Clin Exp Med DOI 10.1007/s10238-014-0313-6
ORIGINAL ARTICLE
Potential survival markers in cancer patients undergoing chemotherapy Krzysztof Roszkowski • Jan Filipiak • Magdalena Wisniewska • Anna Mucha-Malecka Pawel Basta
•
Received: 1 July 2014 / Accepted: 15 September 2014 Ó Springer-Verlag Italia 2014
Abstract Due to the importance of the identification of chemotherapy outcome prognostic factors, we attempted to establish the potential of oxidative stress/DNA damage parameters such as prognostic markers. The aim of the study was to determine whether platinum derivative-based chemotherapy in cancer patients (n = 66) is responsible for systemic oxidatively damaged DNA and whether damage biomarkers, such as 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxo-dG) and the modified base 8-oxo-7,8-dihydroguanine (8-oxo-Gua), in urine and DNA may be used as a prognostic factor for the outcome of chemotherapy. All the aforementioned modifications were analyzed using techniques involving high-performance liquid chromatography/ electrochemical detection (HPLC/EC) or HPLC/gas chromatography–mass spectrometry (GC–MS). Among all the analyzed parameters, the significantly decreased levels of 8-oxo-Gua in urine collected from a subgroup of patients
K. Roszkowski Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Torun, Poland K. Roszkowski (&) Department of Radiotherapy, The F. Lukaszczyk Oncology Center, Bydgoszcz, Poland e-mail: [email protected] J. Filipiak M. Wisniewska Department of Chemotherapy, The F. Lukaszczyk Oncology Center, Bydgoszcz, Poland A. Mucha-Malecka Department of Radiotherapy, Center of Oncology – Maria Skłodowska-Curie Memorial Institute, Krakow, Poland P. Basta I Department of Surgery, Medical College, Jagiellonian University, Krakow, Poland
24 h after the first infusion of the drug, as compared with the baseline levels, correlated with a significantly longer overall survival (OS) (60 months after therapy) than in the subgroup without any decrease of this parameter after therapy (median OS = 24 months, p = 0.007). Moreover, a significantly longer OS was also observed in a group with increased urine levels of 8-oxo-dG after chemotherapy (38.6 vs. 20.5 months, p = 0.03). The results of our study suggest that patients with decreased 8-oxo-Gua levels and increased 8-oxo-dG levels in urine 24 h after the first dose should be considered as better responders to the administered chemotherapy, with a lower risk of death. The conclusion may permit the use of these parameters as markers for predicting the clinical outcome of platinum derivativebased chemotherapy. Keywords Molecular biomarkers Oxidatively damaged DNA Survival markers 8-Oxo-Gua 8-Oxo-dG Chemotherapy Cancer patients
Introduction In the course of some essential metabolic processes occurring in cells, reactive oxygen species (ROS) with potentially genotoxic and mutagenic properties are formed [1]. ROS, such as superoxide, hydrogen peroxide and hydroxyl radical are by-products of cellular respiration, but they may also be generated during inflammatory processes [2, 3]. Similarly, reactive nitrogen species (RNS) generate toxic products in their reactions with cells and free molecules [4]. Normal cells are equipped with enzymatic and nonenzymatic mechanisms limiting the production of ROS; however, abnormally functioning cells are often subjected
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to oxidative stress in which the balance between oxidants and antioxidants becomes disrupted, which results in an increased amount of cellular damage. The consequences of this process include, e.g., peroxidation of membrane lipids or oxidative modifications of proteins and DNA [5, 6]. Eukaryotic organisms are constantly at risk of the effects of ROS that play an important role in the pathogenesis of some diseases in human: tumors, cardiovascular diseases, neurodegenerative diseases and problems related with aging [7, 8]. Characteristics of tumors are the persistent high levels of ROS and permanent oxidative stress [9]. These processes are aided by, e.g., abnormal vascular system of the tumor, infiltration by macrophages and glycolysis [10, 11]. The nitrogenous bases forming DNA are particularly susceptible to the oxidation by ROS. The low oxidoreduction potential of guanine causes this base to be susceptible to such modifications, resulting in the formation of many guanine oxidation products. 8-Oxo-7,8-dihydroguanine (8-oxo-Gua) is often employed as a biomarker of oxidative stress [12]. It is generally accepted that 8-oxo-Gua excised from DNA molecules in the process of cellular DNA repair is excreted with urine in an unmetabolized form [13]. However, the repair processes which contribute to the presence of 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) in urine are still an open question. In general, the evidence suggests that the action of the Nudix hydrolase family of enzymes, sanitizing the deoxyribonucleotide pool via the degradation of 8-oxo-7,8-dihydro-20 -deoxyguanosine-50 -triphosphate and 8-oxo-7,8-dihydro-20 -deoxyguanosine-50 -diphosphate, and yielding mononucleotide products which may then be dephosphorylated to 8-oxodG, probably represents the most logical and primary source of 8-oxo-dG in urine, with a lesser role of other possible DNA repair processes [14]. However, some of the most recent studies report that NER (nucleotide excision repair) and NIR (nucleotide incision repair), transcriptioncoupled repair, mismatch repair and various exonuclease activities may play a greater role in the formation of 8-oxodG excreted in urine than previously presumed [14]. Antitumor chemotherapy acts via various mechanisms characteristic of a given class of medicines, leading to cell death. Some chemotherapeutics, apart from their principal mode of action, affect cells also through the increase of oxidative stress [15–17]. Oxidative stress may possibly emphasize the cytotoxic effect of chemotherapy on tumor cells. Therefore, in our studies we analyzed several parameters describing oxidatively damaged DNA. Apart from 8-oxo-dG excreted with urine, we determined the urine levels of the modified DNA base (8-oxo-Gua), as well as the background level of the modification in cellular DNA.
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Our previous studies have shown that oxidative stress markers such as 8-oxo-dG and 8-oxo-Gua may be predictive factors of individual sensitivity to radiation therapy [18]. Therefore, we decided to check whether the same markers may somehow be responsible for chemotherapy success. The aim of the study was to establish: a.
b.
whether platinum derivative-based chemotherapy (cisplatin and carboplatin) is responsible for systemic oxidatively damaged DNA in cancer patients; whether damage biomarkers excreted in urine, such as 8-oxo-dG or 8-oxo-Gua, as well as the level of oxidatively damaged DNA in leukocytes (all of these parameters reflect systemic oxidatively damaged DNA) may be employed for establishing a prognosis of chemotherapy outcome.
Materials and methods The analysis of daily excretion of 8-oxo-Gua and 8-oxo-dG in urine was conducted in a group of 66 patients with malignant tumors (clinical stage III and IV), undergoing treatment between June 2002 and July 2005. The patients suffered from various malignancies: ovarian cancer (n = 38), testicular cancer (n = 7), lung cancer (n = 9), head and neck cancer (n = 4) and others: stomach cancer, bile duct cancer, bladder cancer (n = 8). All patients received cisplatin-based chemotherapy at appropriate doses. In all patients treated with cisplatin participating in this study, normal values of renal parameters were determined before treatment (it was a necessary condition for chemotherapy). The adopted chemotherapy treatment schemes included: cisplatin with paclitaxel (22 patients, 33%), cisplatin with cyclophosphamide (14 patients, 21%), cisplatin with bleomycin (9 patients, 14%), cisplatin with 5-fluorouracil (6 patients, 9%), cisplatin with etoposide (6 patients, 6%), cisplatin with doxorubicin (9 patients, 14%). The clinical characteristics of the patients are presented in Table 1. The concentrations of basic markers of oxidatively damaged DNA were determined before the administration of systemic therapy (sample I) and 24 h after administering the first dose (sample II). Our earlier studies revealed that in patients undergoing oncological therapy, the levels of biomarkers excreted with urine may either increase or decrease [18, 19]. Therefore, we distinguished subgroups of patients in whom an increase (subgroup A) or a decrease (subgroup B) in the levels of the three studied markers were detected after the first 24 h of therapy. Analysis of overall survival was conducted for both subgroups. The assessment of diurnal excretion of 8-oxo-Gua and 8-oxo-
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dG in urine and the determination of the 8-oxo-dG content in the DNA of peripheral blood leukocytes were performed using previously described methods [20, 21]. Urinary excretion expressed in nmol/kg/24 h can provide more information, e.g., allows the quantification of damage repaired per day per cell [22]. Some researchers collect a single sample of urine and determine the 8-oxodG and 8-oxo-Gua levels with a correction for creatinine concentration (i.e., in the same urine sample, the quantities of oxidatively modified bases and nucleotide levels are determined along with the levels of creatinine) [23]. Urine samples collected in the morning may be used to determine the excretion rate as long as the creatinine excretion remains unchanged. Such a method of data interpretation can be problematic in patients with advanced cancer. The levels of creatinine excreted in urine in these patients may Table 1 Patient and treatment characteristics Patient characteristic
No. of Patients
Total
66
%
Age (years) Median Range
56 34–71
Sex Male
25
38
Female
41
62
vary according to the changes in body weight. In this case, a reasonable and more convenient solution is the determination of oxidatively damaged DNA parameters in urine from diurnal collection before treatment and the day immediately after the administration of the above-mentioned first therapeutic dose (of the medicinal agent). This creates methodological difficulties and forces researchers to follow a certain study regime, but also allows them to obtain more reliable results. Such an approach was adopted in this study. For the above reasons, parameters before treatment and after the first day of treatment were analyzed in the investigated group of patients. The study was approved by the medical ethics committee of The Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland, no. 274/2002; 409/2002 (in accordance with Good Clinical Practice, 1998), and all patients granted their informed consent. All results were expressed as means using the SPSS Statistics software, version 10.0. Statistical analyses were carried out using Student’s t test (for variables with normal distribution levels of oxidatively damaged DNA before and after chemotherapy). The association between overall survival and the urinary excretion of 8-oxo-dG and 8-oxoGua, as well as the level of oxidatively damaged DNA in leukocytes, were estimated using the Kaplan–Meier method and assessed using the log-rank test. The results were considered as statistically significant at p \ 0.05.
ECOG performance status 0–1
52
79
C2
14
21
Ovarian cancer (n = 38)
III
57
Lung cancer (n = 9)
IIIb
14
Stage %
Head & neck (n = 4)
IV
6
Testicular cancer (n = 7)
IIIb
11
Stomach, bile duct, bladder cancers (n = 8)
IV
12
22 14
33 21 14
Chemotherapy Cisplatin ? paclitaxel Cisplatin ? cyclophosphamide Cisplatin ? bleomycin
9
Cisplatin ? 5-fluorouracil
6
9
Cisplatin ? etoposide
6
9
Cisplatin ? doxorubicin
9
14
ECOG Eastern Cooperative Oncology Group
Table 2 The level of 8-oxoGua, 8-oxo-dG in the urine and 8-oxo-dG in the leukocytes’ DNA before infusion of the drug (sample I), 24 h after (sample II)
All patients
Results For the total patient population, the median value of 8-oxoGua concentration in urine before treatment (sample I) was 9.86 nmol/24 h, and interquartile range was 5.47–6.09. After the first infusion of the drug (sample II), the measured concentration increased to 24.23 nmol/24 h; interquartile range 14.38–36.30. This difference was statistically significant (p = 0.001; Table 2). There were no significant differences between samples I and II with respect to the urinary excretion of 8-oxo-dG (the values were 15.17 nmol/24 h, interquartile range 8.92–7.68 and 16.74 nmol/24 h, interquartile range: 7.22–8.27, respectively), while the median 8-oxo-dG content in DNA isolated from leukocytes reached 4.71
Median (interquartile range) Sample I
p
Sample II
Urinary 8-oxo-Gua (nmol/24 h)
9.86 (5.47– 6.09)
24.23 (14.38–36.30)
p = 0.001
Urinary 8-oxo-dG (nmol/24 h)
15.17 (8.92– 7.68)
16.74 (7.22– 8.27)
p = 0.47
4.71 (3.47–5.83)
4.77 (3.76–5.93)
p = 0.26
6
Leukocytes’ 8-oxo-dG/10 dG
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Fig. 2 Kaplan–Meier curves of subgroup of 9 patients, in whom both a decrease in 8-oxo-Gua and an increase in 8-oxo-dG in urine and all other patients
Fig. 1 Kaplan–Meier curves of a 8-oxo-Gua in urine; b 8-oxo-dG in urine; c 8-oxo-dG in the leukocytes’ 24 h after infusion chemotherapy
(3.47–5.83) and 4.77 (3.76–5.93) of 8-oxo-dG per 106 dG, respectively (Table 2). A comparative analysis of overall survival with respect to possible differences between sample I and II was conducted for all the above parameters. A significant increase in overall survival was found in the subgroup of 14 patients with a decrease (sample II) in
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the levels of urinary 8-oxo-Gua (median: 60 months), as compared with the subgroup that responded with an increase (sample I) in the urine levels of this derivative 24 h after infusion, median: 24 months (p = 0.007; logrank test) (Fig. 1a). A statistically significant extension of overall survival was also observed in the subgroup of patients with an increase in urine 8-oxo-dG levels after chemotherapy (Fig. 1b). No significant differences in overall survival were noticed with respect to the 8-oxo-dG content in cellular DNA (data not shown). The selection of patients followed the criteria of the group of patients; these included age, body weight and smoking status. There were no differences among the patients with various stages of the disease development concerning all of the analyzed modifications (data not shown). In a small subgroup of 9 patients, in whom both a decrease in 8-oxo-Gua and an increase in 8-oxo-dG in urine occurred after the first day of drug infusion, exceptionally long overall survival, even up to 78 months, was observed (Fig. 2). Similarly, no significant differences among the studied time points were found with respect to creatinine clearance and creatinine concentration (data not shown). A similar observation was reported by other groups [19, 24].
Discussion The analysis of 8-oxo-Gua and 8-oxo-dG levels patients’ urine, taken before drug administration different times after the start of chemotherapy, provide data indicating either the effectiveness
in the and at should of the
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mechanisms of oxidatively damaged DNA repair or the intensity of systemic oxidative stress. By comparing the level of these modifications with parameters used for the assessment of therapy outcome, such as overall survival, we should be able to determine the potential causal relationship between the prognosis for the patients and the oxidatively damaged DNA induced by chemotherapy, as well as the effectiveness of repair mechanisms. Establishing such a correlation might be one of the parameters considered when deciding on another cycle of chemotherapy or treatment termination in clinically dubious cases. As has been previously revealed, cisplatin therapy causes serious oxidative stress [19, 25]. Therefore, the observed increased urine levels of 8-oxo-Gua and 8-oxodG in most cancer patients 24 h after drug administration may be a result of an increased generation of reactive oxygen species (Table 2). This variability may reflect individual differences in metabolism and DNA repair capacity, as well as, at least partially, in background genetic differences [26, 27]. Other studies also indicate that oxidative stress and ROS generation may be at least partially responsible for the cytotoxicity induced by cisplatin and seem to play a role in cellular resistance to cisplatin [28, 29]. In the investigated subgroup of patients with decreased 8-oxo-Gua levels after the first day of treatment, a better prognosis (median OS = 60 months) was determined, compared to the patients with increased levels of this derivative at the same time point (median OS = 24 months) (Fig. 1a). An interesting interpretation of this effect that we propose is that the decrease in 8-oxoGua levels may reflect the elimination of tumor cells characterized by much higher proliferation rate as a result of cisplatin therapy. If cell death is the cause of the increased levels of the modified nucleoside/base in urine, the most pronounced increase should be expected in patients with better prognosis. However, in the study by Siomek et al. [19], on days when mass death of either normal or cancer cells was observed (usually 7–14 days after the infusion) in patients treated with cisplatin, the urinary excretion of 8-oxo-Gua and 8-oxo-dG decreased compared to the period immediately following the infusion. This may prove that cell death does not contribute to the presence of 8-oxo-Gua and 8-oxo-dG in human urine. The argument was previously proposed by Cooke et al. [13]. However, our studies demonstrated that better prognosis was correlated with a decrease in the levels of the modified base observed 24 h after drug infusion (Fig. 1a). No significant change in the background level of 8-oxo-dG in DNA was observed in either of the two patient subgroups (with increased and decreased urine levels of 8-oxo-Gua)
(Fig. 1c). Therefore, the decreased levels of the modified base excreted in urine, observed in the subgroup with longer overall survival, is probably a measure of a better response to chemotherapy (higher susceptibility to the chemotherapeutic) and also of a decreased efficiency of DNA repair mechanisms in tumor cells in this subgroup of patients [30]. Since 8-oxo-Gua is a product of DNA repair mechanisms, it is plausible that at least in some patients with impaired OGG1 activity, the concurrent decrease in OGG1 repair efficiency and chemotherapy resulted in the stable background level of 8-oxo-Gua in cellular DNA [31, 32]. Due to the apparent decrease in efficiency, DNA repair mechanisms were unable to cope with the additional 8-oxoGua induced by chemotherapy, which led to a greater occurrence of potentially mutagenic/carcinogenic alterations [33]. In this respect, a higher repair efficiency would negatively impact the susceptibility to the chemotherapeutic. It may be inferred that patients with a lower efficiency of the recognition and removal of oxidatively damaged DNA in tumor cells demonstrate a higher susceptibility to platinum derivative-based chemotherapy and have a longer overall survival. Another conclusion of the presented study was the extension of overall survival in the group of patients with an increase in the levels of 8-oxo-dG in urine after chemotherapy (Fig. 1b). In the study by Siomek et al. [19], as in our study, an increase in the urinary excretion of the oxidative stress markers 8-oxo-Gua and 8-oxo-dG was observed in the majority of cancer patients 24 h after drug infusion. However, on days when mass death of normal cells, manifested by a decrease in the morphological parameters of peripheral blood, was observed (usually 7–14 days after the infusion), the urinary excretion of 8-oxo-Gua and 8-oxo-dG decreased compared to the period immediately following the infusion. The authors of this paper did not conduct any evaluation of the results of the clinical treatment in each group of patients; therefore, it is not possible to draw conclusions regarding the prognosis in these patients with reference to the levels of oxidative stress markers. In the study by Erhola et al. [34], as opposed to our study, it was observed that in patients with small cell lung carcinoma and a partial or complete response to chemotherapy, the urine levels of 8-oxo-dG decreased, while in patients with progression of the disease, 8-oxo-dG levels increased during treatment. It should be noted that the levels of the modified nucleotide in urine date may be a marker of oxidative DNA damage and of general oxidative stress (both in tumor cells and in normal tissues) [35]. An interesting possibility could be that the increase in 8-oxo-dG may reflect the degree of
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tumor damage and the elimination of tumor cells induced by chemotherapy. This in turn may suggest the death of tumor cells as the potential cause of the increased 8-oxodG levels in urine [13]. There are individual differences with respect to the formation and removal of the modified bases after chemotherapy. Therefore, it is possible that the observed variability may partially explain the differences in the clinical response to therapy [31, 33, 36]. This variability may also reflect the individual differences in metabolism and DNA repair capacity and has, at least partially, genetic background [37, 38]. However, the measurement of exclusive urinary excretion of 8-oxo-dG may sometimes be misleading because it provides no information on the oxidative steady state (level of damage vs. repair efficiency) within cellular DNA. Therefore, in our study we analyzed a some of parameters describing oxidatively damaged DNA. In addition to urinary 8-oxo-dG, we also determined the levels of the modified base (8-oxo-Gua) in urine, as well as the background level of the modification in cellular DNA. The content of 8-oxo-dG in lymphocytic DNA directly indicates the level of oxidative damage in normal cells [39, 40]. Therefore, the content of 8-oxo-dG in DNA does not necessarily reflect the level of oxidative damage induced by chemotherapy in tumor cells. Moreover, it should be taken into consideration that the quantity and function of lymphocytes in patients undergoing systemic treatment are significantly far from normal. The modification of the base itself, from its oxidation, through damage recognition and initiation of the enzymatic mechanism of base/nucleoside excision, with or without cutting DNA strand or the separation of strand, and the reassembly of the DNA molecule are in correlation, and balance with other mechanisms of DNA repair and cell division block, acting when damage to the nucleic acid, has been detected. Therefore, further studies of the correlation of these processes with the susceptibility of tumors to chemotherapy should be conducted. To conclude, due to the importance of the identification of chemotherapy outcome prognostic factors, we attempted to check whether the parameters describing oxidative stress / DNA damage may be used as such prognostic markers. The results of our study suggest that patients with decreased 8-oxo-Gua levels and increased 8-oxo-dG levels in urine 24 h after the first dose should be considered as better responders to the administered chemotherapy, with a lower risk of death. The conclusion should permit the use of these parameters as markers for predicting the clinical outcome. Acknowledgments I would like to thank Professor Ryszard Olinski, Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, PL for the inspiration to perform
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this work and his making possible laboratory investigations. I would like to acknowledge the contributions of Rafal Rozalski and Daniel Gackowski to this study. Conflicts of interest The authors have no conflicts of interest that are directly relevant to the content of this article.
References 1. Dizdaroglu M. Oxidatively induced DNA damage: mechanisms, repair and disease. Cancer Lett. 2012;327:26–47. 2. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J. 1996;313:17–29. 3. Cho WC, Kwan CK, Yau S, So PP, Poon PC, Au JS. The role of inflammation in the pathogenesis of lung cancer. Expert Opin Ther Targets. 2011;15:1127–37. 4. Nediani C, Raimondi L, Borchi E, Cerbai E. Nitric oxide/reactive oxygen species generation and nitroso/redox imbalance in heart failure: from molecular mechanisms to therapeutic implications. Antioxid Redox Signal. 2011;14:289–331. 5. Cunningham RP. DNA repair: caretakers of the genome? Curr Biol. 1997;7:576–9. 6. Xu DD, Lam HM, Hoeven R, Xu CB, Leung AW, Cho WC. Photodynamic therapy induced cell death of hormone insensitive prostate cancer PC-3 cells with autophagic characteristics. Photodiagnosis Photodyn Ther. 2013;10:278–87. 7. Cooke MS, Olinski R, Evans MD. Does measurement of oxidative damage to DNA have clinical significance? Clin Chim Acta. 2006;365:30–49. 8. Noelker C, Hampel H, Dodel R. Blood-based protein biomarkers for diagnosis and classification of neurodegenerative diseases: current progress and clinical potential. Mol Diagn Ther. 2011;15:83–102. 9. Roszkowski K, Jozwicki W, Blaszczyk P, Mucha-Malecka A, Siomek A. Oxidative damage DNA: 8-oxoGua and 8-oxodG as molecular markers of cancer. Med Sci Monit. 2011;17:329–33. 10. Li N, Tan W, Li J, Li P, Lee S, Wang Y, et al. Glucose metabolism in breast cancer and its implication in cancer therapy. Int J Clin Med. 2011;2:110–28. 11. Cho WC. Nasopharyngeal carcinoma: molecular biomarker discovery and progress. Mol Cancer. 2007;6:1–9. 12. Sinhk SK, Szulik MW, Ganguly M, Khutsishvili I, Stone MP, Marky LA, et al. Characterization of DNA with an 8-oxoguanine modification. Nucleic Acids Res. 2011;39:6789–801. 13. Cooke MS, Evans MD, Dove R, Rozalski R, Gackowski D, Siomek A, Lunec J, Olinski R. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine. Mutat Res. 2005;574:58–66. 14. Evans MD, Saparbaev M, Cooke MS. DNA repair and the origins of urinary oxidized 20 -deoxyribonucleosides. Mutagenesis. 2010;25:433–42. 15. Havelka AM, Berndtsson M, Olofsson MH, Shoshan MC, Linder S. Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: Is acute apoptosis an ‘‘off-target’’ effect? Mini Rev Med Chem. 2007;7:1035–9. 16. Goncalves MS, Silveira AF, Teixeira AR, Hyppolito MA. Mechanisms of cisplatin ototoxicity: theoretical review. J Laryngol Otol. 2013;7:1–6. 17. Weijl NI, Hopman GD, Wipkink-Bakker A, Lentjes EG, Berger HM, Cleton FJ, et al. Cisplatin combination chemotherapy induces a fall in plasma antioxidants of cancer patients. Ann Oncol. 1998;9:1331–7.
Clin Exp Med 18. Roszkowski K, Olinski R. Urinary 8-oxoguanine as a predictor of survival in patients undergoing radiotherapy. Cancer Epidemiol Biomarkers Prev. 2012;21:629–34. 19. Siomek A, Tujakowski J, Gackowski D, Rozalski R, Foksinski M, Dziaman T, et al. Severe oxidatively damaged DNA after cisplatin treatment of cancer patients. Int J Cancer. 2006;119:2228–30. 20. Ravanat JL, Guicherd P, Tuce Z, Cadet J. Simultaneous determination of five oxidative DNA lesions in human urine. Chem Res Toxicol. 1999;12:802–8. 21. Siomek A, Gackowski D, Rozalski R, Dziaman T, Szpila A, Guz J, et al. Higher leukocyte 8-oxo-7,8-dihydro-2-deoxyguanosine and lower plasma ascorbate in aging humans? Antioxid Redox Signal. 2007;9:143–50. 22. Foksinski M, Rozalski R, Guz J, Ruszkowska B, Sztukowska P, Piwowarski M, et al. Urinary excretion of DNA repair products correlates with metabolic rates as well as with maximum life spans of different mammalian species. Free Radic Biol Med. 2004;37(9):1449–54. 23. Poulsen HE, Loft S, Prieme H, Vistisen K, Lykkesfeldt J, Nyyssonen K, et al. Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion. Free Rad Res. 1998;29(6):565–71. 24. Weijl NI, Elsendoorn TJ, Lentjes EG, Hopman GD, WipkinkBakker A, Zwinderman AH, et al. Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study. Eur J Cancer. 2004;40:1713–23. 25. Zamble DB, Lippard SJ. Cisplatin and DNA repair in cancer chemotherapy. Trends Biochem Sci. 1995;20:435–9. 26. Kyrtopoulos SA. Variability in DNA repair and individual susceptibility to genotoxins. Clin Chem. 1995;41:1848–53. 27. Strauss B, Hanawalt P, Swenberg J. Risk assessment in environmental carcinogenesis. An American Association for Cancer Research special conference in cancer research cosponsored by the Environmental Mutagen Society. Cancer Res. 1994;54: 5493–6. 28. Sherman-Baust CA, Becker KG, Wood Iii WH, Zhang Y, Morin PJ. Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin. J Ovarian Res. 2011;4(1):21. doi:10.1186/1757-2215-4-21. 29. Srivastava AN, Gupta A, Srivastava S, Natu SM, Mittal B, Negi MP, Prasad R. Cisplatin combination chemotherapy induces
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
oxidative stress in advance non small cell lung cancer patients. Asian Pac J Cancer Prev. 2010;11(2):465–71. Dziaman T, Banaszkiewicz Z, Roszkowski K, Gackowski D, Wisniewska E, Rozalski R, et al. 8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients. Int J Cancer. 2013;134:376–83. Loft S, Danielsen P, Lohr M, Jantzen K, Hemmingsen JG, Roursgaard M, et al. Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA. Arch Biochem Biophys. 2012;518:142–50. Kuznetsov NA, Koval VV, Fedorova OS. Mechanism of recognition and repair of damaged DNA by human 8-oxoguanine DNA glycosylase hOGG1. Biochemistry (Mosc). 2011;76:118–30. Afzal S, Jensen SA, Sorensen JB, Henriksen T, Weimann A, Poulsen HE. Oxidative damage to guanine nucleosides following combination chemotherapy with 5-fluorouracil and oxaliplatin. Cancer Chemother Pharmacol. 2012;69:301–7. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, et al. Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy-20 -deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment. FEBS Lett. 1997;409:287–91. Poulsen HE, Nadal LL, Broedbaek K, Nielsen PE, Weimann A. Detection and interpretation of 8-oxodG and 8-oxoGua in urine, plasma and cerebrospinal fluid. Biochim Biophys Acta. 2014;1840:801–8. Gupta A, Srivastava S, Prasad R, Natu SM, Mittal B, Negi MP, et al. Oxidative stress in non-small cell lung cancer patients after chemotherapy: association with treatment response. Respirology. 2010;15:349–56. Li X, Xu H, Xu C, Lin M, Song X, Yi F, et al. The yin-yang of DNA damage response: roles in tumorigenesis and cellular senescence. Int J Mol Sci. 2013;14:2431–48. Salehan MR, Morse HR. DNA damage repair and tolerance: a role in chemotherapeutic drug resistance. Br J Biomed Sci. 2013;70:31–40. Collins AR, Cadet J, Moller L, Poulsen HE, Vina J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch Biochem Biophys. 2004;423:57–65. Siomek A, Gackowski D, Rozalski R, Dziaman T, Szpila A, Guz J, et al. Higher leukocyte 8-oxo-7,8-dihydro-20 -deoxyguanosine and lower plasma ascorbate in aging humans? Antioxid Redox Signal. 2007;9:143–50.
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ORIGINAL ARTICLE
Potential survival markers in cancer patients undergoing chemotherapy Krzysztof Roszkowski • Jan Filipiak • Magdalena Wisniewska • Anna Mucha-Malecka Pawel Basta
•
Received: 1 July 2014 / Accepted: 15 September 2014 Ó Springer-Verlag Italia 2014
Abstract Due to the importance of the identification of chemotherapy outcome prognostic factors, we attempted to establish the potential of oxidative stress/DNA damage parameters such as prognostic markers. The aim of the study was to determine whether platinum derivative-based chemotherapy in cancer patients (n = 66) is responsible for systemic oxidatively damaged DNA and whether damage biomarkers, such as 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxo-dG) and the modified base 8-oxo-7,8-dihydroguanine (8-oxo-Gua), in urine and DNA may be used as a prognostic factor for the outcome of chemotherapy. All the aforementioned modifications were analyzed using techniques involving high-performance liquid chromatography/ electrochemical detection (HPLC/EC) or HPLC/gas chromatography–mass spectrometry (GC–MS). Among all the analyzed parameters, the significantly decreased levels of 8-oxo-Gua in urine collected from a subgroup of patients
K. Roszkowski Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Torun, Poland K. Roszkowski (&) Department of Radiotherapy, The F. Lukaszczyk Oncology Center, Bydgoszcz, Poland e-mail: [email protected] J. Filipiak M. Wisniewska Department of Chemotherapy, The F. Lukaszczyk Oncology Center, Bydgoszcz, Poland A. Mucha-Malecka Department of Radiotherapy, Center of Oncology – Maria Skłodowska-Curie Memorial Institute, Krakow, Poland P. Basta I Department of Surgery, Medical College, Jagiellonian University, Krakow, Poland
24 h after the first infusion of the drug, as compared with the baseline levels, correlated with a significantly longer overall survival (OS) (60 months after therapy) than in the subgroup without any decrease of this parameter after therapy (median OS = 24 months, p = 0.007). Moreover, a significantly longer OS was also observed in a group with increased urine levels of 8-oxo-dG after chemotherapy (38.6 vs. 20.5 months, p = 0.03). The results of our study suggest that patients with decreased 8-oxo-Gua levels and increased 8-oxo-dG levels in urine 24 h after the first dose should be considered as better responders to the administered chemotherapy, with a lower risk of death. The conclusion may permit the use of these parameters as markers for predicting the clinical outcome of platinum derivativebased chemotherapy. Keywords Molecular biomarkers Oxidatively damaged DNA Survival markers 8-Oxo-Gua 8-Oxo-dG Chemotherapy Cancer patients
Introduction In the course of some essential metabolic processes occurring in cells, reactive oxygen species (ROS) with potentially genotoxic and mutagenic properties are formed [1]. ROS, such as superoxide, hydrogen peroxide and hydroxyl radical are by-products of cellular respiration, but they may also be generated during inflammatory processes [2, 3]. Similarly, reactive nitrogen species (RNS) generate toxic products in their reactions with cells and free molecules [4]. Normal cells are equipped with enzymatic and nonenzymatic mechanisms limiting the production of ROS; however, abnormally functioning cells are often subjected
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to oxidative stress in which the balance between oxidants and antioxidants becomes disrupted, which results in an increased amount of cellular damage. The consequences of this process include, e.g., peroxidation of membrane lipids or oxidative modifications of proteins and DNA [5, 6]. Eukaryotic organisms are constantly at risk of the effects of ROS that play an important role in the pathogenesis of some diseases in human: tumors, cardiovascular diseases, neurodegenerative diseases and problems related with aging [7, 8]. Characteristics of tumors are the persistent high levels of ROS and permanent oxidative stress [9]. These processes are aided by, e.g., abnormal vascular system of the tumor, infiltration by macrophages and glycolysis [10, 11]. The nitrogenous bases forming DNA are particularly susceptible to the oxidation by ROS. The low oxidoreduction potential of guanine causes this base to be susceptible to such modifications, resulting in the formation of many guanine oxidation products. 8-Oxo-7,8-dihydroguanine (8-oxo-Gua) is often employed as a biomarker of oxidative stress [12]. It is generally accepted that 8-oxo-Gua excised from DNA molecules in the process of cellular DNA repair is excreted with urine in an unmetabolized form [13]. However, the repair processes which contribute to the presence of 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) in urine are still an open question. In general, the evidence suggests that the action of the Nudix hydrolase family of enzymes, sanitizing the deoxyribonucleotide pool via the degradation of 8-oxo-7,8-dihydro-20 -deoxyguanosine-50 -triphosphate and 8-oxo-7,8-dihydro-20 -deoxyguanosine-50 -diphosphate, and yielding mononucleotide products which may then be dephosphorylated to 8-oxodG, probably represents the most logical and primary source of 8-oxo-dG in urine, with a lesser role of other possible DNA repair processes [14]. However, some of the most recent studies report that NER (nucleotide excision repair) and NIR (nucleotide incision repair), transcriptioncoupled repair, mismatch repair and various exonuclease activities may play a greater role in the formation of 8-oxodG excreted in urine than previously presumed [14]. Antitumor chemotherapy acts via various mechanisms characteristic of a given class of medicines, leading to cell death. Some chemotherapeutics, apart from their principal mode of action, affect cells also through the increase of oxidative stress [15–17]. Oxidative stress may possibly emphasize the cytotoxic effect of chemotherapy on tumor cells. Therefore, in our studies we analyzed several parameters describing oxidatively damaged DNA. Apart from 8-oxo-dG excreted with urine, we determined the urine levels of the modified DNA base (8-oxo-Gua), as well as the background level of the modification in cellular DNA.
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Our previous studies have shown that oxidative stress markers such as 8-oxo-dG and 8-oxo-Gua may be predictive factors of individual sensitivity to radiation therapy [18]. Therefore, we decided to check whether the same markers may somehow be responsible for chemotherapy success. The aim of the study was to establish: a.
b.
whether platinum derivative-based chemotherapy (cisplatin and carboplatin) is responsible for systemic oxidatively damaged DNA in cancer patients; whether damage biomarkers excreted in urine, such as 8-oxo-dG or 8-oxo-Gua, as well as the level of oxidatively damaged DNA in leukocytes (all of these parameters reflect systemic oxidatively damaged DNA) may be employed for establishing a prognosis of chemotherapy outcome.
Materials and methods The analysis of daily excretion of 8-oxo-Gua and 8-oxo-dG in urine was conducted in a group of 66 patients with malignant tumors (clinical stage III and IV), undergoing treatment between June 2002 and July 2005. The patients suffered from various malignancies: ovarian cancer (n = 38), testicular cancer (n = 7), lung cancer (n = 9), head and neck cancer (n = 4) and others: stomach cancer, bile duct cancer, bladder cancer (n = 8). All patients received cisplatin-based chemotherapy at appropriate doses. In all patients treated with cisplatin participating in this study, normal values of renal parameters were determined before treatment (it was a necessary condition for chemotherapy). The adopted chemotherapy treatment schemes included: cisplatin with paclitaxel (22 patients, 33%), cisplatin with cyclophosphamide (14 patients, 21%), cisplatin with bleomycin (9 patients, 14%), cisplatin with 5-fluorouracil (6 patients, 9%), cisplatin with etoposide (6 patients, 6%), cisplatin with doxorubicin (9 patients, 14%). The clinical characteristics of the patients are presented in Table 1. The concentrations of basic markers of oxidatively damaged DNA were determined before the administration of systemic therapy (sample I) and 24 h after administering the first dose (sample II). Our earlier studies revealed that in patients undergoing oncological therapy, the levels of biomarkers excreted with urine may either increase or decrease [18, 19]. Therefore, we distinguished subgroups of patients in whom an increase (subgroup A) or a decrease (subgroup B) in the levels of the three studied markers were detected after the first 24 h of therapy. Analysis of overall survival was conducted for both subgroups. The assessment of diurnal excretion of 8-oxo-Gua and 8-oxo-
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dG in urine and the determination of the 8-oxo-dG content in the DNA of peripheral blood leukocytes were performed using previously described methods [20, 21]. Urinary excretion expressed in nmol/kg/24 h can provide more information, e.g., allows the quantification of damage repaired per day per cell [22]. Some researchers collect a single sample of urine and determine the 8-oxodG and 8-oxo-Gua levels with a correction for creatinine concentration (i.e., in the same urine sample, the quantities of oxidatively modified bases and nucleotide levels are determined along with the levels of creatinine) [23]. Urine samples collected in the morning may be used to determine the excretion rate as long as the creatinine excretion remains unchanged. Such a method of data interpretation can be problematic in patients with advanced cancer. The levels of creatinine excreted in urine in these patients may Table 1 Patient and treatment characteristics Patient characteristic
No. of Patients
Total
66
%
Age (years) Median Range
56 34–71
Sex Male
25
38
Female
41
62
vary according to the changes in body weight. In this case, a reasonable and more convenient solution is the determination of oxidatively damaged DNA parameters in urine from diurnal collection before treatment and the day immediately after the administration of the above-mentioned first therapeutic dose (of the medicinal agent). This creates methodological difficulties and forces researchers to follow a certain study regime, but also allows them to obtain more reliable results. Such an approach was adopted in this study. For the above reasons, parameters before treatment and after the first day of treatment were analyzed in the investigated group of patients. The study was approved by the medical ethics committee of The Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland, no. 274/2002; 409/2002 (in accordance with Good Clinical Practice, 1998), and all patients granted their informed consent. All results were expressed as means using the SPSS Statistics software, version 10.0. Statistical analyses were carried out using Student’s t test (for variables with normal distribution levels of oxidatively damaged DNA before and after chemotherapy). The association between overall survival and the urinary excretion of 8-oxo-dG and 8-oxoGua, as well as the level of oxidatively damaged DNA in leukocytes, were estimated using the Kaplan–Meier method and assessed using the log-rank test. The results were considered as statistically significant at p \ 0.05.
ECOG performance status 0–1
52
79
C2
14
21
Ovarian cancer (n = 38)
III
57
Lung cancer (n = 9)
IIIb
14
Stage %
Head & neck (n = 4)
IV
6
Testicular cancer (n = 7)
IIIb
11
Stomach, bile duct, bladder cancers (n = 8)
IV
12
22 14
33 21 14
Chemotherapy Cisplatin ? paclitaxel Cisplatin ? cyclophosphamide Cisplatin ? bleomycin
9
Cisplatin ? 5-fluorouracil
6
9
Cisplatin ? etoposide
6
9
Cisplatin ? doxorubicin
9
14
ECOG Eastern Cooperative Oncology Group
Table 2 The level of 8-oxoGua, 8-oxo-dG in the urine and 8-oxo-dG in the leukocytes’ DNA before infusion of the drug (sample I), 24 h after (sample II)
All patients
Results For the total patient population, the median value of 8-oxoGua concentration in urine before treatment (sample I) was 9.86 nmol/24 h, and interquartile range was 5.47–6.09. After the first infusion of the drug (sample II), the measured concentration increased to 24.23 nmol/24 h; interquartile range 14.38–36.30. This difference was statistically significant (p = 0.001; Table 2). There were no significant differences between samples I and II with respect to the urinary excretion of 8-oxo-dG (the values were 15.17 nmol/24 h, interquartile range 8.92–7.68 and 16.74 nmol/24 h, interquartile range: 7.22–8.27, respectively), while the median 8-oxo-dG content in DNA isolated from leukocytes reached 4.71
Median (interquartile range) Sample I
p
Sample II
Urinary 8-oxo-Gua (nmol/24 h)
9.86 (5.47– 6.09)
24.23 (14.38–36.30)
p = 0.001
Urinary 8-oxo-dG (nmol/24 h)
15.17 (8.92– 7.68)
16.74 (7.22– 8.27)
p = 0.47
4.71 (3.47–5.83)
4.77 (3.76–5.93)
p = 0.26
6
Leukocytes’ 8-oxo-dG/10 dG
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Fig. 2 Kaplan–Meier curves of subgroup of 9 patients, in whom both a decrease in 8-oxo-Gua and an increase in 8-oxo-dG in urine and all other patients
Fig. 1 Kaplan–Meier curves of a 8-oxo-Gua in urine; b 8-oxo-dG in urine; c 8-oxo-dG in the leukocytes’ 24 h after infusion chemotherapy
(3.47–5.83) and 4.77 (3.76–5.93) of 8-oxo-dG per 106 dG, respectively (Table 2). A comparative analysis of overall survival with respect to possible differences between sample I and II was conducted for all the above parameters. A significant increase in overall survival was found in the subgroup of 14 patients with a decrease (sample II) in
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the levels of urinary 8-oxo-Gua (median: 60 months), as compared with the subgroup that responded with an increase (sample I) in the urine levels of this derivative 24 h after infusion, median: 24 months (p = 0.007; logrank test) (Fig. 1a). A statistically significant extension of overall survival was also observed in the subgroup of patients with an increase in urine 8-oxo-dG levels after chemotherapy (Fig. 1b). No significant differences in overall survival were noticed with respect to the 8-oxo-dG content in cellular DNA (data not shown). The selection of patients followed the criteria of the group of patients; these included age, body weight and smoking status. There were no differences among the patients with various stages of the disease development concerning all of the analyzed modifications (data not shown). In a small subgroup of 9 patients, in whom both a decrease in 8-oxo-Gua and an increase in 8-oxo-dG in urine occurred after the first day of drug infusion, exceptionally long overall survival, even up to 78 months, was observed (Fig. 2). Similarly, no significant differences among the studied time points were found with respect to creatinine clearance and creatinine concentration (data not shown). A similar observation was reported by other groups [19, 24].
Discussion The analysis of 8-oxo-Gua and 8-oxo-dG levels patients’ urine, taken before drug administration different times after the start of chemotherapy, provide data indicating either the effectiveness
in the and at should of the
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mechanisms of oxidatively damaged DNA repair or the intensity of systemic oxidative stress. By comparing the level of these modifications with parameters used for the assessment of therapy outcome, such as overall survival, we should be able to determine the potential causal relationship between the prognosis for the patients and the oxidatively damaged DNA induced by chemotherapy, as well as the effectiveness of repair mechanisms. Establishing such a correlation might be one of the parameters considered when deciding on another cycle of chemotherapy or treatment termination in clinically dubious cases. As has been previously revealed, cisplatin therapy causes serious oxidative stress [19, 25]. Therefore, the observed increased urine levels of 8-oxo-Gua and 8-oxodG in most cancer patients 24 h after drug administration may be a result of an increased generation of reactive oxygen species (Table 2). This variability may reflect individual differences in metabolism and DNA repair capacity, as well as, at least partially, in background genetic differences [26, 27]. Other studies also indicate that oxidative stress and ROS generation may be at least partially responsible for the cytotoxicity induced by cisplatin and seem to play a role in cellular resistance to cisplatin [28, 29]. In the investigated subgroup of patients with decreased 8-oxo-Gua levels after the first day of treatment, a better prognosis (median OS = 60 months) was determined, compared to the patients with increased levels of this derivative at the same time point (median OS = 24 months) (Fig. 1a). An interesting interpretation of this effect that we propose is that the decrease in 8-oxoGua levels may reflect the elimination of tumor cells characterized by much higher proliferation rate as a result of cisplatin therapy. If cell death is the cause of the increased levels of the modified nucleoside/base in urine, the most pronounced increase should be expected in patients with better prognosis. However, in the study by Siomek et al. [19], on days when mass death of either normal or cancer cells was observed (usually 7–14 days after the infusion) in patients treated with cisplatin, the urinary excretion of 8-oxo-Gua and 8-oxo-dG decreased compared to the period immediately following the infusion. This may prove that cell death does not contribute to the presence of 8-oxo-Gua and 8-oxo-dG in human urine. The argument was previously proposed by Cooke et al. [13]. However, our studies demonstrated that better prognosis was correlated with a decrease in the levels of the modified base observed 24 h after drug infusion (Fig. 1a). No significant change in the background level of 8-oxo-dG in DNA was observed in either of the two patient subgroups (with increased and decreased urine levels of 8-oxo-Gua)
(Fig. 1c). Therefore, the decreased levels of the modified base excreted in urine, observed in the subgroup with longer overall survival, is probably a measure of a better response to chemotherapy (higher susceptibility to the chemotherapeutic) and also of a decreased efficiency of DNA repair mechanisms in tumor cells in this subgroup of patients [30]. Since 8-oxo-Gua is a product of DNA repair mechanisms, it is plausible that at least in some patients with impaired OGG1 activity, the concurrent decrease in OGG1 repair efficiency and chemotherapy resulted in the stable background level of 8-oxo-Gua in cellular DNA [31, 32]. Due to the apparent decrease in efficiency, DNA repair mechanisms were unable to cope with the additional 8-oxoGua induced by chemotherapy, which led to a greater occurrence of potentially mutagenic/carcinogenic alterations [33]. In this respect, a higher repair efficiency would negatively impact the susceptibility to the chemotherapeutic. It may be inferred that patients with a lower efficiency of the recognition and removal of oxidatively damaged DNA in tumor cells demonstrate a higher susceptibility to platinum derivative-based chemotherapy and have a longer overall survival. Another conclusion of the presented study was the extension of overall survival in the group of patients with an increase in the levels of 8-oxo-dG in urine after chemotherapy (Fig. 1b). In the study by Siomek et al. [19], as in our study, an increase in the urinary excretion of the oxidative stress markers 8-oxo-Gua and 8-oxo-dG was observed in the majority of cancer patients 24 h after drug infusion. However, on days when mass death of normal cells, manifested by a decrease in the morphological parameters of peripheral blood, was observed (usually 7–14 days after the infusion), the urinary excretion of 8-oxo-Gua and 8-oxo-dG decreased compared to the period immediately following the infusion. The authors of this paper did not conduct any evaluation of the results of the clinical treatment in each group of patients; therefore, it is not possible to draw conclusions regarding the prognosis in these patients with reference to the levels of oxidative stress markers. In the study by Erhola et al. [34], as opposed to our study, it was observed that in patients with small cell lung carcinoma and a partial or complete response to chemotherapy, the urine levels of 8-oxo-dG decreased, while in patients with progression of the disease, 8-oxo-dG levels increased during treatment. It should be noted that the levels of the modified nucleotide in urine date may be a marker of oxidative DNA damage and of general oxidative stress (both in tumor cells and in normal tissues) [35]. An interesting possibility could be that the increase in 8-oxo-dG may reflect the degree of
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tumor damage and the elimination of tumor cells induced by chemotherapy. This in turn may suggest the death of tumor cells as the potential cause of the increased 8-oxodG levels in urine [13]. There are individual differences with respect to the formation and removal of the modified bases after chemotherapy. Therefore, it is possible that the observed variability may partially explain the differences in the clinical response to therapy [31, 33, 36]. This variability may also reflect the individual differences in metabolism and DNA repair capacity and has, at least partially, genetic background [37, 38]. However, the measurement of exclusive urinary excretion of 8-oxo-dG may sometimes be misleading because it provides no information on the oxidative steady state (level of damage vs. repair efficiency) within cellular DNA. Therefore, in our study we analyzed a some of parameters describing oxidatively damaged DNA. In addition to urinary 8-oxo-dG, we also determined the levels of the modified base (8-oxo-Gua) in urine, as well as the background level of the modification in cellular DNA. The content of 8-oxo-dG in lymphocytic DNA directly indicates the level of oxidative damage in normal cells [39, 40]. Therefore, the content of 8-oxo-dG in DNA does not necessarily reflect the level of oxidative damage induced by chemotherapy in tumor cells. Moreover, it should be taken into consideration that the quantity and function of lymphocytes in patients undergoing systemic treatment are significantly far from normal. The modification of the base itself, from its oxidation, through damage recognition and initiation of the enzymatic mechanism of base/nucleoside excision, with or without cutting DNA strand or the separation of strand, and the reassembly of the DNA molecule are in correlation, and balance with other mechanisms of DNA repair and cell division block, acting when damage to the nucleic acid, has been detected. Therefore, further studies of the correlation of these processes with the susceptibility of tumors to chemotherapy should be conducted. To conclude, due to the importance of the identification of chemotherapy outcome prognostic factors, we attempted to check whether the parameters describing oxidative stress / DNA damage may be used as such prognostic markers. The results of our study suggest that patients with decreased 8-oxo-Gua levels and increased 8-oxo-dG levels in urine 24 h after the first dose should be considered as better responders to the administered chemotherapy, with a lower risk of death. The conclusion should permit the use of these parameters as markers for predicting the clinical outcome. Acknowledgments I would like to thank Professor Ryszard Olinski, Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, PL for the inspiration to perform
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this work and his making possible laboratory investigations. I would like to acknowledge the contributions of Rafal Rozalski and Daniel Gackowski to this study. Conflicts of interest The authors have no conflicts of interest that are directly relevant to the content of this article.
References 1. Dizdaroglu M. Oxidatively induced DNA damage: mechanisms, repair and disease. Cancer Lett. 2012;327:26–47. 2. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J. 1996;313:17–29. 3. Cho WC, Kwan CK, Yau S, So PP, Poon PC, Au JS. The role of inflammation in the pathogenesis of lung cancer. Expert Opin Ther Targets. 2011;15:1127–37. 4. Nediani C, Raimondi L, Borchi E, Cerbai E. Nitric oxide/reactive oxygen species generation and nitroso/redox imbalance in heart failure: from molecular mechanisms to therapeutic implications. Antioxid Redox Signal. 2011;14:289–331. 5. Cunningham RP. DNA repair: caretakers of the genome? Curr Biol. 1997;7:576–9. 6. Xu DD, Lam HM, Hoeven R, Xu CB, Leung AW, Cho WC. Photodynamic therapy induced cell death of hormone insensitive prostate cancer PC-3 cells with autophagic characteristics. Photodiagnosis Photodyn Ther. 2013;10:278–87. 7. Cooke MS, Olinski R, Evans MD. Does measurement of oxidative damage to DNA have clinical significance? Clin Chim Acta. 2006;365:30–49. 8. Noelker C, Hampel H, Dodel R. Blood-based protein biomarkers for diagnosis and classification of neurodegenerative diseases: current progress and clinical potential. Mol Diagn Ther. 2011;15:83–102. 9. Roszkowski K, Jozwicki W, Blaszczyk P, Mucha-Malecka A, Siomek A. Oxidative damage DNA: 8-oxoGua and 8-oxodG as molecular markers of cancer. Med Sci Monit. 2011;17:329–33. 10. Li N, Tan W, Li J, Li P, Lee S, Wang Y, et al. Glucose metabolism in breast cancer and its implication in cancer therapy. Int J Clin Med. 2011;2:110–28. 11. Cho WC. Nasopharyngeal carcinoma: molecular biomarker discovery and progress. Mol Cancer. 2007;6:1–9. 12. Sinhk SK, Szulik MW, Ganguly M, Khutsishvili I, Stone MP, Marky LA, et al. Characterization of DNA with an 8-oxoguanine modification. Nucleic Acids Res. 2011;39:6789–801. 13. Cooke MS, Evans MD, Dove R, Rozalski R, Gackowski D, Siomek A, Lunec J, Olinski R. DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine. Mutat Res. 2005;574:58–66. 14. Evans MD, Saparbaev M, Cooke MS. DNA repair and the origins of urinary oxidized 20 -deoxyribonucleosides. Mutagenesis. 2010;25:433–42. 15. Havelka AM, Berndtsson M, Olofsson MH, Shoshan MC, Linder S. Mechanisms of action of DNA-damaging anticancer drugs in treatment of carcinomas: Is acute apoptosis an ‘‘off-target’’ effect? Mini Rev Med Chem. 2007;7:1035–9. 16. Goncalves MS, Silveira AF, Teixeira AR, Hyppolito MA. Mechanisms of cisplatin ototoxicity: theoretical review. J Laryngol Otol. 2013;7:1–6. 17. Weijl NI, Hopman GD, Wipkink-Bakker A, Lentjes EG, Berger HM, Cleton FJ, et al. Cisplatin combination chemotherapy induces a fall in plasma antioxidants of cancer patients. Ann Oncol. 1998;9:1331–7.
Clin Exp Med 18. Roszkowski K, Olinski R. Urinary 8-oxoguanine as a predictor of survival in patients undergoing radiotherapy. Cancer Epidemiol Biomarkers Prev. 2012;21:629–34. 19. Siomek A, Tujakowski J, Gackowski D, Rozalski R, Foksinski M, Dziaman T, et al. Severe oxidatively damaged DNA after cisplatin treatment of cancer patients. Int J Cancer. 2006;119:2228–30. 20. Ravanat JL, Guicherd P, Tuce Z, Cadet J. Simultaneous determination of five oxidative DNA lesions in human urine. Chem Res Toxicol. 1999;12:802–8. 21. Siomek A, Gackowski D, Rozalski R, Dziaman T, Szpila A, Guz J, et al. Higher leukocyte 8-oxo-7,8-dihydro-2-deoxyguanosine and lower plasma ascorbate in aging humans? Antioxid Redox Signal. 2007;9:143–50. 22. Foksinski M, Rozalski R, Guz J, Ruszkowska B, Sztukowska P, Piwowarski M, et al. Urinary excretion of DNA repair products correlates with metabolic rates as well as with maximum life spans of different mammalian species. Free Radic Biol Med. 2004;37(9):1449–54. 23. Poulsen HE, Loft S, Prieme H, Vistisen K, Lykkesfeldt J, Nyyssonen K, et al. Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion. Free Rad Res. 1998;29(6):565–71. 24. Weijl NI, Elsendoorn TJ, Lentjes EG, Hopman GD, WipkinkBakker A, Zwinderman AH, et al. Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study. Eur J Cancer. 2004;40:1713–23. 25. Zamble DB, Lippard SJ. Cisplatin and DNA repair in cancer chemotherapy. Trends Biochem Sci. 1995;20:435–9. 26. Kyrtopoulos SA. Variability in DNA repair and individual susceptibility to genotoxins. Clin Chem. 1995;41:1848–53. 27. Strauss B, Hanawalt P, Swenberg J. Risk assessment in environmental carcinogenesis. An American Association for Cancer Research special conference in cancer research cosponsored by the Environmental Mutagen Society. Cancer Res. 1994;54: 5493–6. 28. Sherman-Baust CA, Becker KG, Wood Iii WH, Zhang Y, Morin PJ. Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin. J Ovarian Res. 2011;4(1):21. doi:10.1186/1757-2215-4-21. 29. Srivastava AN, Gupta A, Srivastava S, Natu SM, Mittal B, Negi MP, Prasad R. Cisplatin combination chemotherapy induces
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
oxidative stress in advance non small cell lung cancer patients. Asian Pac J Cancer Prev. 2010;11(2):465–71. Dziaman T, Banaszkiewicz Z, Roszkowski K, Gackowski D, Wisniewska E, Rozalski R, et al. 8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients. Int J Cancer. 2013;134:376–83. Loft S, Danielsen P, Lohr M, Jantzen K, Hemmingsen JG, Roursgaard M, et al. Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA. Arch Biochem Biophys. 2012;518:142–50. Kuznetsov NA, Koval VV, Fedorova OS. Mechanism of recognition and repair of damaged DNA by human 8-oxoguanine DNA glycosylase hOGG1. Biochemistry (Mosc). 2011;76:118–30. Afzal S, Jensen SA, Sorensen JB, Henriksen T, Weimann A, Poulsen HE. Oxidative damage to guanine nucleosides following combination chemotherapy with 5-fluorouracil and oxaliplatin. Cancer Chemother Pharmacol. 2012;69:301–7. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, et al. Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy-20 -deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment. FEBS Lett. 1997;409:287–91. Poulsen HE, Nadal LL, Broedbaek K, Nielsen PE, Weimann A. Detection and interpretation of 8-oxodG and 8-oxoGua in urine, plasma and cerebrospinal fluid. Biochim Biophys Acta. 2014;1840:801–8. Gupta A, Srivastava S, Prasad R, Natu SM, Mittal B, Negi MP, et al. Oxidative stress in non-small cell lung cancer patients after chemotherapy: association with treatment response. Respirology. 2010;15:349–56. Li X, Xu H, Xu C, Lin M, Song X, Yi F, et al. The yin-yang of DNA damage response: roles in tumorigenesis and cellular senescence. Int J Mol Sci. 2013;14:2431–48. Salehan MR, Morse HR. DNA damage repair and tolerance: a role in chemotherapeutic drug resistance. Br J Biomed Sci. 2013;70:31–40. Collins AR, Cadet J, Moller L, Poulsen HE, Vina J. Are we sure we know how to measure 8-oxo-7,8-dihydroguanine in DNA from human cells? Arch Biochem Biophys. 2004;423:57–65. Siomek A, Gackowski D, Rozalski R, Dziaman T, Szpila A, Guz J, et al. Higher leukocyte 8-oxo-7,8-dihydro-20 -deoxyguanosine and lower plasma ascorbate in aging humans? Antioxid Redox Signal. 2007;9:143–50.
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