The cell viability shown in Fig. 1A indicates that 1 J/cm2 treatment condition only induced slight cell death (<40% at 4 hr after PDT). Some of these cells might undergo apoptosis because of caspase-3 activation as shown in Fig. 1B although DNA fragmentation was not seen (Fig. 1C).

To demonstrate that our finding is clinically practical for use in PDT, NADH fluorescence lifetime alone is not enough for this purpose because PDT often induces both necrosis and apoptosis. The change of NADH fluorescence lifetime could only differentiate apoptosis from all other conditions including the controls, slight cytotoxicity, and necrosis (Figs. 3 and 4A), but it could not differentiate necrosis from controls and slight cytotoxicity. Based on many reports showing decreased NADH fluorescence intensity during/after cell death, we performed NADH fluorescence intensity measurement that has potential to differentiate necrosis from controls and slight cytotoxicity. Figure 4B shows that NADH fluorescence intensity decreased significantly (p-value = 0.036 by comparing with controls without light and drug) at light fluence of 6 J/cm2 but did not change at light fluence of 1 J/cm2 within 1 hr after PDT. Data at 0 hr after PDT treatment in Fig. 4B represents results immediately after PDT treatment. NADH fluorescence intensity did not show change between controls without drug and light, light and drug controls (data not shown). Our results suggest that primary apoptotic cells could be identified by measuring the signal of increased NADH fluorescence lifetime, and necrotic cells could be identified by detecting no NADH fluoresce lifetime change but significant decreased NADH fluorescence intensity.

4. Discussion

In the in vivo situation, cells die after PDT through a mix of apoptosis and necrosis. The results of this study suggest that NADH fluorescence lifetime increases only when cell death was primarily apoptotic (e.g., cells treated with 2 J/cm2 showed less than 30% cell viability at 4 hr after PDT, caspase-3 activation and DNA fragmentation in Fig. 1, and increased lifetime in Figs. 3 and 4A), but not when less than 40% cells died 4 hr after PDT and part of them died by apoptosis (e.g., cells treated with 1 J/cm2 showed 62% cell viability at 4 hr after PDT, caspase-3 activation in Fig. 1B, but no lifetime change in Figs. 3 and 4A). We envision that monitoring NADH fluorescence lifetime will be unique but only useful for optimizing the treatment efficacy of apoptosis favored treatments such as metronomic PDT. When cells die pre-dominantly by necrosis, we envision that no NADH fluorescence lifetime but a significant decrease in NADH fluorescence intensity will be detected.

It is generally accepted that cells have the characteristics of shrinkage, round shape and membrane blebbing during apoptotic process [27

27. H. Okada and T. W. Mak, “Pathways of apoptotic and non-apoptotic death in tumour cells,” Nat. Rev. Cancer 4(8), 592–603 (2004). [CrossRef] [PubMed]

,28

28. S. Elmore, “Apoptosis: a review of programmed cell death,” Toxicol. Pathol. 35(4), 495–516 (2007). [CrossRef] [PubMed]

]. We did not observe cell shrinkage but round shape and membrane blebbing for PDT-treated cells with 2 J/cm2 in which caspase-3 activation and DNA fragmentation were elicited in the cell dead of apoptosis. We observed cell shrinkage for control cells treated with drug only and for PDT-cells treated with 1J/cm2, but these 2 groups of cells had different kinetic change in cell size within 2 hr FLIM imaging as mentioned in the Results: 1) PDT-treated cells shrank and then resumed to its control size within 2 hr after the treatment; 2) the mitochondria showed more punctuated pattern in PDT-treated cells than did drug control cells. Based on the cell shrinkage effect of low dose ALA-PDT treatment (1 J/cm2), we suspect that the shrinkage of drug control cells was due to the imaging laser light that introduced some PDT effect.

Acknowledgments

We acknowledge the use of the imaging core facility of National Yang-Ming University in carrying out part of the experiments reported in this study. This work was supported by National Science Council of Taiwan grants NSC94-2321-B-010-004-YC, NSC98-2112-010-002, and NSC97-2320-B-010-013-MY3 and by the Ministry of Education of Taiwan grant 97QC021 multidisciplinary training program for talented college students.

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1.

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2.

B. C. Wilson, M. S. Patterson, and L. Lilge, “Implicit and explicit dosimetry in photodynamic therapy: a New paradigm,” Lasers Med. Sci. 12(3), 182–199 (1997). [CrossRef] [PubMed]

3.

K. K.-H. Wang, S. Mitra, and T. H. Foster, “A comprehensive mathematical model of microscopic dose deposition in photodynamic therapy,” Med. Phys. 34(1), 282–293 (2007). [CrossRef] [PubMed]

4.

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5.

M. J. Niedre, A. J. Secord, M. S. Patterson, and B. C. Wilson, “In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy,” Cancer Res. 63(22), 7986–7994 (2003). [PubMed]

6.

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7.

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A. Bogaards, A. Varma, K. Zhang, D. Zach, S. K. Bisland, E. H. Moriyama, L. Lilge, P. J. Muller, and B. C. Wilson, “Fluorescence image-guided brain tumour resection with adjuvant metronomic photodynamic therapy: pre-clinical model and technology development,” Photochem. Photobiol. Sci. 4(5), 438–442 (2005). [CrossRef] [PubMed]

14.

B. W. Henderson, T. M. Busch, and J. W. Snyder, “Fluence rate as a modulator of PDT mechanisms,” Lasers Surg. Med. 38(5), 489–493 (2006). [CrossRef] [PubMed]

15.

B. W. Henderson, S. O. Gollnick, J. W. Snyder, T. M. Busch, P. C. Kousis, R. T. Cheney, and J. Morgan, “Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors,” Cancer Res. 64(6), 2120–2126 (2004). [CrossRef] [PubMed]

16.

D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res. 65(19), 8766–8773 (2005). [CrossRef] [PubMed]

17.

M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A. 104(49), 19494–19499 (2007). [CrossRef] [PubMed]

18.

H. W. Wang, Y. H. Wei, and H. W. Guo, “Reduced nicotinamide adenine dinucleotide (NADH) fluorescence for the detection of cell death,” Anticancer. Agents Med. Chem. 9(9), 1012–1017 (2009). [PubMed]

19.

H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, “Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells,” J. Biomed. Opt. 13(5), 054011 (2008). [CrossRef] [PubMed]

20.

J. S. Yu, H. W. Guo, C. H. Wang, Y. H. Wei, and H. W. Wang, “Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells,” J. Biomed. Opt. 16(3), 036008 (2011). [CrossRef] [PubMed]

21.

H. W. Guo, Y. H. Wei, and H. W. Wang, “Reduced nicotinamide adenine dinucleotide fluorescence lifetime detected poly(adenosine-5′-diphosphate-ribose) polymerase-1-mediated cell death and therapeutic effect of pyruvate,” J. Biomed. Opt. 16(6), 068001 (2011). [CrossRef] [PubMed]

22.

D. Grebeňová, K. Kuželová, K. Smetana, M. Pluskalová, H. Cajthamlová, I. Marinov, O. Fuchs, J. Souček, P. Jarolím, and Z. Hrkal, “Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells,” J. Photochem. Photobiol. B 69(2), 71–85 (2003). [CrossRef] [PubMed]

23.

N. L. Oleinick, R. L. Morris, and I. Belichenko, “The role of apoptosis in response to photodynamic therapy: what, where, why, and how,” Photochem. Photobiol. Sci. 1(1), 1–21 (2002). [CrossRef] [PubMed]

24.

F. Giuntini, L. Bourré, A. J. MacRobert, M. Wilson, and I. M. Eggleston, “Improved peptide prodrugs of 5-ALA for PDT: rationalization of cellular accumulation and protoporphyrin IX production by direct determination of cellular prodrug uptake and prodrug metabolization,” J. Med. Chem. 52(13), 4026–4037 (2009). [CrossRef] [PubMed]

25.

B. W. Pogue, J. D. Pitts, M. A. Mycek, R. D. Sloboda, C. M. Wilmot, J. F. Brandsema, and J. A. O’Hara, “In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy,” Photochem. Photobiol. 74(6), 817–824 (2001). [CrossRef] [PubMed]

26.

H. W. Guo, C. T. Chen, Y. H. Wei, O. K. Lee, V. Gukassyan, F. J. Kao, and H. W. Wang, “Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies,” J. Biomed. Opt. 13(5), 050505 (2008). [CrossRef] [PubMed]

27.

H. Okada and T. W. Mak, “Pathways of apoptotic and non-apoptotic death in tumour cells,” Nat. Rev. Cancer 4(8), 592–603 (2004). [CrossRef] [PubMed]

28.

S. Elmore, “Apoptosis: a review of programmed cell death,” Toxicol. Pathol. 35(4), 495–516 (2007). [CrossRef] [PubMed]

OCIS Codes
(170.1420) Medical optics and biotechnology : Biology
(170.1610) Medical optics and biotechnology : Clinical applications
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(170.5180) Medical optics and biotechnology : Photodynamic therapy

ToC Category:
Medical Optics and Biotechnology

History
Original Manuscript: August 10, 2011
Revised Manuscript: October 3, 2011
Manuscript Accepted: October 3, 2011
Published: October 10, 2011

Virtual Issues
Vol. 6, Iss. 11 Virtual Journal for Biomedical Optics

Citation
Guan-Chin Su, Yau-Huei Wei, and Hsing-Wen Wang, "NADH fluorescence as a photobiological metric in 5-aminolevlinic acid (ALA)-photodynamic therapy," Opt. Express 19, 21145-21154 (2011)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-19-22-21145


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References

  1. D. E. Dolmans, D. Fukumura, and R. K. Jain, “Photodynamic therapy for cancer,” Nat. Rev. Cancer3(5), 380–387 (2003). [CrossRef] [PubMed]
  2. B. C. Wilson, M. S. Patterson, and L. Lilge, “Implicit and explicit dosimetry in photodynamic therapy: a New paradigm,” Lasers Med. Sci.12(3), 182–199 (1997). [CrossRef] [PubMed]
  3. K. K.-H. Wang, S. Mitra, and T. H. Foster, “A comprehensive mathematical model of microscopic dose deposition in photodynamic therapy,” Med. Phys.34(1), 282–293 (2007). [CrossRef] [PubMed]
  4. M. T. Jarvi, M. J. Niedre, M. S. Patterson, and B. C. Wilson, “Singlet oxygen luminescence dosimetry (SOLD) for photodynamic therapy: current status, challenges and future prospects,” Photochem. Photobiol.82(5), 1198–1210 (2006). [CrossRef] [PubMed]
  5. M. J. Niedre, A. J. Secord, M. S. Patterson, and B. C. Wilson, “In vitro tests of the validity of singlet oxygen luminescence measurements as a dose metric in photodynamic therapy,” Cancer Res.63(22), 7986–7994 (2003). [PubMed]
  6. K. K. Wang, S. Mitra, and T. H. Foster, “Photodynamic dose does not correlate with long-term tumor response to mTHPC-PDT performed at several drug-light intervals,” Med. Phys.35(8), 3518–3526 (2008). [CrossRef] [PubMed]
  7. S. Gross, A. Gilead, A. Scherz, M. Neeman, and Y. Salomon, “Monitoring photodynamic therapy of solid tumors online by BOLD-contrast MRI,” Nat. Med.9(10), 1327–1331 (2003). [CrossRef] [PubMed]
  8. J. C. Finlay and T. H. Foster, “Hemoglobin oxygen saturations in phantoms and in vivo from measurements of steady-state diffuse reflectance at a single, short source-detector separation,” Med. Phys.31(7), 1949–1959 (2004). [CrossRef] [PubMed]
  9. A. A. Stratonnikov and V. B. Loschenov, “Evaluation of blood oxygen saturation in vivo from diffuse reflectance spectra,” J. Biomed. Opt.6(4), 457–467 (2001). [CrossRef] [PubMed]
  10. G. Yu, T. Durduran, C. Zhou, H. W. Wang, M. E. Putt, H. M. Saunders, C. M. Sehgal, E. Glatstein, A. G. Yodh, and T. M. Busch, “Noninvasive monitoring of murine tumor blood flow during and after photodynamic therapy provides early assessment of therapeutic efficacy,” Clin. Cancer Res.11(9), 3543–3552 (2005). [CrossRef] [PubMed]
  11. B. Chen, B. W. Pogue, I. A. Goodwin, J. A. O’Hara, C. M. Wilmot, J. E. Hutchins, P. J. Hoopes, and T. Hasan, “Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor,” Radiat. Res.160(4), 452–459 (2003). [CrossRef] [PubMed]
  12. S. K. Bisland, L. Lilge, A. Lin, R. Rusnov, and B. C. Wilson, “Metronomic photodynamic therapy as a new paradigm for photodynamic therapy: rationale and preclinical evaluation of technical feasibility for treating malignant brain tumors,” Photochem. Photobiol.80(1), 22–30 (2004). [CrossRef] [PubMed]
  13. A. Bogaards, A. Varma, K. Zhang, D. Zach, S. K. Bisland, E. H. Moriyama, L. Lilge, P. J. Muller, and B. C. Wilson, “Fluorescence image-guided brain tumour resection with adjuvant metronomic photodynamic therapy: pre-clinical model and technology development,” Photochem. Photobiol. Sci.4(5), 438–442 (2005). [CrossRef] [PubMed]
  14. B. W. Henderson, T. M. Busch, and J. W. Snyder, “Fluence rate as a modulator of PDT mechanisms,” Lasers Surg. Med.38(5), 489–493 (2006). [CrossRef] [PubMed]
  15. B. W. Henderson, S. O. Gollnick, J. W. Snyder, T. M. Busch, P. C. Kousis, R. T. Cheney, and J. Morgan, “Choice of oxygen-conserving treatment regimen determines the inflammatory response and outcome of photodynamic therapy of tumors,” Cancer Res.64(6), 2120–2126 (2004). [CrossRef] [PubMed]
  16. D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White, and N. Ramanujam, “Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH,” Cancer Res.65(19), 8766–8773 (2005). [CrossRef] [PubMed]
  17. M. C. Skala, K. M. Riching, A. Gendron-Fitzpatrick, J. Eickhoff, K. W. Eliceiri, J. G. White, and N. Ramanujam, “In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia,” Proc. Natl. Acad. Sci. U.S.A.104(49), 19494–19499 (2007). [CrossRef] [PubMed]
  18. H. W. Wang, Y. H. Wei, and H. W. Guo, “Reduced nicotinamide adenine dinucleotide (NADH) fluorescence for the detection of cell death,” Anticancer. Agents Med. Chem.9(9), 1012–1017 (2009). [PubMed]
  19. H. W. Wang, V. Gukassyan, C. T. Chen, Y. H. Wei, H. W. Guo, J. S. Yu, and F. J. Kao, “Differentiation of apoptosis from necrosis by dynamic changes of reduced nicotinamide adenine dinucleotide fluorescence lifetime in live cells,” J. Biomed. Opt.13(5), 054011 (2008). [CrossRef] [PubMed]
  20. J. S. Yu, H. W. Guo, C. H. Wang, Y. H. Wei, and H. W. Wang, “Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells,” J. Biomed. Opt.16(3), 036008 (2011). [CrossRef] [PubMed]
  21. H. W. Guo, Y. H. Wei, and H. W. Wang, “Reduced nicotinamide adenine dinucleotide fluorescence lifetime detected poly(adenosine-5′-diphosphate-ribose) polymerase-1-mediated cell death and therapeutic effect of pyruvate,” J. Biomed. Opt.16(6), 068001 (2011). [CrossRef] [PubMed]
  22. D. Grebeňová, K. Kuželová, K. Smetana, M. Pluskalová, H. Cajthamlová, I. Marinov, O. Fuchs, J. Souček, P. Jarolím, and Z. Hrkal, “Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells,” J. Photochem. Photobiol. B69(2), 71–85 (2003). [CrossRef] [PubMed]
  23. N. L. Oleinick, R. L. Morris, and I. Belichenko, “The role of apoptosis in response to photodynamic therapy: what, where, why, and how,” Photochem. Photobiol. Sci.1(1), 1–21 (2002). [CrossRef] [PubMed]
  24. F. Giuntini, L. Bourré, A. J. MacRobert, M. Wilson, and I. M. Eggleston, “Improved peptide prodrugs of 5-ALA for PDT: rationalization of cellular accumulation and protoporphyrin IX production by direct determination of cellular prodrug uptake and prodrug metabolization,” J. Med. Chem.52(13), 4026–4037 (2009). [CrossRef] [PubMed]
  25. B. W. Pogue, J. D. Pitts, M. A. Mycek, R. D. Sloboda, C. M. Wilmot, J. F. Brandsema, and J. A. O’Hara, “In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy,” Photochem. Photobiol.74(6), 817–824 (2001). [CrossRef] [PubMed]
  26. H. W. Guo, C. T. Chen, Y. H. Wei, O. K. Lee, V. Gukassyan, F. J. Kao, and H. W. Wang, “Reduced nicotinamide adenine dinucleotide fluorescence lifetime separates human mesenchymal stem cells from differentiated progenies,” J. Biomed. Opt.13(5), 050505 (2008). [CrossRef] [PubMed]
  27. H. Okada and T. W. Mak, “Pathways of apoptotic and non-apoptotic death in tumour cells,” Nat. Rev. Cancer4(8), 592–603 (2004). [CrossRef] [PubMed]
  28. S. Elmore, “Apoptosis: a review of programmed cell death,” Toxicol. Pathol.35(4), 495–516 (2007). [CrossRef] [PubMed]

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