1 Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago; 2 Department of Obstetrics and Gynecology, Jichi Medical School Hospital, Ustunomiya, Japan
Received 21 May 2002; revised 26 July 2002; accepted 17 October 2002
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Abstract |
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To determine whether and how apoptosis through the p53Bax pathway affects sensitivity to chemotherapy in cervical cancer.
Materials and methods:
Thirty patients with cervical squamous cell carcinoma, who had human papilloma virus (HPV) and underwent neoadjuvant chemotherapy, were entered in the present study. Tumor specimens were obtained before and after chemotherapy. HPV was detected by polymerase chain reaction. The expression of Ki-67, p53, Bax and Bcl-2 proteins was determined by immunohistochemical staining. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling method.
Results:
Of 30 patients, 18 responded to chemotherapy and 12 did not. The apoptotic index in tumors of responders was significantly higher than in non-responders after chemotherapy. The Ki-67 labeling index (LI) in responders was significantly higher than in non-responders before chemotherapy. Patients with tumors >33% of the LI, which was determined by a receiver operating characteristic curve, had a better survival rate. The incidence of p53 protein expression did not differ between responders and non-responders. After chemotherapy, the expression of Bax protein in responders was more frequent and Bcl-2 protein expression was less frequent than in non-responders.
Conclusions:
Chemosensitivity in cervical cancer may be associated with apoptosis via the p53Bax pathway.
Key words: apoptosis, Bax, cervical carcinoma, chemotherapy, p53, uterus
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Introduction |
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p53 is known to be a cell cycle checkpoint protein playing a regulatory role in the control of cell proliferation and apoptosis [12]. Although cervical squamous cell carcinoma commonly contains a wild-type p53 gene, it is highly correlated with human papilloma virus (HPV) infection. Because the viral oncoprotein E6 binds and inhibits the function of p53 protein, inhibition by HPV may be one cause of chemoresistance in cervical cancer [13, 14]. However, in cervical cancer the relationship between apoptosis through the p53 pathway and chemosensitivity is not clear.
Ki-67, a marker for cellular proliferation, has been applied to study the growth fraction and cell-kinetic activities [15]. Bax, which is regulated by the p53 gene, controls apoptosis, and Bcl-2 opposes Bax function [1618]. We conducted the present study to determine whether and how apoptosis through the p53Bax pathway affects sensitivity to chemotherapy in cervical cancer.
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Materials and methods |
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According to Minagawas protocol [6], all patients received bleomycin 3.5 mg/m2 i.v. on days 15; vincristin 0.7 mg/m2 and mitomycin C 10 mg i.v. on day 5; and 25 mg/m2 cisplatin infused via each internal iliac artery on day 5. Cisplatin was injected for 20 min into both of the internal iliac arteries according to the Seldinger [19] technique.
Twenty-nine patients underwent two cycles whilst the remaining three patients received three cycles. Three weeks after each course of chemotherapy, we evaluated therapeutic efficacy according to the following criteria using both computed tomography and magnetic resonance imaging for all patients. Complete response (CR) was defined as absence of disease; partial response (PR) was defined as a >50% reduction in all measurable lesions without the appearance of new lesions; no change (NC) was defined as a <50% decrease or a <25% increase in all measurable lesions without the appearance of new lesions. Progressive disease was defined as a >25% increase in the measurable disease at a known site or the appearance of new lesions. To obtain a specimen before chemotherapy, a biopsy was carried out under colposcopy. A surgical specimen was used after chemotherapy.
HPV detection
Human papilloma virus DNA was examined by the polymerase chain reaction (PCR). Genomic DNA was extracted from the paraffin-embedded tissue of the most severe lesion of the surgical specimen. In brief, tissue blocks were cut into 10 µm sections using a microtome. Five tissue sections were deparaffinized twice in xylene, hydrated in graded alcohols, and incubated in proteinase K buffer consisting of 10 mM TrisHCl, 10 mM EDTA, proteinase K 100 µg/ml and 0.5% sodium dodecyl sulfate at 37°C, overnight. The supernatant fluid was treated twice with phenol and once with chloroform. A precipitate of DNA was obtained by adding 3 M sodium acetate and 100% ethanol. The precipitate was washed in 70% ethanol and dissolved in distilled water. ß-globin primer was used as an internal control for the suitability of DNA in the samples. We used a pair of consensus primers with the ability to detect HPV types 6, 11, 16, 18, 31, 33, 42, 52 and 58 [20]. The DNA sequences of the consensus primer pairs were 5'-CGTAAACGTTTTCCCTATTTTTTT-3' and 5'-TACCCTAAATACTCTGTATTG-3'. Human papilloma virus DNA was amplified in 50 µl of a reaction mixture containing 0.5 µg of sample DNA, 50 mM potassium chloride, 10 mM TrisHCl (pH 8.8), 1.5 mM magnesium chloride, 0.1% Triton X-100, 200 µM of deoxyribonucleoside triphosphate, 0.5 µM of each primer, and 1 U of Taq DNA polymerase (Wako, Osaka, Japan). The sample was subjected to 35 cycles of amplification on a PCR processor (PC-700; Astec, Fukuoka, Japan). Each cycle consisted of DNA denaturing for 1.5 min at 95°C, annealing for 1.5 min at 48°C, and extension for 2 min at 72°C. An aliquot of 10 µl of the reaction mixture was electrophoresed on 4% agarose gel with ethidium bromide staining. As a result, HPV DNA was detected in 30 of 32 patients (93.7%). We examined 30 patients with HPV-positive tumor in a further study.
Immunohistochemistry
Immunohistochemical studies were performed to detect Ki-67, p53, Bax and Bcl-2 proteins. Formalin-fixed, paraffin-embedded tissue sections were mounted on silane-coated glass slides. Deparaffinized and rehydrated samples were heated in a microwave oven for 15 min at 94°C in citrate buffer solution. The slides were cooled and endogeneous peroxidase was blocked with 3% hydrogen peroxide (H2O2) in methanol for 20 min at room temperature, followed by rinsing in distilled water. To detect Ki-67, p53, Bax and Bcl-2 proteins, the sections were incubated overnight at 4°C with anti-Ki-67 monoclonal antibody, MIB-1 (1:50 dilution; Immunotech, Marseille, France), anti-p53 monoclonal antibody, DO-7 (1:50 dilution; DAKO HS, Glostrup, Denmark), anti-Bax polyclonal antibody, N-20 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-bcl-2 monoclonal antibody, Ab-3 (1:100 dilution; Calbiochem, San Diego, LA), respectively. The primary antibody was visualized using the Histofine Simple Stain PO kit (Nichirei, Tokyo, Japan) according to the instruction manual. For the negative controls, the primary antibodies were replaced with phosphate-buffered saline. The slides were counterstained with hematoxylin.
For Ki-67 staining slides, the labeled and unlabeled cells were counted in five high-power fields (x400). A total of 500 cells were counted in each specimen. The Ki-67 labeling index (LI) was determined using the following formula: LI (%) = 100 x labeled cells/total cells. For expression of p53, Bax and Bcl-2 proteins, a positive case was defined as staining of the tumor cells and a negative case was defined as no staining of any tumor cells.
Detection of apoptosis
Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method using the Apop Taq in situ detection kit (Oncor Inc., Gaithersburg, MD). Dewaxed and rehydrated specimens were incubated in proteinase K 40 µg/ml for 1 h at 37°C and were treated with 3% H2O2 in methanol for 30 min at room temperature. After adding equilibration buffer for 5 min at room temperature, terminal deoxynucleotidyl transferase (TdT) enzyme was pipetted onto the sections and incubated at 37°C for 2 h. The reaction was stopped by incubating the sections in stop buffer for 30 min at 37°C. Anti-digoxigenin peroxidase was added to the slides, followed by incubation for 30 min at 37°C. Slides were stained with diaminobenzine for 10 min and counterstained with hematoxylin. A total of 500 cells were counted in each specimen. The apoptotic index (AI) was defined as follows: AI (%) = 100 x apoptotic cells/total cells.
Statistical analysis
Patient survival distribution was calculated using the KaplanMeier method. The significance of the survival distribution in each group was tested by a log-rank test. Values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using the Stat View Version 5.0-J program (Hulinks Inc., Tokyo Japan). A value of P <0.05 was considered statistically significant.
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Results |
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Discussion |
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The aim of the present study was to determine whether and how apoptosis through the p53Bax pathway affects sensitivity to chemotherapy in cervical cancer. We examined cervical cancer patients with HPV-positive tumor. Because the present study had no control group, no real comment could be made. Human papilloma virus is detected in >90% of cervical cancers and E6 protein, encoded by HPV, inhibits the function of p53 protein. This suggests that HPV may be directly related to pathways regulating apoptosis [32].
We failed to find a correlation between p53 protein expression by immunohistochemical staining and chemotherapy-induced apoptosis. Previously, overexpression of the p53 protein was believed to be caused by an underlying abnormal p53 gene, leading to the expression of an abnormal and stabilized protein [33]. In the literature, the expression of p53 protein did not affect apoptosis [34]. These findings suggest that immunohistochemical staining is not an appropriate technique to assess p53 function. In addition, p53 expression may be suppressed by HPV in cervical cancer.
To determine p53Bax pathway-mediated apoptosis, we examined the expression of Bax and Bcl-2 proteins. p53 is a direct transcriptional activator of the Bax gene, but Bcl-2 blocks both p53-dependent and p53-independent pathways. In the literature, the proportion of Bax-positive cells was significantly higher in responders with advanced cervical cancer treated by cisplatin-based chemotherapy, but no significant difference was found in Bcl-2 protein expression between responders and non-responders [35]. In contrast, Tjalma et al. showed a strong relationship between Bcl-2 protein expression and overall survival in cervical cancer [36]. However, it is noteworthy that these findings were seen only before treatment. Interestingly, the present study showed that the expression of Bax protein was observed more frequently and Bcl-2 protein expression less frequently in responders after chemotherapy. Harima et al., investigating the expression of Bax and Bcl-2 proteins before and during the course of radiation therapy in cervical cancer, found that better tumor control was accompanied by increased Bax protein expression [26]. An in vitro study showed that expression of p53 and Bax proteins increased and expression of Bcl-2 protein decreased after exposure to DNA-damaging agents [37].
In conclusion, the present study suggests that apoptosis via the p53Bax pathway is associated with response to cisplatin-based chemotherapy in cervical cancer.
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Footnotes |
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References |
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