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Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed. Introduction mosomal translocation, gene amplification, intragenic Growing cancers often acquire an increasing number of mutation, and gene silencing, are responsible for the acti- genetic alterations. Such genetic changes, including chro- vation of oncogenes and the inactivation of tumour-sup- Page 1 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 pressor genes [1]. How cancer cells acquire genetic (Dako) was used afterward. Staining was revealed by instability remains unclear. Exposure of cells to adverse using diaminobenzidine tetra-hydrochloride substrate conditions like hypoxia can lead to genome alterations, (DAB Chromogen; Dako), followed by light counterstain- enhancing the progression potential of tumor cells and ing with Harris hematoxylin as previously described [10]. resistance to oncological treatments [1]. Hypoxia may Data of p53 and angiogenesis, estimated by CD-31 stain- lead to conditions that causes increased spontaneous ing, were obtained from our files [2]. Paraffin-embedded damage to DNA or inhibit DNA repair processes, impair tissues from tumor biopsies were available from all DNA repair and cause tumor progression by altered p53 patients, and the most representative tumor block was expression and increased angiogenesis [2,3]. Deregulation used for immunohistochemical analysis. Blocks were han- of DNA repair pathways can contribute to the phenome- dled as previously described and then incubated for the non of hypoxia-induced genetic instability within the specific secondary antibody (p53, Clon:DO-7, Novocastra tumor [4]. Hypoxia is measured in clinical tumors by sev- Laboratories Ltd., Newcastle upon Tyne, UK; CD-31 eral techniques, including the Eppendorf polarographic Clon:JC/70A, Dako, Carpintería, CA, USA) [2]. The pri- method [2,5]. In cervical cancer patients, hypoxia is com- mary antibody was omitted in one section as a negative monly associated to a lesser response to treatment and control in each set of slides. As a positive control, a strong lower survival rates [6,7]. Hypoxic tumors have a signifi- positive tumor for the oncoprotein was used. Tumor oxy- cant higher probability of relapse and death [7] and they genation was measured by an Eppendorf device following are resistant to chemotherapy [8]. Chemo-resistance standard criteria as previously described [2,12] using a would be mediated by up-regulation of Major Vault Pro- polarographic probe system "pO2 Histograph" (Eppen- tein (MVP) through the Hypoxia-inducible factor 1 (HIF- dorf AG, Hamburg, Germany). For each set of measure- 1) as shown in previously studies performed in vitro [9]. ments obtained from tumor, 200 single pO2 values were Hypoxia inhibits the non-homologous end joining recorded using at least 6 different electrode tracks. Tumor (NHEJ) DNA repair through down-regulating Ku70/80 hypoxia data were reanalyzed for detecting cases of severe expression, combined with increased angiogenesis and hypoxia and the percentage of pO2 values < 2.5 mmHg altered p53 expression [2]. Cervical tumors over-express- were obtained from the pooled data and for each individ- ing MVP also showed down-regulation of Ku70/80 and ual. Assessment of immunostaining or tumor oxygena- BAX [10]. MVP over-expression has been associated with tion result was blinded to knowledge of the clinical a suppression of NHEJ repair, and subsequent genomic outcome of the patient. Statistical analysis was performed instability [10]. These mechanisms would be responsible by SPSS 15.0 software. for tumor progression in cervical carcinoma. Moreover, MVP over-expression was associated to reduced long-term Results local control in patients who achieved clinical complete All immunohistochemical markers and hypoxia values response to radio-chemotherapy [11]. The aim of the were known in all 43 cases (Figure 1). MVP expression was present study was to assess the relation between the considered low (negative/slightly positive) in 23 cases expression of the Major Vault Protein and tumor hypoxia and high (strongly positive) in 20 cases. Data of mean vas- in clinical cervical tumors. cular density (MVD) and p53 expression were obtained from our files [2] (Table 1). MVD was 49.62 ± 33.98% Methods (median 41%, range 0–160). P53 expression showed a Forty-three consecutive patients suffering from localized mean value of 39.15 ± 27.62% (median 35%, range 0– cervix carcinoma were prospectively included in this study 92%). Tumor hypoxia was also known in all patients. from July 1997 to September 2001 [2]. Patients were diag- Mean tumor hypoxic fraction <2.5 mmHg (HF 2.5) values nosed and treated by definitive radiation at the Hospital were 35.89 ± 26.80 (median 35.20%, range 0–91.30%). Universitario Materno-Infantil, at the Hospital Universi- MVP expression was independent of clinical and histolog- tario Dr. Negrín and at the Hospital Universitario Insular ical variables, except for adenocarcinoma tumors. In fact in Las Palmas de Gran Canaria (Spain). Written informed adenocarcinoma tumors (5 cases) included in the present consent was given previously. The study was approved by study over-expressed MVP versus 15 out of 38 squamous the Research and Ethics Committee of our institution. The cancers (p = 0.011). Besides, high MVP expression was mean age of the patients was 49.48 ± 12.79 years (median related to severe hypoxia as determined by higher hypoxic 48, range 29–81 years). Fourteen patients had stage I dis- fractions HF (2.5) (45.82 ± 28.00%) compared to low ease, 22 stage II and 7 stage III-IVA. MVP expression was MVP expressing tumors (27.26 ± 22.96%) (p = 0.022) studied by immunohistochemistry in paraffin-embedded (Figure 2a). Tumors over-expressing MVP also showed 4 μm sections incubated for the specific primary antibody increased angiogenesis (65.41 ± 38.38) compared to low (MVP, Neomarkers CA, USA). A secondary biotinated expressing cases (35.89 ± 22.55) (p = 0.003) (Figure 2b). antibody (Dako Detection Kit, LSBA) was incubated for MVP expression was independent of p53 protein expres- 30 minutes, and peroxidase-streptavidin-biotin complex sion. Page 2 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 Table 1: Characteristics of the patients in the study Characteristics All patients MVP low MVP high P value (n = 43) (n = 23) (n = 20) Age 49.48 ± 12.79 49.47 ± 13.68 49.50 ± 12.04 (29–81) (29–81) (32–72) 0.325 Stage I14 5 9 II 22 13 9 III 7 5 2 0.228 Histology Epidermoid 382315 Adenocarcinoma 5 0 5 0.011 Grade I 532 II 19 10 9 III 19 10 9 0.952 p53 39.15 ± 27.62 37.53 ± 28.04 41.02 ± 27.74 (0–92) (0–92) (0–81) 0.685 Vascular density 49.62 ± 33.98 35.89 ± 22.55 65.41 ± 38.38 (0–160) (0–113) (12–160) 0.003 Hypoxic fraction 35.89 ± 26.80 27.26 ± 22.96 45.82 ± 28.00 (0–91.30) (0–66.30) (0–91.30) 0.022 Median pO 7.61 ± 8.98 7.84 ± 7.85 7.36 ± 10.34 (0–41.90) (0–24.30) (0–41.90) 0.863 Mean ± standard deviation and range are included as well as p53, vascular density, hypoxic fraction and median of pO Discussion ing to the relationship between MVP expression and drug In this study we show for the first time that severe tumor resistance in clinical oncology [19-22]. The role of MVP in hypoxia is related to high MVP expression in clinical cer- clinical outcome after radio-chemotherapy in cervical car- vical tumors. MVP is ubiquitously expressed and, besides cinoma [11] and other cancers [23] has been reported. chemotherapy resistance, it has been implicated in the MVP seems to down-regulate the pro-apoptotic gene BAX regulation of several cellular processes including transport through its relation with Ku70/80. Ku70/80 are key genes mechanisms, signal transmissions and immune responses in the NHEJ repair pathway for radiation-induced DNA [13]. Previous studies have demonstrated that vaults are double strand breaks. Expression of Ku70/80 has been up-regulated in different multidrug resistant cancer cell related to survival in patients treated with x-rays [24,25]. lines [14] and resistance models [15,16]. Increased levels Ku70/80 is a central regulator of apoptosis by interacting of MVP have been reported in numerous cell lines after with BAX [26] and BCL-2, which in turn has been shown selection with a wide panel of cytostatic drugs (e.g. doxo- to suppress Ku, thus inhibiting NHEJ repair [27]. In the rubicin, methotrexate, vincristine or cisplatin) [17]. By clinical setting, up-regulation of MVP by hypoxia is of crit- contrast, tumour necrosis factor-either applied externally ical relevance because chemotherapy is currently a stand- or after gene transduction, led to down-regulation of MVP ard treatment for those patients. In the other hand, transcription [18]. There are several publications concern- hypoxia inhibits the NHEJ DNA repair through down-reg- Repr Figure 1 esentative immunostaining of MVP (a), p53 (b) and micro-vessels (c) Representative immunostaining of MVP (a), p53 (b) and micro-vessels (c). Page 3 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 Relationship Figure 2 between (a) MVP and hypoxic fraction (HF 2.5) and (b) mean vascular density Relationship between (a) MVP and hypoxic fraction (HF 2.5) and (b) mean vascular density. ulating Ku70/80 expression [2]. Preclinical studies about cers) depends, at least in part, of those parameters. An the role of hypoxia in cancer cells showed that reduction increased genetic instability, oncogenic properties, resist- of pO is a favoring factor to increase chemo-resistance ance to treatment and increased ability to metastization [8,28]. In cancer, hypoxia is an adverse prognostic indica- are expected. tor associated with tumor progression and resistance to therapy [29]. Cellular drug delivery and uptake in hypoxic From our results it could be suggested that hypoxia not areas are affected by hypoxia. Some chemotherapeutic only induces increased genetic instability, oncogenic drugs require oxygen to generate free radicals that contrib- properties and metastatization, but through the correla- ute to cytotoxicity. Hypoxia induces cellular adaptations tion observed with MVP expression, another pathway of that compromise the effectiveness of chemotherapy. chemo-resistance could be developed. Moreover, the expression of several genes controlling tumor cell survival is regulated by hypoxia (e.g., growth Abbreviations factors governing the formation of new blood vessels and HIF-1: Hypoxia-inducible factor 1; MVD: Mean vascular hypoxia-responsive transcription factors modulating the density; MVP: Major Vault Protein; NHEJ: non-homolo- expression of genes). The transcription factor Hypoxia- gous end joining. inducible factor 1 (HIF-1) is one of the principal media- tors of homeostasis in human tissues exposed to hypoxia. Conflict of interests The authors declare that they have no competing interests. It is implicated in virtually every process of rapid gene expression in response to low oxygen levels [30]. HIF- 1alpha is over-expressed in the majority of common Authors' contributions human cancers and their metastases, due to the presence PCL has been involved in conception and design of the of intratumoral hypoxia and as a result of mutations in study as well as in drafting the manuscript, and has given genes encoding oncoproteins and tumor suppressor genes final approval of the version to be published. MLl has [31,32]. Whether in clinical tumors this chemo-resistance made the measurements of tumour hypoxia and has can be reverted by HIF-1 inhibitors deserves to be studied treated all patients. BC has made the measurements of [9]. Pharmacologic manipulation of HIF-1 levels may pro- tumour hypoxia. RMA has made the angiogenesis studies. vide a novel therapeutic approach to diseases like cancer, LAHH has been involved in the writing of the manuscript especially in combination with anti-angiogenic agents and type of packaging likewise in the submission process. [33] that would further reduce tumour oxygenation. Our EB has made the MVP studies. FF has made the p53 stud- previously clinical results showed a close relation of clini- ies. AR has reviewed and overlooked all the immunohis- cal hypoxia to increased angiogenesis and in a lesser tochemistry experiments. extent to p53 oncoprotein alteration [2]. Clinical outcome in patients suffering different types of tumours mainly treated by radiation (i.e., cervical and head & neck can- Page 4 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 18. Stein U, Walther W, Laurencot CM, Scheffer GL, Scheper RJ, Shoe- Acknowledgements maker RH: Tumor necrosis factor-alpha and expression of the This work was subsidized by grants: FIS 1035/98, 0855/01. Henríquez- multidrug resistance-associated genes LRP and MRP. J Natl Hernández LA, Bordón E and Fontes F were supported by an educational Cancer Inst 1997, 89:807-813. grant from the Instituto Canario de Investigación del Cáncer (ICIC). 19. Mossink MH, van Zon A, Scheper RJ, Sonneveld P, Wiemer EA: Vaults: a ribonucleoprotein particle involved in drug resist- ance? Oncogene 2003, 22:7458-7467. References 20. Suprenant KA: Vault ribonucleoprotein particles: sarcophagi, 1. Huang LE, Bindra RS, Glazer PM, Harris AL: Hypoxia-induced gondolas, or safety deposit boxes? Biochemistry 2002, genetic instability – a calculated mechanism underlying 41:14447-14454. tumor progression. J Mol Med 2007, 85:139-148. 21. Steiner E, Holzmann K, Elbling L, Micksche M, Berger W: Cellular 2. Lara PC, Lloret M, Clavo B, Apolinario RM, Bordon E, Rey A, Falcon functions of vaults and their involvement in multidrug resist- O, Alonso AR, Belka C: Hypoxia downregulates Ku70/80 ance. Curr Drug Targets 2006, 7:923-934. expression in cervical carcinoma tumors. Radiother Oncol 2008, 22. Izquierdo MA, Scheffer GL, Schroeijers AB, de Jong MC, Scheper RJ: 89:222-226. Vault-related resistance to anticancer drugs determined by 3. Wood RD: DNA repair in eukaryotes. Annu Rev Biochem 1996, the expression of the major vault protein LRP. Cytotechnology 65:135-167. 1998, 27:137-148. 4. Bindra RS, Crosby ME, Glazer PM: Regulation of DNA repair in 23. Silva P, West CM, Slevin N, Valentine H, Ryder WD, Hampson L, Bibi hypoxic cancer cells. Cancer Metastasis Rev 2007, 26:249-260. R, Sloan P, Thakker N, Homer J, Hampson I: Tumor expression of 5. Tatum JL, Kelloff GJ, Gillies RJ, Arbeit JM, Brown JM, Chao KS, Chap- major vault protein is an adverse prognostic factor for radi- man JD, Eckelman WC, Fyles AW, Giaccia AJ, Hill RP, Koch CJ, otherapy outcome in oropharyngeal carcinoma. Int J Radiat Krishna MC, Krohn KA, Lewis JS, Mason RP, Melillo G, Padhani AR, Oncol Biol Phys 2007, 69:133-140. Powis G, Rajendran JG, Reba R, Robinson SP, Semenza GL, Swartz 24. Beskow C, Kanter L, Holgersson A, Nilsson B, Frankendal B, Avall- HM, Vaupel P, Yang D, Croft B, Hoffman J, Liu G, Stone H, Sullivan D: Lundqvist E, Lewensohn R: Expression of DNA damage Hypoxia: importance in tumor biology, noninvasive meas- response proteins and complete remission after radiother- urement by imaging, and value of its measurement in the apy of stage IB-IIA of cervical cancer. Br J Cancer 2006, management of cancer therapy. Int J Radiat Biol 2006, 94:1683-1689. 82:699-757. 25. Wilson CR, Davidson SE, Margison GP, Jackson SP, Hendry JH, West 6. Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P: Asso- CM: Expression of Ku70 correlates with survival in carcinoma ciation between tumor hypoxia and malignant progression of the cervix. Br J Cancer 2000, 83:1702-1706. in advanced cancer of the uterine cervix. Cancer Res 1996, 26. Amsel AD, Rathaus M, Kronman N, Cohen HY: Regulation of the 56:4509-4515. proapoptotic factor Bax by Ku70-dependent deubiquityla- 7. Fyles AW, Milosevic M, Wong R, Kavanagh MC, Pintilie M, Sun A, tion. Proc Natl Acad Sci USA 2008, 105:5117-5122. Chapman W, Levin W, Manchul L, Keane TJ, Hill RP: Oxygenation 27. Wang Q, Gao F, May WS, Zhang Y, Flagg T, Deng X: Bcl2 negatively predicts radiation response and survival in patients with cer- regulates DNA double-strand-break repair through a nonho- vix cancer. Radiother Oncol 1998, 48:149-156. mologous end-joining pathway. Mol Cell 2008, 29:488-498. 8. Sasabe E, Zhou X, Li D, Oku N, Yamamoto T, Osaki T: The involve- 28. Cosse JP, Michiels C: Tumour hypoxia affects the responsive- ment of hypoxia-inducible factor-1alpha in the susceptibility ness of cancer cells to chemotherapy and promotes cancer to gamma-rays and chemotherapeutic drugs of oral squa- progression. Anticancer Agents Med Chem 2008, 8:790-797. mous cell carcinoma cells. Int J Cancer 2007, 120:268-277. 29. Shannon AM, Bouchier-Hayes DJ, Condron CM, Toomey D: 9. Lelong-Rebel I, Brisson C, Fabre M, Bergerat JP, Rebel G: Effect of Tumour hypoxia, chemotherapeutic resistance and hypoxia- pO2 on antitumor drug cytotoxicity on MDR and non-MDR related therapies. Cancer Treat Rev 2003, 29:297-307. variants selected from the LoVo metastatic colon carcinoma 30. Adams JM, Difazio LT, Rolandelli RH, Lujan JJ, Hasko G, Csoka B, cell line. Anticancer Res 2008, 28:55-68. Selmeczy Z, Nemeth ZH: HIF-1: a key mediator in hypoxia. Acta 10. Lloret M, Lara PC, Bordon E, Fontes F, Rey A, Pinar B, Falcon O: Physiol Hung 2009, 96:19-28. Major vault protein may affect nonhomologous end-joining 31. Semenza GL: Expression of hypoxia-inducible factor 1: mecha- repair and apoptosis through Ku70/80 and bax downregula- nisms and consequences. Biochem Pharmacol 2000, 59:47-53. tion in cervical carcinoma tumors. Int J Radiat Oncol Biol Phys 32. Zhong H, De Marzo AM, Laughner E, Lim M, Hilton DA, Zagzag D, 2009, 73:976-979. Buechler P, Isaacs WB, Semenza GL, Simons JW: Overexpression 11. Lloret M, Lara PC, Bordon E, Rey A, Falcon O, Apolinario RM, Clavo of hypoxia-inducible factor 1alpha in common human can- B, Ruiz A: MVP expression is related to IGF1-R in cervical car- cers and their metastases. Cancer Res 1999, 59:5830-5835. cinoma patients treated by radiochemotherapy. Gynecol Oncol 33. Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D: Effects of 2008, 110:304-307. angiogenesis inhibitors on multistage carcinogenesis in mice. 12. Clavo B, Perez JL, Lopez L, Suarez G, Lloret M, Morera J, Macias D, Science 1999, 284:808-812. Martinez JC, Santana M, Hernandez MA, Robaina F, Gunderoth M: Influence of haemoglobin concentration and peripheral mus- cle pO2 on tumour oxygenation in advanced head and neck tumours. Radiother Oncol 2003, 66:71-74. 13. Berger W, Steiner E, Grusch M, Elbling L, Micksche M: Vaults and the major vault protein: novel roles in signal pathway regu- lation and immunity. Cell Mol Life Sci 2009, 66:43-61. Publish with Bio Med Central and every 14. Izquierdo MA, Shoemaker RH, Flens MJ, Scheffer GL, Wu L, Prather scientist can read your work free of charge TR, Scheper RJ: Overlapping phenotypes of multidrug resist- ance among panels of human cancer-cell lines. Int J Cancer "BioMed Central will be the most significant development for 1996, 65:230-237. disseminating the results of biomedical researc h in our lifetime." 15. Izquierdo MA, Scheffer GL, Flens MJ, Shoemaker RH, Rome LH, Scheper RJ: Relationship of LRP-human major vault protein to Sir Paul Nurse, Cancer Research UK in vitro and clinical resistance to anticancer drugs. Cytotech- Your research papers will be: nology 1996, 19:191-197. 16. Siva AC, Raval-Fernandes S, Stephen AG, LaFemina MJ, Scheper RJ, available free of charge to the entire biomedical community Kickhoefer VA, Rome LH: Up-regulation of vaults may be nec- peer reviewed and published immediately upon acceptance essary but not sufficient for multidrug resistance. Int J Cancer 2001, 92:195-202. cited in PubMed and archived on PubMed Central 17. Lange C, Walther W, Schwabe H, Stein U: Cloning and initial anal- yours — you keep the copyright ysis of the human multidrug resistance-related MVP/LRP gene promoter. Biochem Biophys Res Commun 2000, 278:125-133. BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

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Springer Journals
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Copyright © 2009 by Lara et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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10.1186/1748-717X-4-29
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19660100
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Abstract

Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed. Introduction mosomal translocation, gene amplification, intragenic Growing cancers often acquire an increasing number of mutation, and gene silencing, are responsible for the acti- genetic alterations. Such genetic changes, including chro- vation of oncogenes and the inactivation of tumour-sup- Page 1 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 pressor genes [1]. How cancer cells acquire genetic (Dako) was used afterward. Staining was revealed by instability remains unclear. Exposure of cells to adverse using diaminobenzidine tetra-hydrochloride substrate conditions like hypoxia can lead to genome alterations, (DAB Chromogen; Dako), followed by light counterstain- enhancing the progression potential of tumor cells and ing with Harris hematoxylin as previously described [10]. resistance to oncological treatments [1]. Hypoxia may Data of p53 and angiogenesis, estimated by CD-31 stain- lead to conditions that causes increased spontaneous ing, were obtained from our files [2]. Paraffin-embedded damage to DNA or inhibit DNA repair processes, impair tissues from tumor biopsies were available from all DNA repair and cause tumor progression by altered p53 patients, and the most representative tumor block was expression and increased angiogenesis [2,3]. Deregulation used for immunohistochemical analysis. Blocks were han- of DNA repair pathways can contribute to the phenome- dled as previously described and then incubated for the non of hypoxia-induced genetic instability within the specific secondary antibody (p53, Clon:DO-7, Novocastra tumor [4]. Hypoxia is measured in clinical tumors by sev- Laboratories Ltd., Newcastle upon Tyne, UK; CD-31 eral techniques, including the Eppendorf polarographic Clon:JC/70A, Dako, Carpintería, CA, USA) [2]. The pri- method [2,5]. In cervical cancer patients, hypoxia is com- mary antibody was omitted in one section as a negative monly associated to a lesser response to treatment and control in each set of slides. As a positive control, a strong lower survival rates [6,7]. Hypoxic tumors have a signifi- positive tumor for the oncoprotein was used. Tumor oxy- cant higher probability of relapse and death [7] and they genation was measured by an Eppendorf device following are resistant to chemotherapy [8]. Chemo-resistance standard criteria as previously described [2,12] using a would be mediated by up-regulation of Major Vault Pro- polarographic probe system "pO2 Histograph" (Eppen- tein (MVP) through the Hypoxia-inducible factor 1 (HIF- dorf AG, Hamburg, Germany). For each set of measure- 1) as shown in previously studies performed in vitro [9]. ments obtained from tumor, 200 single pO2 values were Hypoxia inhibits the non-homologous end joining recorded using at least 6 different electrode tracks. Tumor (NHEJ) DNA repair through down-regulating Ku70/80 hypoxia data were reanalyzed for detecting cases of severe expression, combined with increased angiogenesis and hypoxia and the percentage of pO2 values < 2.5 mmHg altered p53 expression [2]. Cervical tumors over-express- were obtained from the pooled data and for each individ- ing MVP also showed down-regulation of Ku70/80 and ual. Assessment of immunostaining or tumor oxygena- BAX [10]. MVP over-expression has been associated with tion result was blinded to knowledge of the clinical a suppression of NHEJ repair, and subsequent genomic outcome of the patient. Statistical analysis was performed instability [10]. These mechanisms would be responsible by SPSS 15.0 software. for tumor progression in cervical carcinoma. Moreover, MVP over-expression was associated to reduced long-term Results local control in patients who achieved clinical complete All immunohistochemical markers and hypoxia values response to radio-chemotherapy [11]. The aim of the were known in all 43 cases (Figure 1). MVP expression was present study was to assess the relation between the considered low (negative/slightly positive) in 23 cases expression of the Major Vault Protein and tumor hypoxia and high (strongly positive) in 20 cases. Data of mean vas- in clinical cervical tumors. cular density (MVD) and p53 expression were obtained from our files [2] (Table 1). MVD was 49.62 ± 33.98% Methods (median 41%, range 0–160). P53 expression showed a Forty-three consecutive patients suffering from localized mean value of 39.15 ± 27.62% (median 35%, range 0– cervix carcinoma were prospectively included in this study 92%). Tumor hypoxia was also known in all patients. from July 1997 to September 2001 [2]. Patients were diag- Mean tumor hypoxic fraction <2.5 mmHg (HF 2.5) values nosed and treated by definitive radiation at the Hospital were 35.89 ± 26.80 (median 35.20%, range 0–91.30%). Universitario Materno-Infantil, at the Hospital Universi- MVP expression was independent of clinical and histolog- tario Dr. Negrín and at the Hospital Universitario Insular ical variables, except for adenocarcinoma tumors. In fact in Las Palmas de Gran Canaria (Spain). Written informed adenocarcinoma tumors (5 cases) included in the present consent was given previously. The study was approved by study over-expressed MVP versus 15 out of 38 squamous the Research and Ethics Committee of our institution. The cancers (p = 0.011). Besides, high MVP expression was mean age of the patients was 49.48 ± 12.79 years (median related to severe hypoxia as determined by higher hypoxic 48, range 29–81 years). Fourteen patients had stage I dis- fractions HF (2.5) (45.82 ± 28.00%) compared to low ease, 22 stage II and 7 stage III-IVA. MVP expression was MVP expressing tumors (27.26 ± 22.96%) (p = 0.022) studied by immunohistochemistry in paraffin-embedded (Figure 2a). Tumors over-expressing MVP also showed 4 μm sections incubated for the specific primary antibody increased angiogenesis (65.41 ± 38.38) compared to low (MVP, Neomarkers CA, USA). A secondary biotinated expressing cases (35.89 ± 22.55) (p = 0.003) (Figure 2b). antibody (Dako Detection Kit, LSBA) was incubated for MVP expression was independent of p53 protein expres- 30 minutes, and peroxidase-streptavidin-biotin complex sion. Page 2 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 Table 1: Characteristics of the patients in the study Characteristics All patients MVP low MVP high P value (n = 43) (n = 23) (n = 20) Age 49.48 ± 12.79 49.47 ± 13.68 49.50 ± 12.04 (29–81) (29–81) (32–72) 0.325 Stage I14 5 9 II 22 13 9 III 7 5 2 0.228 Histology Epidermoid 382315 Adenocarcinoma 5 0 5 0.011 Grade I 532 II 19 10 9 III 19 10 9 0.952 p53 39.15 ± 27.62 37.53 ± 28.04 41.02 ± 27.74 (0–92) (0–92) (0–81) 0.685 Vascular density 49.62 ± 33.98 35.89 ± 22.55 65.41 ± 38.38 (0–160) (0–113) (12–160) 0.003 Hypoxic fraction 35.89 ± 26.80 27.26 ± 22.96 45.82 ± 28.00 (0–91.30) (0–66.30) (0–91.30) 0.022 Median pO 7.61 ± 8.98 7.84 ± 7.85 7.36 ± 10.34 (0–41.90) (0–24.30) (0–41.90) 0.863 Mean ± standard deviation and range are included as well as p53, vascular density, hypoxic fraction and median of pO Discussion ing to the relationship between MVP expression and drug In this study we show for the first time that severe tumor resistance in clinical oncology [19-22]. The role of MVP in hypoxia is related to high MVP expression in clinical cer- clinical outcome after radio-chemotherapy in cervical car- vical tumors. MVP is ubiquitously expressed and, besides cinoma [11] and other cancers [23] has been reported. chemotherapy resistance, it has been implicated in the MVP seems to down-regulate the pro-apoptotic gene BAX regulation of several cellular processes including transport through its relation with Ku70/80. Ku70/80 are key genes mechanisms, signal transmissions and immune responses in the NHEJ repair pathway for radiation-induced DNA [13]. Previous studies have demonstrated that vaults are double strand breaks. Expression of Ku70/80 has been up-regulated in different multidrug resistant cancer cell related to survival in patients treated with x-rays [24,25]. lines [14] and resistance models [15,16]. Increased levels Ku70/80 is a central regulator of apoptosis by interacting of MVP have been reported in numerous cell lines after with BAX [26] and BCL-2, which in turn has been shown selection with a wide panel of cytostatic drugs (e.g. doxo- to suppress Ku, thus inhibiting NHEJ repair [27]. In the rubicin, methotrexate, vincristine or cisplatin) [17]. By clinical setting, up-regulation of MVP by hypoxia is of crit- contrast, tumour necrosis factor-either applied externally ical relevance because chemotherapy is currently a stand- or after gene transduction, led to down-regulation of MVP ard treatment for those patients. In the other hand, transcription [18]. There are several publications concern- hypoxia inhibits the NHEJ DNA repair through down-reg- Repr Figure 1 esentative immunostaining of MVP (a), p53 (b) and micro-vessels (c) Representative immunostaining of MVP (a), p53 (b) and micro-vessels (c). Page 3 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 Relationship Figure 2 between (a) MVP and hypoxic fraction (HF 2.5) and (b) mean vascular density Relationship between (a) MVP and hypoxic fraction (HF 2.5) and (b) mean vascular density. ulating Ku70/80 expression [2]. Preclinical studies about cers) depends, at least in part, of those parameters. An the role of hypoxia in cancer cells showed that reduction increased genetic instability, oncogenic properties, resist- of pO is a favoring factor to increase chemo-resistance ance to treatment and increased ability to metastization [8,28]. In cancer, hypoxia is an adverse prognostic indica- are expected. tor associated with tumor progression and resistance to therapy [29]. Cellular drug delivery and uptake in hypoxic From our results it could be suggested that hypoxia not areas are affected by hypoxia. Some chemotherapeutic only induces increased genetic instability, oncogenic drugs require oxygen to generate free radicals that contrib- properties and metastatization, but through the correla- ute to cytotoxicity. Hypoxia induces cellular adaptations tion observed with MVP expression, another pathway of that compromise the effectiveness of chemotherapy. chemo-resistance could be developed. Moreover, the expression of several genes controlling tumor cell survival is regulated by hypoxia (e.g., growth Abbreviations factors governing the formation of new blood vessels and HIF-1: Hypoxia-inducible factor 1; MVD: Mean vascular hypoxia-responsive transcription factors modulating the density; MVP: Major Vault Protein; NHEJ: non-homolo- expression of genes). The transcription factor Hypoxia- gous end joining. inducible factor 1 (HIF-1) is one of the principal media- tors of homeostasis in human tissues exposed to hypoxia. Conflict of interests The authors declare that they have no competing interests. It is implicated in virtually every process of rapid gene expression in response to low oxygen levels [30]. HIF- 1alpha is over-expressed in the majority of common Authors' contributions human cancers and their metastases, due to the presence PCL has been involved in conception and design of the of intratumoral hypoxia and as a result of mutations in study as well as in drafting the manuscript, and has given genes encoding oncoproteins and tumor suppressor genes final approval of the version to be published. MLl has [31,32]. Whether in clinical tumors this chemo-resistance made the measurements of tumour hypoxia and has can be reverted by HIF-1 inhibitors deserves to be studied treated all patients. BC has made the measurements of [9]. Pharmacologic manipulation of HIF-1 levels may pro- tumour hypoxia. RMA has made the angiogenesis studies. vide a novel therapeutic approach to diseases like cancer, LAHH has been involved in the writing of the manuscript especially in combination with anti-angiogenic agents and type of packaging likewise in the submission process. [33] that would further reduce tumour oxygenation. Our EB has made the MVP studies. FF has made the p53 stud- previously clinical results showed a close relation of clini- ies. AR has reviewed and overlooked all the immunohis- cal hypoxia to increased angiogenesis and in a lesser tochemistry experiments. extent to p53 oncoprotein alteration [2]. Clinical outcome in patients suffering different types of tumours mainly treated by radiation (i.e., cervical and head & neck can- Page 4 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 18. 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Radiation OncologySpringer Journals

Published: Aug 6, 2009

References