Increased singlet oxygen-induced secondary ROS production in the serum of cancer patients

Journal of Photochemistry and Photobiology B: Biology 107 (2012) 14–19

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Increased singlet oxygen-induced secondary ROS production in the serum of cancer patients Edith Bigot c, Regis Bataille b, Thierry Patrice a,⇑ a

Cancer Photobiology, Laennec Hospital, 44093 Nantes, France Anti Cancer Center René Gauducheau (CRLCC), 44800 Saint Herblain, France c Biochemistry, Laënnec Hospital, 44093 Nantes, France b

a r t i c l e

i n f o

Article history: Received 28 September 2011 Received in revised form 8 November 2011 Accepted 9 November 2011 Available online 22 November 2011 Keywords: Singlet oxygen Reactive Oxygen Species (ROS) Cancer Oxidative stress Anti-oxidant status Photodynamic therapy

a b s t r a c t Photodynamic therapy (PDT) generates singlet oxygen (1O2) and Reactive Oxygen Species (ROS) that are counteracted by patient’s defenses. As cancer treatments are among the most important PDT applications the aim of this pilot study was to determine whether the serum of cancer patients produces more or less secondary ROS or peroxides after a photoreaction as compared to healthy persons. Fifty-three volunteers and 105 cancer patients were recruited. The capacity of 1O2 or secondary oxidant production was found to be increased in 6 healthy donors and 36 cancer patients (23/69 women and 13/31 men p < 0.007 and p < 0.04) with a mean value of 1.52 as compared to 1.29 in the healthy subjects (p < 0.05) when considering values higher than the normal range (norm = 1 ± 10%) or 1.1 vs. 0.85 (p < 0.01) in the whole cohort. This increase correlated with a poor prognosis, TNM and SBR classification. Serum 1O2 deactivation capacity was impaired and secondary ROS were more produced during cancer progression. Although it is currently unclear whether this is the cause or effect of cancer, this finding may hold interest as a potential marker of cancer severity. It would also support the interest of PDT as an adjuvant for cancer treatment, even for aggressive tumors particularly when associated to surgery for bulk removal. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Cancer photodynamic therapy (PDT) is based on the production of singlet oxygen and hydroxyl radicals generating secondary Reactive Oxygen Species (ROS) and peroxides that we shall gather under the acronym SOS [1]. Cancers are prone to inducing oxidative stress and at any step of their development from induction to metastasis [2,3] including during treatments such as radiotherapy. Cancer growth also induces inflammatory reactions, which again contribute to generating singlet oxygen (1O2) [4]. 1O2 is produced in vivo by activated neutrophils [5,6] or eosinophils [7], during various biochemical reactions [8] including energy production, and may be generated indirectly by ionizing radiations and by photoreactions [8,9] during PDT. Singlet oxygen, is strongly oxidant for many biological targets, acting either directly or through the successive formation of various oxidative species arising during its deactivation and may react with various targets and/or generate SOS with a much longer half-life [10–13]. In a previous paper, we demonstrated that resistance to 1O2 was significantly decreased in patients with diabetes mellitus [14]. To determine whether ⇑ Corresponding author. Address: Laboratoire de Photobiologie des Cancers, Département Laser, 44093 Nantes, France. Tel.: +33 2 40 16 56 75/53 37; fax: +33 2 40 16 59 35. E-mail address: [email protected] (T. Patrice). 1011-1344/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2011.11.003

serum from patients diagnosed with cancers of various origins had the same capacity of SOS production after an initial 1O2 production as that from healthy volunteers, we used a standardized photoreaction as a source of 1O2, with light delivery immediately followed by the addition of DCFH, a marker that becomes fluorescent upon oxidation. The aim of the present paper was thus to determine whether cancer growth, which is likely to increase chronically ROS production, modifies response i.e. resistance to photo produced 1O2 and SOS, without presuming of the cause or the metabolic pathway leading to it. We also sought to identify any correlation between 1O2 or ROS neutralization and tumor histology or patient characteristics in order to determine whether it could modify tumor responses to PDT.

2. Materials and methods The study was conducted according to the protocol (NTS 200602) established between the Etablissement Français du Sang, Nantes anti-cancer center (CLRCC Nantes) and the Nantes University Hospital (NUH), in accordance with the Helsinki declaration (1964/2000). The protocol was approved by the NUH ethics committee. Blood samples obtained from 53 healthy blood donors were analyzed (Table 1) on a per donor basis or after sample pooling. None of the healthy donors had ever been previously diagnosed

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E. Bigot et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 14–19 Table 1 Singlet oxygen deactivation capacity of sera from healthy or cancer patients. Control

Age Ratio

H M L Mean Cancer localization

Cancer

Total

Men

Women

Total

Men

Women

40.1/53 (21–65)

42.8/31 (22–65)

36.2/22 (21–65)

60.6/100 (28–85)

58.9/31 (28–80)

61.6/69 (28–85)

1.29/6, 11% (0.14) 0.98/13 (24%) (0.05) 0.74/34, 64% (0.12) 0.85 (0.22)

1.28/4, 12% (0.16) 0.97/10, 32% (0.06) 0.75/17, 54% (0.13) 0.87 (0.23)

1.31/2, 9% (0.11) 1.00/4, 18%

(0.09) 0.83 (0.2)

1.52/36, 36% (0.5) 1.00/32, 32% (0.05) 0.74/32, 32% (0.17) 1.10 (0.48)

1.40/13, 41% (0.29) 1.01/5, 16% (0.06) 0.68/13, 41% (0.17) 1.03 (0.40)

1.59/23, 33% (0.65) 1.00/27, 39% (0.05) 0.74/19, 27% (0.17) 1.12 (0.52)

Breast Upper GI tract Lower GI tract Ovary Thyroid Kidney

– 1.21/2 0.97/13 – 0.63/3 –

1.07/41 1.17/3 0.94/8 1.33/7 0.81/2 0.94/1

Prostate Lung Testicle Pancreas Uterus Others or X?

1.21/5 1.34/3 0.81/3 1.13/1 – 1

– – – 2.58/2 0.85/2 3

Mean ages (extremes in brackets) and serum 1O2 deactivation capacity (Standard Deviation in brackets) of 53 healthy donors and 100 cancer patients according to gender and initial histology. 1O2 was produced by a standardized photo-reaction and secondary Reactive Oxygen Species (ROS) detected by fluorescence of oxidized fluorescein (DCF) at 525 nm for 66 min. Ratios are areas under the curve of DCF fluorescence divided by a control value previously established on 75 healthy male donors. Percentage refers to the number of samples measured as compared to the mean a given cohort ±10%. H: high production. M: medium. L: low production of secondary ROS. Numbers after/represents cohort size.

with a severe disease. Ten milliliters of blood were drawn and hemolysis was avoided as much as possible by using sterile clotactivator dry tubes (TerumoÒ Venosafe VF-054SP). Serum was collected avoiding hemoglobin aspiration under sterile conditions, separated into two aliquots and frozen at 20 °C until processed. The time period between blood sampling and freezing did not exceed 40 min. No donor had eaten or smoked 2 h before blood sampling. One-hundred and five patients were consecutively recruited and blood samples taken in the same conditions as for the healthy volunteers. Of these, 100 were included (69 women, mean age 61.6 years, range 28–85 and 31 men, mean age 58.9 years, range 28–80) as 5 patients were excluded due to an excessive rate of hemolysis [15–17]. The patients had been diagnosed with cancer between 1987 and 2007. Their clinical characteristics are described in Table 1. After the diagnosis and TNM and SBR for breast cancer staging, patients entered into treatment and follow-up protocols. Generally TNM classification was preferred in order to enable a minimum degree of comparison between tumors types. SBR was preferred to more recent staging methods because the latter did not apply to all patients included, given the sometimes long time interval between diagnosis and blood sampling. Disease progression was defined as tumor size increase and/or appearance of metastasis and/or Karnofsky score decrease. A disease free or remission status was defined as an absence of detectable tumor at the primary site or of metastasis, with a stable general health status for at least 12 months. Blood sampling was carried out during admission to hospital in 2008. Donors and patients were assayed for viral status (HIV and Hepatitis B), HDL-C, LDL-C, total cholesterol, triglycerides, Na, K, Ca, total proteins and albumin, glucose, urea, creatinine, uric acid, total and conjugated bilirubin, and C-reactive protein. ACE, CA 199, CA 15-3, CA 12-5, and expression of other more specific markers was determined when relevant for certain localizations, including SRB1 and 2 and hormonal receptors for breast cancers. Blood samples sent to our laboratory were identified only by numbers so that sample processing could be performed without knowing the patient characteristics. The study could thus be considered as semi blinded. The singlet oxygen-induced secondary ROS production that reflects resistance to 1O2 resistance assay has been described previously [15]. Briefly, absorption spectra were obtained with a

TechcompÒ 8500 absorption spectrophotometer for each patient sample and each control, after dilution in water for injections (5% serum). The level of hemolysis was evaluated from the 413 nm peak absorption and the baseline by the 650 nm absorption where there is normally a minimal amount of absorption. The baseline value was subtracted to provide a ‘‘corrected value’’ using a previously determined formula [15]. The principle of the measurement, as described previously [15], is to analyze the speed of neutralization of SOS induced by photodynamically produced 1O2, by means of the DCFH/DCF system. Activated DCFH is added to each assayed sample immediately after the end of light delivery (514 nm, 500 mW, 20 J/sqcm) and in a standardized manner. Rose Bengal (U 1O2: 0.75) [18] used as a source of 1O2 under light exposure may also produce small amounts of other oxidants such as superoxide ions. The Area Under the Curve (AUC) for the change in DCF fluorescence over time (Exitation 488nm, emission 525nm) was measured for each donor and patient serum. The corrected value for each patient was divided by the DCF fluorescence AUC value obtained with a pool of serum samples prepared from healthy volunteers and used as a reference. The reference ratio was equal to 1, a value higher than 1 indicated a greater AUC fluorescence than the reference pool used and thus a lower capacity for the given serum to neutralize 1O2 and a greater SOS production over time, whereas a lower value indicated a higher capacity to neutralize 1O2 and a lower SOS production. In an absence of other data, a normal range of values had been established from a precedent cohort of 75 presumed healthy male donors [15] at 1 ± 10% thus an interval of 20%. Data were tested for normal distribution using the Kolmogorov–Smirnov test. The Chi square test was then used for group comparisons, the Mann–Whitney U test for data comparisons and the Pearson test for correlations. P values p < 0.05 were considered statistically significant. Cohorts of at least 8 individuals were included for statistical analyses.

3. Results The profiles of DCF fluorescence evolution as well as the AUC values were similar to previous data [15]. Although variations existed within the AUC recorded for each subject (Fig. 1A), these were not linked to differences in the extinction coefficient as they had

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E. Bigot et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 14–19 4

4

A

3,5

Corrected Ratio

3 2,5 2 1,5

B

3 2,5 2 1,5 1

0,5

0,5

0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Corrected Ratio

3,5

Donors

Patients 4

4

C

D

3

Corrected Ratio

3 2,5 2 1,5

2,5

1,5

22

21

20

19

18

17

16

15

14

13

12

11

9

10

8

7

6

0 5

0 4

1 0,5

3

1

2

« 63 »

2

0,5

1

Corrected Ratio

3,5

3,5

Donors

Patients

Fig. 1. Corrected ratios represent patient values divided by the reference (pooled sera from healthy volunteers) of Area Under the Curve (AUC) of DCF fluorescence observed after addition of DCFH to photo-treated (514 nm, 20 J cm 2) sera in water (5%) containing RB (5 lg/mL). Individual variations of the serum resistance to singlet oxygen in: (A) healthy male donors. (B) Male patients. (C) Healthy female donors. (D) Female patients. For (B) and (D), black bars indicate healthy individuals or patients in remission, white bars with diagonal black lines indicate patients with a progressive form of cancer, gray bars indicate a non-progressive cancer but with an unknown etiology and gray bars with diagonal black lines indicate that cancers (No. 26) and (No. 63) are in progression but with an unknown etiology. Horizontal gray lines represent the normal interval of corrected ratio.

been corrected for absorption differences at 413 nm. The means calculated from the whole cohort of healthy donors (0.85, range 0.44–1.51) for a mean optical density of 0.096 at 413 nm did not significantly differ when they were compared to the range of variations measured on a per donor basis (AUC) for men (mean 0.87, SD 0.23, range 0.44–1.51) or women (mean 0.83, SD 0.2, range 0.54–1.38). They slightly differed (0.85 instead of 1) from the reference, which was also composed of healthy pooled sera, because the values were corrected for randomly occurring hemolysis whereas the reference was not. Six donors (4 men) had been found above the ‘‘normal’’ interval and 34 under. Age had only a slight influence on the capacity of serum to produce secondary oxidants after exposure to 1O2 and ROS within the range of ages assayed, although this capacity slightly increased between 40 and 60 years of age [15]. No correlation was noted with individual AUC-measured DCF fluorescence and the donor’s viral status, biochemical and hematological parameters (all of which were within the normal range). The profile of DCF fluorescence evolution was also similar to those recorded for healthy donors (unpublished data). The mean capacity of serum to deactivate secondary oxidants after exposure to 1O2 and ROS was similar between cancer patients and healthy individuals in both the male and female cohort (Table 1). The influence of gender was similar with 1O2 and ROS deactivation capacity for women as compared to men (1.00 versus 1.01). 23 of 69 women and 13 of 31 men had a reduced capacity to deactivate 1O2 and ROS. Similarly 19 women and 13 men had a better capacity than normal to deactivate 1O2 and ROS. This did not correlate with serum absorption, particularly at the absorption wavelength of 413 nm for hemoglobin released from hemolysis following blood sampling (data not shown). The highest observed fluorescence values in cancer patients were greater than the highest values

measured in presumed healthy individuals and the number of individuals above the ‘‘normal’’ range was significantly greater: 6/53 (11%) vs. 36/100 (36%), (p < 0.02) (Fig. 1). Both the series of scores of resistance to 1O2 (Mann–Whitney U test) and the number of patients (Chi square test) significantly differed for all of them from their corresponding healthy counterparts (0.001 < p < 0.05). There was no relationship with resistance to 1O2 according to age in cancer patients. Moreover, no correlation was found between SOS production and the initial tumor histology, and no subgroup was associated to a particularly abnormal value. Nine out of 26 patients (11 women, 21 adenocarcinomas of the lower GI tract) had a GI cancer associated to a decreased 1O2 deactivation capacity (36%) thus the same rate as in the whole cohort (36/100). When considering tumors of the upper GI tract an equally reduced capacity was noted in men and women. On the other hand, for patients with a lower GI tract tumor, a decreased capacity was noted in 4/13 men (31%) but in only 1/8 women (12%). In 41 women with breast cancers, 14 (34%) displayed an increased SOS production after 1O2 delivery. However, no correlation with breast tumor histology was noted as among the 32 tubular type tumors, 10 had a decreased resistance to 1O2 (31%), 14 had a normal resistance and 8 had an increased resistance. The capacity of serum to deactivate 1O2 appeared to be associated, although not significantly (p < 0.06), with hormonal expression as estrogen (12/ 14 patients) and progesterone (9/14 patients) receptor expression was the strongest when the singlet oxygen-induced SOS production or 1O2 deactivation capacity was the lowest (Table 2). No correlation had been noted with expression of specific or non-specific markers, as ACE was associated to a low value in 13% of cases, CA 19-9 in 16%, CA 15-3 in 13% and CA 125 in 30% (4 of 13 patients had an abnormal value). Among the 36 patients with a reduced

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E. Bigot et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 14–19 Table 2 Singlet oxygen deactivation capacity of sera from patients with breast cancer.

90

Ratio H: 1.42 (0.42)/14 M: 1.01 (0.05)/16 L: 0.73 (0.20)/11

Mean

Mean

Est. R

SBR

T

12 (86%) 10 (62%) 6 (54%)

Prog. R 9 (64%) 7 (43%) 5 (45%)

2.36 2.19 1.80

2.2 1.56 1.73

N 10 6 5

Sera capability of 1O2 deactivation of 41 patients bearing breast cancers according to number of tumors expressing estrogen (est) or progesterone (prog) receptors and staging. Ratios are the ratio of areas under the curve of DCF fluorescence after a standardized production of 1O2 divided by a control value previously established on 75 healthy male donors (Standard Deviation), number of patients. Percentage refers to the number of samples measured as compared to a given cohort H: high production, M: medium, L: low production of secondary ROS. SBR: Scarff–Bloom– Richardson classification, T: tumor, N: number of patients with at least one node detected (TNM).

deactivating capacity, 17 patients had at least one elevated marker. However, 9 out of 23 women with a decreased 1O2 deactivation capacity and 10 out of 13 men did not display elevated levels of any of these markers. Finally the strongest correlation was noted between the capacity to deactivate 1O2 and the clinical status of the cancer (Fig. 2). Twenty-five out of 36 (69%) patients with a low 1O2 deactivating capacity were classified as displaying disease progression as compared to 9 out of 64 patients (13%) with a normal or high 1O2 deactivating capacity, p < 0.001. This was also true when considering only the male cohort (10 out of 13 (77%) versus 4 out of 18 (22%), p < 0.048 or the female cohort only (15 out of 23 (65%) as compared to 5 out of 46 (11%), p < 0.007. When considering the breast cancer subgroup, 6 out of 14 (43%) were in progression as compared to 3 out of 27 (11%) with a high 1O2 deactivating capacity, p < 0.041. This correlated well with the SBR score for which a mean of 2.36, 2.19 and 1.80 was found for patients, or with TNM classification, with, respectively, a low, medium or high 1O2 deactivating capacity (Table 2). Two female patients remained disease-free of tubular breast cancer with a follow-up of 10 years and 5 years despite displaying a lower, albeit relatively normal, 1 O2 deactivating capacity. A third female patient with a much lower deactivating capacity at the time of blood sampling is still disease free of a T3N0M0, SerB2 negative, tubular breast cancer after 4 years, although it is worth noting that she had been diagnosed with heart failure at the time of blood sampling, which could itself explain the low measured 1O2 deactivating capacity. Several biochemical parameters for a given patient were found to be abnormal both in the low and high groups, which could explain the measured values. However no statistical significance was reached since multiple and different parameters, likely to influence inversely SOS production, varied simultaneously in sera of patients, including in the normal 1O2 deactivating capacity group. This had been noted previously [14]. 4. Discussion From the ‘‘treatment side’’, PDT efficacy is determined by the balance between photo oxidations induced by 1O2 and SOS versus their elimination by the scavenging activity of tissue antioxidant systems. In addition PDT damages may up-regulate antioxidant systems [19,20]. From the ‘‘target side’’, it had been demonstrated that the various steps during cancer growth evolution are all accompanied by an increased level of oxidative stress arising from an imbalance between ROS production and the deactivation processes that limit their deleterious physiological effects [21,22]. By measuring deactivation time of a standardized amount of 1O2 produced by a short photoreaction, we recently demonstrated that secondary ROS may vary from one person to another and even

Number of patients

80

Number of tumors

High secondary ROS production

70

Low secondary ROS production

60 50

Progression

64

40 30

36

46

20 10

23 25 9

10

P<0.001

14 15

4

0

Total

27

18

13

5

6

3

Men

Women

Breast

P<0.048

P<0.007

P<0.041

Fig. 2. Distribution of patients according to the corrected ratio values and cancer progression. The corrected ratio was calculated by dividing the Area Under the Curve (AUC) of a tested serum by the AUC of the reference, then correcting for hemolysis impairment if necessary. Patients with values higher than 1.1 are high secondary ROS producers whereas values below 0.9 represent a low production.

from one mouse strain to another [23]. We also demonstrated that a chronic imbalance of ROS production during diabetes mellitus leads to a decreased capability to deactivate 1O2 [14]. The procedure we used involves an internal control based on sera from presumed healthy donors. These are sampled blindly so that we cannot exclude some diseased persons among them since 6 of donors (Fig. 1) are above the normal range. From preliminary studies we noted that a single cigarette was likely to modify the SOS production. Samples for controls were thus obtained in rigorously similar conditions. In addition to their direct oxidative effects, SOS have to be deactivated by anti-oxidative molecules. This system creates a kind of vicious circle as the more ROS are produced the more oxidation will occur and the greater the need for ROS to be deactivated, inducing the consumption of more antioxidants. This phenomenon may arise in cancers, as metabolic abnormalities have been identified in many cancers, for which treatments are strongly oxidative [24]. 1O2, which is produced during PDT and radiotherapy, could represent finally a model that may, to a certain extent, account for what happens during inflammatory reactions involving PMNL [25] and during cancer growth inducing also poorly understood inflammatory reactions. For all these reasons estimating accurately and globally the effective defenses against ROS in patients with cancers is thus pivotal for pro-oxidant cancer therapies efficacy [26]. The direct measurement of ROS, ultimately 1O2-induced, is nearly impossible on a routine basis or in vivo [27,28]. Moreover, the separate measurement of anti-oxidant molecules is difficult as their anti-oxidant effects are randomly additive and depend on the targets or the type and quantity of ROS. The total anti-oxidant status (TAS) has been used to assess anti-oxidative patterns [28,29] resulting from the action of very different molecules: vitamins, albumin or more specific molecules such as glutathione [30], but also non-specific compounds with anti-oxidant properties such as uric acid or bilirubin [31]. The DCFH–DCF system is routinely used to detect SOS generated, regardless of their source, with a reasonable accuracy [16,32]. The technique is non-specific and can be used to detect not only primary radicals and radical ions, but also the whole family of oxi-radicals and peroxides [11]. Using 1O2 bypasses the spin interdiction that renders oxygen unable to directly oxidize biological molecules i.e. glucose or lipids. Since triplet ground state oxygen is excited into singlet state, it can directly oxidize targets and then the deactivation will simply follow the physiological routes of deactivation, something impossible with a test that would need the addition of a chemical likely to react with a given kind of compound i.e. lipids.

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E. Bigot et al. / Journal of Photochemistry and Photobiology B: Biology 107 (2012) 14–19

The effects of oxidative reactions after 1O2 production will depend to a large extent on the capacity of each individual’s serum to neutralize 1O2 and the SOS produced. Singlet oxygen may react with many targets producing ROS that will in turn deactivate, progressively producing peroxides, which themselves may produce ROS. The lifetime and the radius of action of 1O2 are short, ranging from 3 ls in water to 30 ls in lipids [33,34]. Secondarily-induced ROS or peroxides can have a much longer half-life [13]. In addition, 1 O2 can reactivate from superoxide anion via the Russel reaction [35] or from hypochlorous acid. Our results suggest that tumors with a poor prognosis should be more sensitive to oxidative treatments since SOS are produced more after 1O2. However our results also suggest that deactivation pathways of ROS may also be impaired systemically in patients with more aggressive tumors. This could have deleterious consequences on the general health of the patient, besides complications linked to the tumor itself, and may also stimulate cancer progression. This in agreement with recent suggestions [36] and findings showing that estrogen promotes carcinogenesis through oxidative stress damage. Our findings that a correlation exists between estrogen receptors and a decreased resistance to 1O2 also supports it [37]. Since cancer may enhance oxidative stress through various pathways, it had been suggested that a supplementation in antioxidants may have a preventive action. Several long-term studies have been performed but the subjects received supplements in a blinded manner, without determining whether any need for antioxidants existed. As such, the impact was shown to be very limited or even negative, stimulating cancer occurrence. In the present study the percentage of patients above or under the normal range (arbitrarily decided) was higher to presumably healthy donors, 1O2 deactivation capacity was significantly decreased and the rate of SOS production after 1O2 production increased in patients with cancers. This pattern was not dependent on the gender but rather on the severity of the cancer. This was true when considering the type of cancer, although the number of patients per histological type was small in this opened pilot study, but it was also true for breast cancers. To our knowledge, the present observation, which correlates well with findings in other medical fields [14], had never been reported before. The question that remains relates to cause or effect: does the decrease in resistance to 1O2, i.e. during aging [38], precede the cancer appearance, or even simply an ‘‘aggressive’’ transformation of a slow growing tumor, or does the ‘‘progressive’’ phenotype induce a decreased resistance to 1O2? This is a key point to determine the potential interest of restoring a normal resistance to 1O2 that could help the patients to defend themselves against cancer or better resist to cancer treatments [2], of course without stimulating tumor growth thanks to an appropriate combination and administration schedule of anti-oxidants. A better understanding of the role of ROS, SOS and antioxidants during PDT, a highly oxidative treatment, could lead to a better efficacy against cancer tissues and a greater preservation of healthy tissues in the vicinity. Finally it could support the use of PDT for aggressive tumor as an adjuvant treatment during surgery, providing the patient with an additional procedure to reach the last cancer cell, without having to destroy the whole mass with PDT. 5. Abbreviations

1

O2 AUC DCF DCF-DA

singlet oxygen Area Under the Curve dichlorofluorescein (oxidized, fluorescent) dichlorofluorescein diacetate

DCFH PDT PMNL RB ROS SD SOS TNM SBR

dichlorofluorescein (reduced, non-fluorescent) photodynamic therapy polymorphonuclear leukocytes Rose Bengal Reactive Oxygen Species Standard Deviation secondary reactive oxygen species and peroxides Tumor–Nodes–Metastasis classification Scarff–Bloom–Richardson classification

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