Relation Between ERCC1, XPC Proteins And Curative Effect Of Neoadjuvant Chemotherapy In Locally Advanced Cervical Cancers

I . BACKGROUNDInvasive cervical cancer(ICC) is the most frequent gynecological cancer. In the more gynecological cancer, incidence of it is the second. Invasive cervical cancer is the leading cause of gynecological cancer death in China . Disease incidence is 9.98/100,000, and the mortality is 3.25/100,000. Treatment for early stage invasive cervical cancer is surgery generally and for locally advanced cervix cancer( LACC) is radiotherapy in combination with chemotherapy or chemoradiation plus surgery. According to recent report , the therapeutic effect of radiotherapy in combination with chemotherapy and neoadjuvant chemotherapy plus surgery is the same. Treatment with chemoradiation plus surgery , the patient can obtain remaining ovaries function. Thus, treatment with chemoradiation plus surgery is the first choice to young sufferer. The platinum is the most effective medicine in the neoadjuvant chemotherapy and its chemotherapy sensibility direct influence prognosis, but lack of adequate screening means to detect espond of chemotherapy. Understanding the factors that determine the chemoresistance of tumor cells is therefore an important area of investigation . DNA is thought to be the main target for the toxicity of platinum . The reactive complex binds to DNA to form inter - and intrastrand, DNA-protein cross-links, resulting in inhibition of both DNA replication and RNA transcription , arrest at the G2 phase of the cell cycle and /or programmed cell death . The survival of cells after exposure to platinum is governed by complex interatlons between a largen number of proteins involved in DNA damage recognition DNA repair, and the response of cells to unrepaired or misrepaired DNA damage.Recently , using multiple analytical approaches , we have identified at leastfive changes : 1) decrease drug accumulation; 2 ) increased cellular glutathione; 3 ) increased repair of platinum-induced DNA damage at the level of the whole genome and in specific DNA sequence; 4 ) altered types of DNA lesions formed by platinum ; 5 ) altered amounts of platinum DNA damage required to kill resistant as compared to sensitive cells.One of the clearly important resistance mechanisms is the ability of resistant cells to repair platinum-induced DNA damage . The repair of platinum-DNA lesions is believed to occur primarily by the process of nucleotide excision repair (NER). Xeroderma pigmentosum (XP) is an autosomal recessive disorder characterized by a genetic predisposition to sunlight-induced skin cancer; it is commonly due to deficiencies in DNA repair enzymes. The most frequent mutations are found in the XP genes from group A through G and group V, which encode for nucleotide excision repair (NER) proteins. NER provides versatile DNA repair mechanisms to ensure the proper functioning of all cells. The majority of patients with XP carry mutations in either the XPA or XPC genes, which encode proteins involved in the recognition of damaged DNA. The gene encoding human XPC maps to chromosome 3p25, and is expressed as a 125 kDa protein. XPC forms a complex with HHR23B (human homologue of the yeast protein RAD23 B) or HR23A, both of which stabilize XPC, and excises thymine dimers from damaged DNA. Specifically, the carboxy terminus of XPC is required for HR23B and DNA binding, and, subsequently, mutations leading to carboxy terminal truncations result in nonfunctional XPC proteins. XPF, which is also designated ERCC4 or ERCC11, is a 115 kDa protein that associates directly with the excision repair cross-complementing 1 (ERCC1) factor. ERCCl, a functional homolog of Rad10 in S. cerevisiae, is a component of a structure-specific endonuclease that is responsible for 5' incisions during DNA repair. The ERCC1-XPF endonuclease preferentially cleaves one strand of DNA between duplex and singlestranded regions near borders of the stem-loop structure and, thereby, contributes to the initial steps of the nucleotide excision repair process.Excision repair involves recognition of DNA damage, incision of DNA on either side of the lesion , generation of a new DNA strand to replace the excised segment, ligation of the new segment to the exsiting strand of DNA, and restoration of the normal chromatin confonnation . Platinum-DNA adducts make the cells arrest at G2 of the cell cycles; on the contrary, XPC and ERCC1 let this procession continue as show the above. Based on this fact, we studied whether there are the differences of protein expression levels of XPC and ERCC1 between the sensitivity and the resistance to platinum-based chemotherapy in the locally advanced cervicalcancers. There are characteristic differences in the nuclear architectures of cancer cells , compared with normal cells, and some anticancer treatments restore normal nuclear structure and function . Therefore, the study was to analysis the protein expression of ERCC1, XPC and evaluate correlations among clinical, pathological, chemotherapy response variables and their prognostic value in locally advanced cervical cancer.II. MATERIALS AND METHODSPatients. 77 continuous patients who was certified to be locally advanced cervical cancer were included. These patients underwent surgery after neoadjuvant chemotherapy from 1999 to 2006 at the Women Hospital, Zhejiang University, suffering from FIGO stage Ib2 or IIb disease of locally advanced cervical cancer. Furthermore, each patient was treated by platinum-based chemotherapy follow by surgery.Tissue Specimens. Cervix tissues prospectively collected by the pathologist were obtained from the department of pathology of Women Hospital, Zhejiang University. Tissue blocks of all patients from the formalin-fixed, paraffin-embedded cervical cancer specimens were cut in approximately 4μm sections, fixed on glass slides, and shipped to the authors' laboratory for further processing. Slides were individeually immunostained , analyzed by using a blinded coding system such that staining procedures and microscopic assessments were performed without knowledge of the histopathological diagnosis and response to chemotherapy.Discriminance criteria. According to WHO criteria ( 1979 ), complete response was defined as complete eradication of all evaluable disease, confirmed by perineoscopy. Partial response was defined as a > 50 % reduction in the measurable lesions lasting at least 4 weeks. Progressive disease was a > 25 % increase in the sum of the products of perpendicular diameters of all measurable lesions or the appearance of new lesions. Stable disease included those clinical circumstances that did not fit the definitions of objective response or progression. Progressive disease and stable disease patients were included in the non-responder category. Complete response and partial response were included in the responder category.Antibody Preparation. XPC (D-18): sc-22535 is an affinity purified goat polyclonal antibody raised against a peptide mapping near the C-terminus of XPC of human origin. ERCC1 (D-10):sc-17809 is a mouse monoclonal antibody raised against amino acids 1-297 representing full length DNA excision repair protein 1 (ERCC1) of human origin. The two production were obtained from Santa CruzBiotechnology, Inc. Tissues determined previously to express XPC and ERCC1 were used as positive controls. The XPC and ERCC1 protein were also expressed in the nuclear of the tumor cells. Negative expression was defined as no detectable nuclear staining in tumor cells .Immunohistochcmistry. Immunohistochemical staining of XPC and ERCC1 protein expression were performed on paraffin-embedded tissue sections. Sections with 4- micrometer thick were dewaxed and rehydrated using xylene and alcohol. Antigen retrieval was performed with a 2 min boiling of high temperature of 110 oC treatment in 10 mM citrate buffer, pH 6.00 and endogenous peroxidase was blocked by dipping the sections in 3 % aqueous H₂O₂ for 10 min . Following antigen retrieval, sections were incubated individually at room temperature for 60 min with a goat polyclonal antibody to the XPC protein ( 1 : 50 dilution ) or a mouse monoclonal antibody to the ERCC1 protein ( 1 : 50 dilution ), and lightly counterstained with haematoxylin . Immunostaining was performed using the avidin-biotin peroxidase complex technique, using diaminobenzidine as a chromogen . Sections were analyzed using light microscopy by the same gynecologic pathologist for the percentage of nuclei staining positive for XPC or ERCC 1 and the intensity of staining on a 0-3+ scale. The pathologist was blinded to clinical data. Any appreciable brown staining was considered positive. The score of each section was on the basis of the immunoreactive scoring (IRS) by Remmele and Stegnerde, and graded as follows: 0, almost no staining of tumor cell nuclei; 1 , barely detectable staining ; 2, easily seen fine gradules were present diffusely throughout the nucleus ; 3 , staining was strong Calculate the percentage of positive cells occupy in 1000 cells of ten high power field, then score calculation as follows: 0, 0%; 1 , 1 — 10%; 2, 11—50%; 3 , >50% then cross product 0, -; 1 2, +; 3 5, + +; 6 9, +++ . According to immunoreactive scoring by Remmele and Stegnerde, the staining results are defined as "-"(negative), "+"(weak positive), "++" (positive), and "+++" (strong positive). The IRS (immunoreactive scoring) was used in this study.Statistical analysis . Results were expressed as mean ± SD and statistical analysis is processed in SPSS 11.5. Paired samples test, t test, Row Column Chi-square test were used to perform two or more groups difference analysis respectively. Spearman correlation analysis was used to test two groups correlations. The statistical analysis tests were two- sided, P < 0 . 05 is regarded as significant difference.III. RESULTSXPC proteins expressed in the locally advanced cervical cancer cell nucleus. XPC proteins expression in the resistance for platinum-based chemotherapy of locally advanced cervical cancer were higher than those seen in the sensitivity for platinum-based chemotherapy of locally advanced cervical cancer, with X² equal to 8.741 and P equal to 0.015, r_s equal to -0.277 and P equal to 0.015, regarded as significant difference. ERCC1 proteins overexpressed in the locally advanced cervical cancer cell nucleus. ERCC1 proteins expression in the resistance for platinum-based chemotherapy of locally advanced cervical cancer were higher than those seen in the sensitivity for platinum-based chemotherapy of locally advanced cervical cancer, with X² equal to 12.616 and P equal to 0.006, r_s equal to -0.329 and P equal to 0.003, regarded as significant difference. However , their mean ages were not significantly different. XPC and ERCC1 proteins pression in the advanced cervix cancer among the different Clinical and pathology parameter were not significantly difference.Coordinate expression of XPC and ERCC1 in the locally advanced cervical cancer. This relationship approximated a straight line with a Kappa of 0.368 and P=0.000. This suggested coordinate expression of these two genes.IV. CONCLUSIONS1. The effective rate of neoadjuvant chemotherapy in locally advanced cervix cancer was 77.9%, and that indicated the neoadjuvant chemotherapy had clinical application value. BIP or PVB were the program of neoadjuvant chemotherapy, and their curative effect is equation.2. Expression of XPC and ERCC1 proteins in locally advanced cervix cancer of resistance to platinum- based neoadjuvant chemotherapy were higher than in sensitivity.