Cervical cancer (CXCA) is the second most deadly cancer in women worldwide. The most important risk factor in the development of CXCA is persistent infection with a high-risk human papillomavirus (HPV). HPV types 16 and 18 are generally acknowledged to cause about 70% of cancer cases. The standard regimen for treatment of advanced-stage CXCA is radiotherapy. However, individuals show different patterns of radiotherapy response, in which one-third of cured patients become resistant during or after complete course of the treatment. Concurrent radiotherapy and chemotherapy improve patient survival by 30–50%, and 12% of CXCA patients have persistent or recurrent disease after treatment. Although the combination of radiotherapy and cisplatin-based chemotherapy is better than radiotherapy alone, 5-year overall survival remains only 52%, suggesting that intrinsic and acquired tumor resistance impede the improvement of standard treatment.

Mechanism of radiotherapy and chemotherapy is to induce DNA damage and cell suicide through apoptotic signaling. Apoptosis is triggered by 2 major pathways designated as the intrinsic and extrinsic pathways. Tumor necrosis factor receptor superfamily member 6 (FAS) and tumor necrosis factor-related apoptosis-inducing ligand receptor-1 (TRAILR1) are death receptors belonging to the tumor necrosis factor (TNF) family, which induces apoptosis via the extrinsic pathway. Binding of these receptors with its ligand activates initiator caspase 8 (CASP8), in which the downstream effector caspases are subsequently processed. The intrinsic pathway is activated by intracellular signals, ie, DNA damage, which activates p53, leading to transcriptional induction of the p53-up-regulated modulator of apoptosis and phorbol-12-myristate-13-acetate-induced protein 1. These proteins then drive proapoptotic members, such as Bcl-2 associated X protein (BAX) and Bcl-2 antagonist killer 1 which engage the mitochondrial membrane to release cytochrome c. Binding of cytochrome c to the caspase activator, APAF1, results in binding to and activation of pro-caspase 9. In healthy cells, antiapoptotic family members, such as B-cell lymphoma 2, B-cell lymphoma-extra large, or myeloid cell leukemia 1, bind to BAX and Bcl-2 antagonist killer 1 to prevent their activation. The 2 apoptotic pathways can be linked via CASP8-mediated activation of BCL-2 homology 3 (BH3) interacting domain death agonist. Cleavage of BCL-2 homology 3 (BH3) interacting domain death agonist by CASP8 results in small molecules that cooperate with BAX to form openings in the outer mitochondrial membrane, leading to the release of cytochrome c to the cytoplasm. Survivin is the smallest of the inhibitors of apoptosis proteins, containing a single baculoviral inhibitor of apoptosis protein repeat domain. Previous studies showed markedly increased expression of Survivin messenger RNA and protein levels in many cancers, including CXCA. Interestingly, Survivin is rarely expressed in normal cells, suggesting its importance as a targeted therapy. Death-associated protein kinase (DAPK) is a proapoptotic protein involved in apoptosis initiated by TNF alpha, interferon gamma, and FAS. DAPK is a Ca ²⁺ calmodulin-regulated serine/threonine kinase known to play a role in apoptosis downstream of FAS-associated via death domain/mediator of receptor-induced toxicity and CASP8.

Genetic and/or epigenetic defects of apoptotic signaling genes can cause many cancers in human beings and even acquired cancer resistance to therapy. Epigenetic silencing is a reversible mechanism; silence genes in apoptotic pathway can be restored with demethylating agents such as 5′-aza-2-deoxycytidine, leading to an efficacy of cancer treatment. To date the molecular events involved in acquired therapeutic resistance of CXCA are not well understood. We addressed whether aberrant DNA methylation of genes involved in apoptotic signaling could cause acquired CXCA resistance to therapy. Methylation profiling of apoptotic genes was investigated in CXCA patients, including response and nonresponse to therapy. This finding will provide better understanding of molecular mechanisms underlying the apoptotic process, which will benefit CXCA patients who acquire therapeutic resistance under standard regimen.

Materials and Methods

Cervical tissue samples

Cervical tissues were collected from patients who attended Tumor and Colposcopic Clinic, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. This study was approved by the university ethical committee. Each patient was provided with research formation and informed consent was obtained. All cases were histologically examined and confirmed by pathologists. CXCA was staged according to the International Federation of Gynecology and Obstetrics staging system. There were 66 cases of squamous cell carcinoma (SCC) and 19 of adenocarcinoma (AC), for which age, histologic type, grading, staging, size of lesion, type of treatment, and response to therapy are summarized in Figure 1 . Twenty-eight normal cervical tissues obtained from surgical resections of myoma patients or colposcopic biopsies were included in this study. After specimen collection, all patients underwent treatment under standard regimen. Pelvic examination, Pap smear, and colposcopic examination were monitored in follow-up cases for 3 months to assess response to primary therapy. Patients whose lesions existed during or after treatment were defined as therapeutic nonresponse, while patients who showed no lesion or recurrence during or after treatment were defined as therapeutic response. Under this criterion, we were able to divide SCC patients into 2 groups, as follows: 34 nonresponse cases and 32 response cases.

Clinical data of 85 cervical cancer patients

AC , adenocarcinoma; CCRT , concurrent chemoradiotherapy; G , grade; LCNK , large cell nonkeratinizing; RHND , radical hysterectomy with pelvic lymph node dissection; SCC , squamous cell carcinoma; TAH , transabdominal hysterectomy; XRT , radiotherapy.

Chaopatchayakul. Aberrant DNA methylation of apoptotic signaling genes in patients with cervical carcinoma. Am J Obstet Gynecol 2010.

Analysis of DNA methylation

Sequences of methylated and unmethylated primers of APAF1, CASP8, DAPK, FAS, Survivin, and TRAILR1 were derived from previous studies, as shown in Table 1 . After bisulfite modification, DNA methylation of CpG islands of these genes was performed using methylation-specific polymerase chain reaction (MSP). In brief, each 25-μL reaction contained 1X PCR buffer (16.6 mmol/L ammonium sulfate, 67 mmol/L Tris pH 8.8), 200 μmol/L each of deoxynucleoside triphosphates, varied concentration of MgCl ₂ , 10 mmol/L 2-mercaptoethanol, 5 pmol of each primer, and 100 ng of bisulfite-modified DNA. Reactions were hot-started at 95°C for 5 minutes before adding 1.5 U of Taq polymerase. Amplifications were taken for 35 cycles (30 seconds at 95°C, 30 seconds at annealing temperature, and 30 seconds at 72°C), followed by final extension at 72°C for 10 minutes. Then, 10 μL of each PCR product was loaded onto 2% agarose gel, stained with ethidium bromide, and visualized under UV illumination.

TABLE 1

Sequences of primers for methylation-specific polymerase chain reaction

Gene		Forward primer (5′→3′)	Reverse primer (5′→3′)	Size
APAF1	M a	TTT CGG GTA AAA GGG ATA GAA TTA GA	TAT AAC GCC CTT CCC CCG ACG ACG	140
	U b	GAG GTT TAG TTA TGT TTT GTT TGT GG	CCA CTC ACT ACC TCT TCT TCT CC	215
CASP8	M a	TAG GGG ATT CGG ACA TTG CGA	CGT ATA TCT ACA TTC GAA ACG A	320
	U c	TAG GGG ATT TGG AGA TTG TGA	CCA TAT ATC TAC ATT CAA AAC AA	321
DAPK	M a	GGA TAG TCG GAT CGA GTT AAC GTC	CCC TCC CAA ACG CCG A	98
	U a	GGA GGA TAG TTG GAT TGA GTT AAT GTT	CAA ATC CCT CCC AAA CAC CAA	106
FAS	M a	GAA AGG GTA GGA GGT CGG TTT TCG AG	CAC TCT TAC GCG AAA TCA AAA ACG AAC TCA	269
	U c	AGA AAG GGT AGG AGG TTG	CAT CAC TCT TAC ACA AAA TC	270
Survivin	M a	TCT GTA TAT TTC GCG TCG TTT C	AAA CCG AAC AAT CTC ACC CGC T	280
	U d	GGT GGG AGG ATT ATA ATT TTT G	CCA CCA CCA CCA CCT CTA C	163
TRAILR1	M e	CCT TTT CGA AAC ACC TAC GAC	AAA GGT TGA GGT TAA TTT TCG ATC	184
	U e	CCC CTT TTC AAA ACA CCT ACA	GAA AGG TTG AGG TTA ATT TTT GAT T	187

 

APAF1 , apoptotic protease activating factor 1; CASP8 , caspase 8; DAPK , death-associated protein kinase; FAS , tumor necrosis factor receptor superfamily member 6; M , methylated primers; TRAILR1 , tumor necrosis factor-related apoptosis-inducing ligand receptor-1; U , unmethylated primers.

Primer sequences for methylation-specific polymerase chain reaction amplification were obtained from:

Chaopatchayakul. Aberrant DNA methylation of apoptotic signaling genes in patients with cervical carcinoma. Am J Obstet Gynecol 2010.

a DNA methylation analysis PCR primer database;

b Yu et al;

c Hopkins-Donaldson et al;

d Yu et al; and

e Bae et al.

Human placental DNA treated in vitro with SssI methyltransferase (New England Biolabs, Ipswich, MA) served as a positive control for methylated DNA, while untreated human placental DNA was used as a control for unmethylated allele.

Statistical analysis

DNA methylation status was analyzed for association with clinicopathologic data of CXCA patients and therapeutic response using χ ² test, Fisher’s exact test, and the odds ratio (OR). The association between genes involved in apoptotic signaling was calculated using Fisher’s exact test. P < .05 was considered statistically significant.