Prognostic value of xanthine oxidoreductase expression in patients with non-small cell lung cancer

Lung Cancer 71 (2011) 186–190

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Prognostic value of xanthine oxidoreductase expression in patients with non-small cell lung cancer Anthony W. Kim a,∗ , Marta Batus c , Ronald Myint c , Mary J. Fidler c , Sanjib Basu c , Philip Bonomi c , L. Penfield Faber b , Sean C. Wightman b , William H. Warren b , Maria McIntire d , Leonidas D. Arvanitis d , Paolo Gattuso d , Xiulong Xu e , Michael J. Liptay b a

Section of Thoracic Surgery, Yale University, United States Division of Thoracic Surgery, Rush University Medical Center, Chicago, IL, United States c Section of Medical Oncology, Rush University Medical Center, Chicago, IL, United States d Department of Pathology, Rush University Medical Center, Chicago, IL, United States e Department of General Surgery, Rush University Medical Center, Chicago, IL, United States b

a r t i c l e

i n f o

Article history: Received 7 December 2009 Received in revised form 18 April 2010 Accepted 4 May 2010 Keywords: Non-small cell lung cancer Xanthine oxidoreductase Surgery

a b s t r a c t Background: Xanthine oxidoreductase (XOR) is a rate-limiting enzyme in the purine metabolism pathway. Lack of XOR expression is associated with unfavorable clinical outcomes. The objective of this study was to correlate XOR expression with prognosis in surgically resected non-small cell lung cancer (NSCLC). Methods: Immunohistochemical staining was performed on deparaffinized specimens from 82 patients with stage I–IV NSCLC using a polyclonal anti-XOR rabbit antibody. Cytoplasmic XOR staining was scored on frequency and intensity scales from 0 to 4 with low expression defined as 0–1 and high expression defined as ≥2–4. XOR immunostaining was correlated with clinical characteristics and outcomes and analyzed using Kaplan–Meier and Cox proportional hazard methods. Results: Positive XOR expression was observed in 53/82 cases (65%). Patients with high XOR frequency had a longer median survival of 3053 days (95% CI: 2190–3916) vs. 592 days (95% CI: 492–692 days) for patients with low XOR frequency, p = 0.0089, HR 0.47. Neither XOR intensity nor the overall score of XOR frequency multiplied by XOR intensity demonstrated any significant association with survival. Surgical resection was performed on 61 patients of which 34 (56%) received adjuvant chemotherapy. Patients who received adjuvant chemotherapy with low XOR expression, 15/34 (44%) had a shortened median survival compared with patients who received adjuvant chemotherapy with high XOR expression (543 days vs. 2023 days, respectively, p = 0.007 and HR = 0.33). Conclusion: Low XOR expression was associated with shortened survival and also conferred a worse prognosis for patients with NSCLC who received adjuvant chemotherapy. Further studies of the XOR pathway are warranted to validate and mechanistically explain these outcomes. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer deaths worldwide [1]. In the United States there are projected to be approximately 219,400 new cases of lung cancer with an estimated 159,000 deaths. Approximately 80% of cases of lung cancer are of the non-

∗ Corresponding author at: 330 Cedar St., BB 205, Section of Thoracic Surgery, Yale University School of Medicine, New Haven, CT 06520, United States. Tel.: +1 203 785 4931; fax: +1 203 737 2163. E-mail address: [email protected] (A.W. Kim). 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.05.006

small cell lung cancer (NSCLC) type. Adjuvant chemotherapy has become the standard of care for stage II and stage III surgically resected NSCLC and is often given for stage I tumors greater than 4 cm [2]. Cisplatin-based doublet chemotherapy is the most studied regimen for adjuvant chemotherapy, but only results in a modest benefit in the reduction of death with absolute risk reductions ranging from 5 to 13% [2,3]. Immunohistochemical markers such as ERCC1 and the human MutS homolog 2 (MSH2) protein have been correlated with cisplatin sensitivity and prognosis [4,5]. Despite this new knowledge, the mechanisms behind chemoresistance, and how this information can be utilized to adjust therapy have yet to

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Fig. 1. Immunohistochemical staining showing (a) negative XOR expression in normal lung tissue, (b) strongly XOR-positive adenocarcinoma, (c) weakly XOR-positive adenocarcinoma, and (d) XOR-negative adenocarcinoma.

be fully elucidated. One marker, xanthine oxidoreductase (XOR), is another enzyme and potential marker that may be of interest in further understanding chemoresistance. XOR is a rate-limiting enzyme in the purine metabolism pathway [6]. XOR plays an important role in the degradation of DNA, RNA and high-energy phosphates [7–9]. Lack of XOR expression is associated with unfavorable clinical outcomes as it is thought to be an indicator of tumor aggressiveness in other types of malignancies [6,10–13]. The objective of this study was to determine if XOR was differentially expressed in NSCLC, and whether expression of XOR correlated with differences in survival. 2. Methods

citrate buffer (pH 6.0) in a microwave for 3 min. After blocking with normal goat serum in 5% phosphate buffered saline (PBS), sections were stained with anti-XOR rabbit antiserum (Rockland, Gilbertsville, PA) using a 1:50 dilution. This was then followed by a goat anti-rabbit antibody–biotin conjugate (diluted at 1:100 in PBS containing 5% normal goat serum). Normal rabbit IgG was included as a negative control. The specificity of anti-XOR antibody was confirmed by Western blot analysis of a rat liver lysate that gives rise to an expected molecular mass. HRP–avidin conjugate (diluted 1:100 in PBS with 5% normal goat serum) was then added. Color development was carried out by using diaminobenzidine (DAB). The sections were counterstained with Mayer’s hematoxylin followed by washing and dehydration. The sections were mounted and examined under a light microscope.

2.1. Tissue specimens and associated clinical information Paraffin-embedded tissue specimens, collected from 1998 to 2004, from 82 patients with known NSCLC were analyzed from an archived thoracic oncology tissue repository at Rush University Medical Center. All patients with stage I, II, and IIIA diseases underwent an anatomic resection. The majority underwent either a lobectomy or pneumonectomy. Patients who underwent wedge resections as their definitive surgical procedure were not included. Specimens from stage IIIB and IV patients were from resections or biopsies used to diagnose their disease. Specimens from patients who received neoadjuvant chemotherapy or radiation therapy were excluded. Patients from whom the specimens were studied had their clinical information extracted from their inpatient and outpatient medical records. Institutional Review Board approval was obtained for this study with a waiver of individual consent. 2.2. Immunohistochemical staining The sections of the paraffin-embedded blocks were deparaffinized and rehydrated. Antigen retrieval was done using a 0.01 M

2.3. Scoring Cytoplasmic XOR staining was reviewed and scored by a single pathologist (P.G.). The reviewer was blinded to the clinical information regarding the patients whose specimen was being scored. The percentage of cells staining positive for XOR expression designated as the frequency of XOR expression was also scored. Frequency of XOR also was graded on a scale of 0 through 4: 0, <10% staining; 1, 10–25% staining; 2, 26–50% staining; 3, 51–75% staining; or 4, >75% staining. XOR frequency of 0–1 was classified as low frequency and 2–4 was classified as high frequency. In general, XOR positivity was defined as 10% or more of cells staining positive with the XOR antibody. In addition to the frequency of XOR expression, XOR intensity was graded on a scale of 0 through 4: 0, no staining; 1, weak staining, no granular cytoplasmic staining; 2, weak staining, fine granular cytoplasmic staining; 3, well distinct, granular cytoplasmic staining; or 4, sharp prominent dense granular cytoplasmic (Fig. 1). With respect to both XOR frequency and intensity, scores of 0–1 were classified as low expression and 2–4 were classified as high expression.

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Table 1 XOR expression and median survival of patients. The individual stages and number of patients who demonstrated high and low XOR expression (as measured by XOR frequency) are displayed with their associated median survival in days. The difference in median survival in days also was expressed in boldface for the entire cohort. The p-values were from log-rank tests of Kaplan–Meier survival curves. The estimated survival for stage I patients in the high XOR expression group was >0.50 at the end of follow-up, thereby making an estimation of the median survival for this subgroup not possible. Patients

Median survival for XOR (0–1) [N]

Median survival for XOR (2–4) [N]

p-Value

Hazard ratio

STAGE I STAGE II STAGE IIIA STAGE IIIB/IV

25 21 15 21

1867 [7] 943.5 [6] 275 [7] 215 [9]

NR [18] 2465 [15] 3402 [8] 424.5 [12]

0.07 0.53 0.06 0.70

0.3 0.7 0.24 0.82

Total

82

592 [29]

3053 [53]

0.0089

0.47

NR, not reached.

2.4. Statistical analysis Individually, XOR frequency, XOR intensity, and an overall score of XOR frequency × XOR intensity (XOR FXI score) were correlated with clinical characteristics and outcomes. An XOR intensity or XOR frequency that was assigned a value of 0–1 or an XOR FXI score that was calculated to be 0–1 were considered low. An XOR intensity or XOR frequency that was assigned a value of 2–4 or XOR FXI score that was calculated to be 2–16 was classified as high. A calculated XOR FXI score that was between 12 and 16 was given a special designation of extremely high. Survival curves were generated by the Kaplan–Meier method. Survivals between different groups were compared with the log-rank test. The survival figures that were generated represented 5-year overall survival. Hazard ratios were obtained using the Cox proportional hazards analysis. Statistical analysis was performed using SAS v9.1 software (SAS Institute Inc., Cary, NC). 3. Results

days), p = 0.0089, HR 0.47 (Fig. 2; Table 1). Low XOR intensity also was associated with a trend toward worse survival. Patients with low XOR intensity experienced a similar survival of 1236 days (95% CI: 0–2981 days) compared to those with high XOR intensity, 2465 days (95% CI: 593–4336 days), p = 0.354, HR 0.765. Interestingly, when multiplying XOR frequency by intensity for an overall XOR FXI score, only a non-significant trend with decreased overall survival was observed. Patients with a low XOR FXI score (0–1) experienced had a median survival of 599 days (95% CI: 0–1332 days) that approached significance when compared to those with a high XOR FXI score (2–16), 2803 days (95% CI: 1237–4373 days), p = 0.093, HR = 0.603. When comparing the extremes of the XOR FXI scores, the difference in survival of patients with a low XOR FXI score (0–1) only approached significance, again, when compared to the overall survival of patients with an extremely high XOR FXI score (12–16), 3139 days (95% CI: incalculable), p = 0.105, HR = 0.489. 3.4. Correlation with XOR expression (frequency) and the survival of patients treated with chemotherapy

3.1. Clinical data Based upon the inclusion and exclusion criteria listed in Section 2, 82 specimens were retrospectively selected for this study. There were 46 female and 36 male patients with mean age of 63 ± 10 years (range: 32–85 years). Histopathologic diagnosis included: adenocarcinoma—41 (50%), squamous carcinoma—31 (38%), poorly differentiated/moderately differentiated NSCLC—8 (10%), and bronchoalveolar—2 (2%). The specimens were from patients with a range of stages and were as follows: 25 (30%) stage I, 21 (26%) stage II, 15 (18%) resected stage IIIA, and 21 (26%) from stage IIIB/IV.

Of the 82 total patients, 34 (41.5%) had stage IIB or higher, and these patients received chemotherapy in the adjuvant setting. Of these 34 patients, 15 (44%) demonstrated low XOR expression. These 15 patients with low XOR expression had a shorter median

3.2. Relationship between XOR expression and histopathology or stage Positive XOR expression was observed in 53/82 (65%) patients (Fig. 1). These were compared against normal lung epithelium and the normal rabbit epithelium that was negative for XOR expression. A normal rabbit IgG was included as a negative control and no non-specific staining was observed. Positive XOR expression was observed in 35/41 (85%) of the adenocarcinomas, 16/31 (52%) of the squamous cell carcinomas, 7/8 (87.5%) of the poorly/moderately differentiated, and none of the bronchoalveolar cancers. XOR frequency, XOR intensity, or the XOR FXI score did not correlate with disease stage, age, or gender. 3.3. Low XOR frequency is associated with a worse survival There were 29/82 (35%) specimens that exhibited low XOR intensity. Patients with low XOR frequency experienced a shorter median survival of 592 days (95% CI: 492–692 days) as compared to those with high XOR frequency, 3053 days (95% CI: 2190–3916

Fig. 2. Survival curves demonstrating a significantly shorter survival [days], for the 29/82 (35%) patients with specimens that had low XOR expression, XOR (0–1)—592 days compared to the survival for the 53/82 (65%) patients with specimens that had high XOR expression, XOR (2–4)—3053 days, p = 0.0089.

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Table 2 Median survival in days of patients undergoing and not undergoing postoperative chemotherapy. The p-values were generated by comparing Kaplan–Meier survival curves between the CHEMO(+) and the CHEMO(−) groups using log-rank tests. The estimated survival for the CHEMO(−) and XOR (2–4) group was >0.50 at the end of follow-up, thereby making an estimation of the median survival for this subgroup not possible.

CHEMO(+) CHEMO(−)

Patients

Median survival for XOR (0–1) [N]

Median survival for XOR (2–4) [N]

p-Value

Hazard ratio

34 48

543 [15] 1577 [14]

2023 [19] NR [34]

0.007 0.29

0.33 0.58

NR, not reached.

survival of 543 days (95% CI: 118–968), compared to the 19 (56%) of patients with high XOR expression (median survival 2023 days, 95% CI: 634–3412). This difference was strongly significant (log-rank p = 0.007, HR = 0.33) (Fig. 3; Table 2). 3.4.1. Smoking status is not associated XOR expression The smoking status was known in 60 of the 82 patients from whom the specimens were immunostained. Of these 60 patients, 50 (83%) were current or former smokers and 10 (17%) were neversmokers. Of these same 60 patients, 30 (50%) were actively smoking and the other 30 (50%) were not actively smoking (including the 10 never-smokers). In general, smoking status was not associated significantly with XOR intensity (p = 0.163), XOR frequency (p = 0.073), or an overall XOR FXI score (p = 0.256). Similarly, an active smoking status was not associated with XOR intensity (p = 0.790), XOR frequency (p = 0.417), or an overall XOR FXI score (p = 0.539). 4. Discussion XOR is expressed in the cytoplasm of hepatocytes, intestinal epithelial cells, vascular endothelial cells, breast acinar and ductal epithelial cells [6,14,15]. Two studies published approximately four decades ago showed a progressive decrease of XOR activity during murine mammary carcinogenesis [16,17]. Downregulation of XOR expression and activity also occurs in rat hepatomas and human renal cell carcinomas [11–13]. A more recent study demonstrated that XOR expression is modestly decreased in about 50% of breast cancers and that these patients with XOR-negative tumors have a two-fold higher risk of distant recurrence [10]. Our study is supportive of other studies which demonstrate that decreased XOR expression is associated with a more aggressive form of cancer [6,10–13,18]. Until presently, there has not been a study published demonstrating decreased survival in NSCLC associated with decreased XOR expression. The survival difference noted in our analysis was not dependent on the clinicopathologic stage though this may have been because our data was insufficiently powered to observe significant differences. Although there was a subset analysis performed on the

Fig. 3. Kaplan–Meier survival curves for the 34 patients who underwent chemotherapy demonstrating that high XOR expression, XOR (2–4) was associated with improved survival over low XOR expression XOR (0–1).

patients who underwent chemotherapy that included, primarily, the stage IIB and higher patients, stage I patients did not have significant difference in survival when stratified by XOR expression. We acknowledge that the pool of patients from which the analysis was performed included those with different types of histology and, therefore, ostensibly, may have represented a heterogeneous group of patients. However, the majority (88%) of the tissue specimens were either adenocarcinoma or squamous carcinoma suggesting that reasonable conclusions could be drawn in light of the addition of the poorly differentiated and bronchiolar alveolar histology in a minority of the specimens (12%). Furthermore, these latter two histologies are still technically classified as NSCLC. Lastly, the tumors in the poorly differentiated group (10%) were more likely to be adenocarcinomas or squamous cell carcinomas and, thus, reasonable to include in the analysis. Although this study was only an exploratory analysis, it is interesting to note that differences in survival between stage I cancers with high and low XOR expression approached significance. None of these patients went on to receive chemotherapy, suggesting poor prognostic implications of decreased XOR expression. Significant differences between survivals by XOR expression were not demonstrated in stage II, III, or IIIB/IV patients. This might be explained by the small sample size or the heterogeneous nature of the stage IIIB/IV patients. Alternatively, the absence of a significant difference may have to do with the fact there was some variability in the patients who received adjuvant chemotherapy following surgical treatment among some of the stage II patients. All stage IIIA patients received some form of adjuvant therapy. Despite the decreased survival associated with downregulated XOR expression, it remains unknown exactly how the attenuated presence of this enzyme contributes to the aggressive behavior and shorter patient survival. It is possible that cancer cells are able to shunt the substrates for purine synthesis to a more efficient purine salvage pathway rather than through a less efficient de novo regeneration pathway [10]. This has been suggested in animal models of cancer that have demonstrated decreased XOR activity [12,19–21] and increased hypoxanthine phosphoribosyltransferase (HPRT) activity (HPRT—an enzyme in the purine regeneration pathway) [22]. The role of uric acid (UA), the other product of the XOR pathway, in cancer progression also remains incompletely defined. Despite earlier theories that antioxidant effects of hyperuricemia were protective against the development of various cancers [23], several epidemiologic studies have not corroborated this theory [24–26]. However, it is possible that the amount of XOR in the natural state may allow for a zero-sum gain physiologically in the normal individual. The decreased UA production secondary to decreased XOR expression in malignancies may result in a relative hypouricemia which in turn may serve as a marker for its deficiency. A subset analysis within this study demonstrated that among the high XOR expression patients who underwent chemotherapy, XOR expression was associated with an improved survival when compared to those who had low XOR expression. This observation suggests that XOR may have a role in modulating chemosensitivity. It has been long believed that chemotherapy and radiation therapy can exert their antitumor activity by targeting the rapidly

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replicating genomic DNA of tumor cells. However, recent studies suggest that chemotherapy and radiation therapy can also activate a number of intracellular signaling pathways that contribute to their tumoricidal effect by inducing apoptosis [27,28]. One such cellular response is the activation of the mitogen activated protein (MAP) kinase pathway [16,7,29,30]. Production of reactive oxygen species (ROS) following the administration of chemotherapeutic agents has been thought to play a critical role in activating the MAP kinase pathway, mainly p38 MAP kinase pathway [16,17,29,30]. We speculate that XOR may facilitate the activation of the MAP kinase pathway through the creation of ROS during purine metabolism, subsequently contributing to the tumoricidal effect of chemotherapy. We acknowledge the fact that while we scored XOR frequency and intensity into four subgroups we ultimately regrouped and analyzed those new groups as those that had low versus high XOR expression (XOR immunostaining 0–1 vs. 2–4, respectively). It is possible that if the four subgroups were larger, there might be an appropriate decreased gradation in survival commensurate with the frequency and intensity of immunostaining observed. In particular, the graded decrease in XOR expression could potentially correlate with decreased chemosensitivity or increased chemoresistance, but at present, our data cannot definitively establish this pattern. In summary, our present study demonstrates that the lack of XOR expression is associated with a worse survival in NSCLC. Our observations suggest that additional studies evaluating XOR expression and survival and chemotherapy sensitivity in NSCLC are warranted. These observations also suggest that the status of XOR expression in NSCLC may have prognostic value and, therefore, further studies should focus on elucidating its underlying molecular mechanisms. Conflict of interest The authors of this manuscript have no conflict of interests. References [1] Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 2008;359(13):1367–80. [2] Pignon JP, Tribodet H, Scagliotti GV, Douillard JY, Shepherd FA, Stephens RJ, et al. Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J Clin Oncol 2008;26(21):3552–9. [3] Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP, Vansteenkiste J. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med 2004;350(4):351–60. [4] Fouret P, Planchard D, Mendiboure J, Kamal NS, Olaussen KA, Bertrand P, et al. MSH2 and adjuvant cisplatin-based chemotherapy in non-small cell lung cancer. J Clin Oncol 2009;27(18S: Suppl.), abstr CRA7502. [5] Olaussen KA, Dunant A, Fouret P, Brambilla E, Andre F, Haddad V, et al. DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 2006;355(10):983–91. [6] Harrison R. Structure and function of xanthine oxidoreductase: where are we now? Free Radic Biol Med 2002;33(6):774–97.

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