Chemosensitivity and p53–Bax pathway-mediated apoptosis in patients with uterine cervical cancer

H. Sultana1, J. Kigawa1,+, Y. Kanamori1, H. Itamochi1, T. Oishi1, S. Sato1, S. Kamazawa1, M. Ohwada2, M. Suzuki2 and N. Terakawa1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives:

To determine whether and how apoptosis through the p53–Bax 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 p53–Bax pathway.

Key words: apoptosis, Bax, cervical carcinoma, chemotherapy, p53, uterus


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Platinum-based chemotherapy has recently proved to be an effective therapy for cervical cancer [1, 2]. Several studies have shown that chemoradiation improved survival rate in patients with cervical cancer [3, 4]. Previously published work also suggests that intra-arterial chemotherapy may improve the prognosis of patients with advanced cervical cancer [5, 6], but is limited by chemoresistance. Many mechanisms have been postulated to explain chemoresistance, including decreased drug accumulation inside tumor cells, increased cellular detoxification, and increased DNA repair activity [79]. Recent studies indicate that apoptosis also contributes to chemosensitivity [10, 11].

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 p53–Bax pathway affects sensitivity to chemotherapy in cervical cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 32 patients with cervical squamous cell carcinoma, who received primary cisplatin-based neoadjuvant chemotherapy followed by radical hysterectomy between 1990 and 2000 at Tottori University Hospital and Jichi Medical School Hospital, were entered in the present study. According to the International Federation of Gynecology and Obstetrics (FIGO) staging system, six cases were stage IB, 18 cases were stage II (three stage IIA and 15 stage IIB ), and eight cases were stage IIIA. The mean age for patients was 49.2 years (range 24–66 years). Informed consent was obtained from all patients.

According to Minagawa’s protocol [6], all patients received bleomycin 3.5 mg/m2 i.v. on days 1–5; 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 Tris–HCl, 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 Tris–HCl (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 Kaplan–Meier 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 30 patients, 18 responded to chemotherapy (PR) and 12 did not (NC). Responders were significantly younger than non-responders (P = 0.012). There were no differences in histological grade and FIGO stage between the two groups (Table 1). The 5-year survival rate for responders was significantly better than for non-responders (92.9% versus 46.9%, P <0.001).


View this table:
[in this window]
[in a new window]
 
Table 1. Patient characteristics
 
Figure 1 shows a representative case of TUNEL and immunohistochemical stainings for Ki-67, Bax and Bcl-2 proteins. The AI did not differ between responders and non-responders before chemotherapy. After chemotherapy, the AI was significantly increased in both groups (P = 0.03; Table 2), and was significantly higher for responders than for non-responders (P = 0.018). Before chemotherapy, the Ki-67 LI in the tumors of responders was significantly higher compared with non-responders (P <0.001). The Ki-67 LI for responders was significantly decreased after chemotherapy (P <0.01), but that for non-responders was not. The receiver operating characteristic curve demonstrated that the cut-off value of Ki-67 LI before chemotherapy was 33.0% [21]. The response rate for patients with tumors >33.0% LI was significantly higher than for those with tumors <33.0% LI (94.1% versus 15.4%). Patients with tumors >33.0% LI showed a better survival rate (Figure 2).



View larger version (202K):
[in this window]
[in a new window]
 
Figure 1. TUNEL and immunohistochemical stainings in a patient after chemotherapy. (A) TUNEL, (B) Ki-67, (C) Bax (positive), and (D) Bcl-2 (positive).

 

View this table:
[in this window]
[in a new window]
 
Table 2. Apoptotic index and Ki-67 labeling index before and after chemotherapy
 


View larger version (15K):
[in this window]
[in a new window]
 
Figure 2. Estimated survival rate. With the cut-off value of Ki-67 LI before chemotherapy set at 33.0%, patients with a >33.0% LI had a better survival rate.

 
The incidence of p53 expression did not differ between responders and non-responders either before or after chemotherapy. Neither Bax nor Bcl-2 expression differed between responders and non-responders before chemotherapy. After, but not before, chemotherapy, the expression of Bax protein was observed more frequently and Bcl-2 protein expression was observed less frequently in responders, compared with non-responders (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. The expression of Bax and Bcl-2 before and after chemotherapy
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several authors examined the prognostic value of tumor proliferation and apoptosis in patients with carcinoma of the uterine cervix, but the results have been controversial [2227]. In patients with cervical cancer undergoing radiotherapy, the Ki-67 LI was a significant factor for disease-free survival, but the AI was not [22]. One author showed that the Ki-67 LI was not related to outcome of radiotherapy; however, a high AI proved to be an indicator for poor outcome [23, 24]. On the other hand, in patients treated with primary surgery, neither the AI nor the Ki-67 LI had prognostic significance [24]. The differential prognostic relevance of the AI for cervical cancer treated with radiotherapy or surgery may be explained by the evidence that tumors with a high AI are hypoxic. Hypoxia is known to be associated with radiation ineffectiveness [28]. In the present study with chemotherapy, the AI did not differ between responders and non-responders before treatment. In contrast, before chemotherapy, the Ki-67 LI for responders was significantly higher than for non-responders. It is known that rapidly proliferating cells are more sensitive to cytotoxic agents than slowly proliferating cells [29, 30]. In another study, intracellular drug accumulation decreased in resting cells [31]. Accordingly, the Ki-67 LI may be a parameter for chemo-response and prognosis in cervical cancer.

The aim of the present study was to determine whether and how apoptosis through the p53–Bax 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 p53–Bax 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 p53–Bax pathway is associated with response to cisplatin-based chemotherapy in cervical cancer.


    Footnotes
 
+ Correspondence to: Dr J. Kigawa, Department of Obstetrics and Gynecology, Tottori University School of Medicine, 36-1 Nisimachi Yonago, 6838504 Japan. Tel: +81-0859-34-8127; Fax: +81-0859-34-8089; E-mail: kigawa{at}grape.med.tottori-u.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. . Panici PB, Greggi S, Scambia G et al. High-dose cisplatin and bleomycin neoadjuvant chemotherapy plus radical surgery in locally advanced cervical carcinoma: preliminary report. Gynecol Oncol 1991; 41: 212–216.[CrossRef][ISI][Medline]

2. . Sardi J, Sananes C, Giaroli A et al. Neoadjuvant chemotherapy in locally advanced carcinoma of the cervix uteri. Gynecol Oncol 1990; 38: 486–493.[CrossRef][ISI][Medline]

3. . Keys HM, Bundy BN, Stehman FB et al. Cisplatin, radiation, and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage IB cervical carcinoma. N Engl J Med 1999; 340: 1154–1161.[Abstract/Free Full Text]

4. . Thomas GM. Improved treatment for cervical cancer—concurrent chemotherapy and radiotherapy. N Engl J Med 1999; 340: 1198–1200.[Free Full Text]

5. . Kigawa J, Minagawa Y, Ishihara H et al. The role of neoadjuvant intra-arterial infusion chemotherapy with cisplatin and bleomycin for locally advanced cervical cancer. Am J Clin Oncol 1996; 19: 255–259.[CrossRef][ISI][Medline]

6. . Minagawa Y, Kigawa J, Irie T et al. Radical surgery following neoadjuvant chemotherapy for patients with stage IIIB cervical cancer. Ann Surg Oncol 1998; 5: 539–543.[Abstract]

7. . Andrews PA, Velury S, Mann SC, Howell SB. cis-Diaminedichloroplatinum (II) accumulation in sensitive and resistant human ovarian carcinoma cells. Cancer Res 1988; 48: 68–73.[Abstract]

8. . Lautier D, Canitrot Y, Deeley RG, Cole SP. Multidrug resistance mediated by the multidrug resistance protein (MRP) gene. Biochem Pharmacol 1996; 52: 967–977.[CrossRef][ISI][Medline]

9. . Kigawa J, Minagawa Y, Cheng X, Terakawa N. Gamma-glutamyl cysteine synthetase up-regulates glutathione and multidrug resistance-associated protein in patients with chemoresistant epithelial ovarian cancer. Clin Cancer Res 1998; 4: 1737–1741.[Abstract]

10. . Righetti SC, Della Torre G, Pilotti S et al. A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Res 1996; 56: 689–693.[Abstract]

11. . Minagawa Y, Kigawa J, Itamochi H et al. Cisplatin-resistant HeLa cells are resistant to apoptosis via p53-dependent and -independent pathways. Jpn J Cancer Res 1999; 90: 1973–1979.

12. . Williams GT, Smith CA. Molecular regulation of apoptosis: genetic controls on cell death. Cell 1993; 74: 777–779.[ISI][Medline]

13. . Villa LL. Human papilloma viruses and cervical cancer. Adv Cancer Res 1997; 71: 321–341.[ISI][Medline]

14. . Scheffner M. Ubiquitin, E6-AP, and their role in p53 inactivation. Pharmacol Ther 1998; 78: 129–139.[CrossRef][ISI][Medline]

15. . Mochen C, Giardini R, Costa A, Silvestrini R. MIB-1 and S-phase cell fraction predict survival in non-Hodgkin’s lymphomas. Cell Prolif 1997; 30: 37–47.[CrossRef][ISI][Medline]

16. . Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995; 80: 293–299.[ISI][Medline]

17. . Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74: 609–619.[ISI][Medline]

18. . Boise LH, Gonzalez-Garcia M, Postema CE et al. Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993; 74: 597–608.[ISI][Medline]

19. . Seldinger SI. Catheter replacement of the needle in percutaneous arteriography. Acta Radiol 1953; 39: 368–376.[ISI]

20. . Yoshikawa H, Kawana T, Kitagawa K et al. Detection and typing of multiple genital human papillomaviruses by DNA amplification with consensus primers. Jpn J Cancer Res 1991; 82: 524–531.[ISI][Medline]

21. . Metz CE. Basic principles of ROC analysis. Semin Nucl Med 1978; 8: 486–498.

22. . Tsang RW, Wong CS, Fyles AW et al. Tumour proliferation and apoptosis in human uterine cervix carcinoma II: correlations with clinical outcome. Radiother Oncol 1999; 50: 93–101.[CrossRef][ISI][Medline]

23. . Levine EL, Renehan A, Gossiel R et al. Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma. Radiother Oncol 1995; 37: 1–9.[CrossRef][ISI]

24. . Levine EL, Davidson SE, Roberts SA et al. Apoptosis as predictor of response to radiotherapy in cervical carcinoma. Lancet 1994; 344: 472.[CrossRef][ISI][Medline]

25. . Hockel M, Schlenger K, Hockel S, Vaupel P. Hypoxic cervical cancers with low apoptotic index are highly aggressive. Cancer Res 1999; 59: 4525–4528.[Abstract/Free Full Text]

26. . Harima Y, Nagata K, Harima K et al. Bax and Bcl-2 protein expression following radiation therapy versus radiation plus thermoradiotherapy in stage IIIB cervical carcinoma. Cancer 2000; 88: 132–138.[ISI][Medline]

27. . Yuki H, Fujimura M, Yamakawa Y et al. Detection of apoptosis and expression of apoptosis-associated proteins as early predictors of prognosis after irradiation therapy in stage IIIb uterine cervical cancer. Jpn J Cancer Res 2000; 91: 127–134.[ISI][Medline]

28. . Gray LH, Conger AD, Ebert M et al. The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol 1953; 26: 638–648.[ISI]

29. . Bonetti A, Zaninelli M, Rodella S et al. Tumor proliferative activity and response to first-line chemotherapy in advanced breast carcinoma. Breast Cancer Res Treat 1996; 383: 289–297.

30. . O’Reilly SM, Camplejohn RS, Rubens RD, Richards MA. DNA flow cytometry and response to preoperative chemotherapy for primary breast cancer. Eur J Cancer 1992; 28: 681–683.[Medline]

31. . Dimanche-Boitrel MT, Pelletier H, Genne P et al. Confluence-dependent resistance in human colon cancer cells: role of reduced drug accumulation and low intrinsic chemosensitivity of resting cells. Int J Cancer 1992; 50: 677–682.[ISI][Medline]

32. . Shoji Y, Saegusa M, Takano Y et al. Correlation of apoptosis with tumour cell differentiation, progression, and HPV infection in cervical carcinoma. J Clin Pathol 1996; 49: 134–138.[Abstract]

33. . Kraiss S, Spiess S, Reihsaus E, Montenarh M. Correlation of metabolic stability and altered quanternacy structure of oncoprotein p53 with cell transformation. Exp Cell Res 1991; 192: 157–164.[ISI][Medline]

34. . Sato S, Kigawa J, Minagawa Y et al. Chemosensitivity and p53-dependent apoptosis in epithelial ovarian carcinoma. Cancer 1999; 86: 1307–1313.[ISI][Medline]

35. . Kokawa K, Shikone T, Otani T, Nakano R. Apoptosis and the expression of Bax and Bcl-2 in squamous cell carcinoma and adenocarcinoma of the uterine cervix. Cancer 1999; 85: 1799–1809.[ISI][Medline]

36. . Tjalma W, De Cuyper E, Weyler J et al. Expression of bcl-2 in invasive and in situ carcinoma of the uterine cervix. Am J Obstet Gynecol 1998; 178: 113–117.[ISI][Medline]

37. . Zhan Q, Bieszczad CK, Bae I et al. Induction of BCL-2 family member MCL1 as an early response to DNA damage. Oncogene 1997; 14: 1031–1039.[CrossRef][ISI][Medline]