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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Such genetic changes, including chro- vation of oncogenes and the inactivation of tumour-sup- Page 1 of 5 (page number not for citation purposes) Radiation Oncology 2009, 4:29 http://www.ro-journal.com/content/4/1/29 pressor genes . 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 . resistance to oncological treatments . 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 . 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 . 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) . 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  and they genation was measured by an Eppendorf device following are resistant to chemotherapy . 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 . 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 . 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 . 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 . 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 . 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  (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 . 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  and other cancers  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 . 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  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  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 . In the rubicin, methotrexate, vincristine or cisplatin) . 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 . 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 . 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 . 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 . 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 . 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.  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 . 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 Oncology – Springer Journals
Published: Aug 6, 2009
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