Introduction

The gene product of ABCB1 (ATP-binding cassette, sub-family B, member 1, also named MDR1, multidrug resistance gene 1), P-glycoprotein, (P-gp) is an efflux pump protein of 170 kDa. It belongs to ABC membrane transporters superfamily of [1]. Overexpression of P-gp in tumour cells leads to multidrug resistance against antineoplastic agents [1–5]. P-gp is expressed in the apical membranes of excretory tissues, such as liver, kidney and intestine. This contributes to the elimination of toxic exogenous substances or metabolites and drugs into bile and urine or limits drug absorption from the gastrointestinal tract [6, 7]. P-gp was implicated in the system regulating cell differentiation, proliferation and survival [4].

Relation between the presence of different single-nucleotide polymorphisms (SNPs) and the risk of colon cancer development is being investigated in over 35 different genes [8]. One of them is ABCB1 which is already known to accompany colon cancer development in cases with high microsatellite instability characteristics (MSI-H) [9].

Multiple mutations in the ABCB1 gene have been identified. Analysis of all 28 exons of the ABCB1 gene demonstrated 40 SNPs, including promoter and the intron–exon region [10]. The most frequent SNP ABCB1 2677G>T/A in exon 21 (refSNP ID: rs2032582), leading to amino acid exchange from Ala to Ser or Thr. The silent mutation in exon 26 ABCB1 3435C>T (refSNP ID: rs1045642) is associated with altered protein function [11]. Subjects with the TT3435 genotype had higher plasma concentration of digoxin, which is P-gp substrate, compared to subjects with the wild-type genotype [12]. Of all SNPs, the ABCB1 3435C>T is best characterised for its association with expression and function in the tissues [13, 14]. The third common polymorphism of ABCB1 gene is mutation in exon 12 ABCB1 1236C>T (refSNP ID: rs1128503). This SNP similarly to ABCB1 3435C>T mutation is silent. Relation between SNPs of ABCB1 gene is not clear. Maybe these three polymorphisms are closely related to disequilibrium (LD), but unknown genetic variant is located on the same LD block or haplotype [10, 15]. The study performed by Kim has pointed that there is a correlation between bioavailability of drugs and mutations in positions of ABCB1 3435C>T, ABCB1 2677G>T/A and ABCB1 1236C>T [16].

P-gp plays an important role in the detoxification systems of normal tissues. Several studies have shown that polymorphisms of ABCB1 gene can influence susceptibility to cancer. Siegsmund and Jamroziak suggested that ABCB1 3435C>T SNP combined with susceptibility to renal epithelial tumours and acute lymphoblastic leukaemia [17, 18]. Such SNP was also reported in colorectal patients [9, 19–22].

Colon and rectum cancer are two of the most frequent neoplasms and are the main reasons of high mortality ratio among all suffering from different types of cancer [23]. Genetic factors together with influence of xenobiotics which are present in a diet, cigarette smoke, drugs, bacterial toxins and other biological and chemical factors may increase the risk of colon and rectum cancer development. A significant relation between the risk of colon cancer development determined by genetic variants of ABCB1 gene and environmental factors such as cigarette smoking was indicated by Osswald [22].

The role of P-gp in carcinogenesis was described in animal models of colon [24], breast [25] and liver [26] cancers. P-gp overexpression was connected with apoptosis inhibition and increasing possibility of neoplasm transformation in an mdr1 mouse model [24]. The implications of genetically determined differences in P-gp function for drug disposition, therapeutic outcome and risk for development of certain diseases are being intensively studied.

The potential mechanism of the ABCB1 polymorphisms affecting susceptibility to colorectal cancer has not been satisfactorily described yet. The aim of this study was to determine the significance of three SNPs of ABCB1, namely ABCB1 1236C>T, ABCB1 2677G>T/A and ABCB1 3435C>T in the development of colorectal cancer. Also, haplotype structure analysis of this gene in colorectal cancer group comparison and frequencies of haplotypes between the studied group was carried out. In this study, we investigated the impact of ABCB1 gene polymorphisms on surviving patients after adjuvant combined chemotherapy with 5-fluorouracil/leucovorin (5-FU/LV).

Materials and methods

Tissues

Tissue samples were obtained from 95 colorectal carcinomas patients in 43 cases with III or IV pTNM (Table 1) operated on in the Oncological Center of Lodz, Poland. Colorectal cancer was diagnosed by histopathological examination using established clinical criteria at the Department of Pathology, Medical University of Lodz, Poland. Primary colorectal carcinoma and normal colorectal mucosa taken from a site distant several centimetres from the tumour were used in the study. Samples were frozen in liquid nitrogen immediately after surgical resection and stored at −80°C until processed. The control group constituted of 95 unrelated peripheral blood samples (46 men and 49 women, ratio 1:0.94, the controls were matched to patients by gender) from Lodz region in central Poland. All subjects were of Slavic origin. All experiments were carried out with the local ethical committee approval.

Table 1 Detailed information for the colorectal cancer group
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DNA isolation

The studying group: DNA was isolated according to protocol “Genomic DNA Prep Plus” (A&A Biotechnology, Gdynia, Poland) from the frozen tissue slides of colon cancers.

“Blood Mini Kit” (A&A Biotechnology) was used for DNA isolation from blood. The purity and concentration of DNA samples were estimated spectrophotometrically. The samples were stored at −20°C until analysis.

Polymerase chain reaction

Polymerase chain reaction (PCR) reaction was conducted according to protocol “AccuTaq™ LA DNA Polymerase Kit” (Sigma Aldrich, Germany). The reaction mixture for PCR amplification consisted of 50 ng of DNA template, 0.5 μM of each primer, 10× AccuTaq Buffer, 0.5 U of AccuTaq LA DNA Polymerase Mix, 0.2 mM each deoxyribonucleotide triphosphate (dNTP) and water to a final volume of 20 μl. Negative control was included in each experiment (sample without DNA template). The primers design were based on published sequences for genotyping procedure of ABCB1 2677G>T/A and ABCB1 3435C>T polymorphisms using genomic DNA [11, 27]. The primer sequences for genotyping procedure of ABCB1 1236C>T was planned by using software Primer3: WWW primer tool (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and GeneBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html). Selected primer sequences and conditions of PCR reaction are summarised in Table 2.

Table 2 Primer sequences, number of cycles and PCR conditions
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Restriction fragment length polymorphism

After checking PCR product in 2% agarose gel (Fig. 1a), DNA fragments were cut by restriction enzyme MboI (Fermentas, Vilnius, Lithuania) for ABCB1 3435C>T mutation during 16 h at 37°C. DNA fragments generated after digestion were separated on 2% agarose gel and visualised with ethidium bromide. Electrophoretic pattern showed two bands (130 and 76 bp) for homozygous wild-type C allele, one band (206 bp) for homozygous mutant T allele and three bands (206, 130 and 76 bp) for heterozygous CT genotype (Fig. 1b).

Fig. 1
figure 1

Electrophoretic pattern of PCR products containing ABCB1 1236C>T. ABCB1 2677G>T/A and ABCB1 3435C>T polymorphic sits (a) and ABCB1 3435C>T polymorphism evaluated by PCR-RFLP (b)

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Sequencing analysis

Genotyping of ABCB1 1236C>T and ABCB1 2677G>T/A was performed by automated sequencing. Sequencing PCR reaction was performed according to “SequiTherm EXCEL™ II DNA Sequencing Kit-LC” protocol (Epicentre Technologies, Madison, USA).

The reaction mixture for sequencing-PCR amplification consisted of DNA fragments generated after sequencing, 0.2 μM of primer, 3.5× Sequencing Buffer, 5 U of SequiTherm EXCEL™ II DNA Polymerase, 0.2 mM each dNTP/ddNTP and distilled water to a final volume of 11 μl. Sequencing primers were labelled by IRD 700 or IRD 800 on 5′ end. Stop/Loading Buffer was used after sequencing-PCR amplification. The primers sequences for automated sequencing genotyping procedure of ABCB1 1236C>T and ABCB1 2677G>T/A was planned by using software Primer3: WWW primer tool (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) and GeneBank database (http://www.ncbi.nlm.nih.gov/Genbank/index.html; Table 2). SeqPCR products after denaturation were separated in polyacrylamide gels. Sequencing was performed with the use of automated sequencer LI-COR®4000. Example of ABCB1 1236C>T sequencing analysis are summarised in Fig. 2.

Fig. 2
figure 2

Sequencing analysis of ABCB1 1236C>T polymorphism. Wild-type genotype CC1236 (a). Heterozygous CT1236 (b) and homozygous for the mutation TT1236 (c)

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Statistical analysis

Statistical significance of the observed genotype frequencies compared to genotype frequencies expected according to Hardy–Weinberg rule was evaluated. Data were analysed using STATISTICA version 8.0. (data analysis software system, StatSoft). The differences in allele or genotype frequencies between studying group (colorectal cancer samples) and control group (blood samples) were calculated using chi-square test. Survival probability of colorectal cancer patients according to genotypes was estimated on the basis of the Kaplan–Meier method and compared between groups by the log-rank test. Haplotypes were statistically inferred using the PHASE v. 2.1 software. Program PHASE implements a Bayesian statistical method for reconstructing haplotypes from population genotype data [28]. Linkage disequilibrium (LD) was estimated according to EMLD software [29] with the Expectation-Maximization Algorithm.

For all analyses, p values at the level of 0.05 were considered as statistically significant.

Result

Analysing for three SNPs of ABCB1 by PCR-restriction fragment length polymorphism (RFLP) method or automated sequencing was successful in all specimens (Figs. 1 and 2).

In our recent study for ABCB1 gene genotyping, we have used DNA isolated from frozen tissue (the colorectal cancer group, Table 1), and for the control group, DNA was isolated from leukocytes [18]. Recent studies have proven that genotyping based on DNA isolated from frozen tissues is equal to DNA isolated from blood [30].

The observed genotype frequency distribution did not show significant deviation from Hardy–Weinberg equilibrium (Table 3). The comparison of results from genotyping studies in colorectal cancer and control group is presented in Table 4. Higher frequency of T1236 allele (genotype CT1236 or TT1236 vs CC1236) in the control than colorectal cancer group was found (70.5% and 56.8%, respectively; p = 0.0499, OR = 0.55, measure of correlation: Fi–Yule coefficient = 0.14). Moreover, the ABCB1 1236C>T wild-type genotype (CC1236) was observed in 43.2% of patients, whereas 36.8% was heterozygous (CT1236) and 20.0% patients were homozygous for the mutation (TT1236). In healthy population, the frequencies of ABCB1 1236C>T genotypes were different compared with colorectal cancer group: 29.5% (CC1236), 56.8% (CT1236) and 13.7% (TT1236) for controls (p = 0.00043). In addition, significant differences in ABCB1 2677G>T/A genotype distribution were found (p = 0.04). The GT2677 was detected in 41.1% subjects of colorectal cancer and 52.6% subjects of healthy population. The frequencies of TT2677 and GG2677 were higher in colorectal cancer (22.1% and 36.8%, respectively) in comparison with control group (14.8% and 32.6%). Statistical analysis did not reveal any differences in ABCB1 3435C>T genotype/allele frequencies between investigated populations.

Table 3 Statistical significance of observed genotype frequencies in investigated populations
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Table 4 ABCB1 genotype and allele frequencies in investigated groups
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Figure 3 shows pair-wise linkage disequilibrium patterns for the ABCB1 gene. We detected a strong LD between ABCB1 1236C>T and ABCB1 2677G>T/A SNPs (D′ = 0.621, r 2 = 0.318). We also observed remains pair-wise LD between ABCB1 1236C>T and ABCB1 3435C>T (D′ = 0.394, r 2 = 0.104) as well as ABCB1 2677G>T/A and ABCB1 3435C>T SNPs (D′ = 0.384, r 2 = 0.120). All SNPs were located in one haplotype block.

Fig. 3
figure 3

Pair-wise linkage disequilibrium of ABCB1 gene (D′ Lewontin coefficient)

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Each of the eight possible haplotypes was noted with frequencies above 3%. However, the frequencies of T1236–T2677–T3435 haplotype were higher in colorectal cancer in comparison with control group (24.0% vs 3.0%, OR = 9.61). There were significant differences in haplotype distributions between colorectal cancer patients and healthy population (p = 0.03; Table 5).

Table 5 Frequencies of haplotypes of ABCB1 gene in investigated populations (combinations of the three SNPs of ABCB1 gene)
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We estimated influence of ABCB1 genotypes on outcome of colorectal cancer therapy.

Analysis included 32 patients subjected to adjuvant combine chemotherapy with 5-FU/LV. Cases with the presence of metastases were excluded from survival analysis despite the fact that they were also treated with adjuvant chemotherapy.

No significant prognostic influence of ABCB1 1236C>T and ABCB1 2677G>T/A SNPs in terms of surviving patients' treatment with adjuvant chemotherapy was detected. However, significant differences in survival probability of colorectal cancer patients according to allele of ABCB1 3435C>T were observed (Fig. 4). Survival probability of patients with wild-type C3435 allele was higher than among patients without this allele (p = 0.04572; Fig. 4b). Higher survival probability of patients without mutant T3435 allele than patients with this allele was detected (p = 0.08605; Fig. 4a).

Fig. 4
figure 4

Adjusted survival probability of colorectal cancer patients treatment chemotherapy according to ABCB1 alleles: T3435 (genotype CT3435 or TT3435 vs CC3435) (a) and C3435 (genotype CC3435 or CT3435 vs TT3435) (b). *log-rang test

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Discussion

It is well documented that P-gp plays an important role in the maintenance of intestinal homeostasis. The interaction between toxic agents (for example bacterial toxin or carcinogens) contained in food and intestinal epithelium can play a significant role in susceptibility to the development of ulcerative colitis and colon cancerogenesis. It is well established that ulcerative colitis predisposes to tumorgenesis. P-gp as a plasma membrane pump may be involved in the clearance of carcinogens within intestinal epithelium. Xenobiotic-dependent modification of cellular defence against dietary carcinogens via ABCB1 expression might differ in ABCB1 genotypes [12, 22]. SNPs in transporters for xenobiotic agents may contribute to the susceptibility and progression of colorectal cancer [31].

Genotype distribution showed genetic stability and no distortion from Hardy–Weinberg rule, which suggests representative sampling for investigated populations. Furthermore, the allele/genotype frequency of the polymorphisms of ABCB1 gene in healthy population was in agreement to other Caucasian populations from Europe [11, 12, http://www.ncbi.nlm.nih.gov/SNP/]. Widely reported ethnical differences in ABCB1 allele/genotype frequencies were seen especially in African, Asian and African-American populations [32, http://www.ncbi.nlm.nih.gov/SNP/]. Much higher frequencies of the CC3435 genotype in African population (Ghanaian and Kenyan) compared with Caucasian population are due to an advantage offered by this SNP (ABCB1 3435C>T) against gastrointestinal tract infections [33]. Variable susceptibility to cancer incidences can be connected with inter-ethnic differences in frequencies of the ABCB1 genotypes [17]. One of the reasons for development of neoplastic diseases is described mutations. Probably, changes of function and structure of genes simultaneously with influence of environmental factors lead to colon cancerogenesis [34]. Significant differences in ABCB1 1236C>T and ABCB1 2677G>T/A genotype distribution between colorectal cancer group and healthy population were found. In addition, significant differences in frequency of T1236 allele in investigated populations were found. There was a tendency for a higher frequency of the T1236 allele, CT1236 and GT2677 genotypes in healthy population compared to colorectal patients (results statistically significant). Several reports have suggested that SNPs of ABCB1 are a risk factor for cancer development, including colorectal cancer [9, 17–22]. Extensive studies have been carried out for ABCB1 3435C>T since the Hoffmeyer report [12]. Changed function of P-gp could be a risk factor of colon cancer due to facilitated intracellular penetration of DNA damaging factor of both exo- and endogenous origin which, in consequence, may lead to the development of colon cancer [21]. In this study, it was found that ABCB1 3435C>T was not associated with colorectal cancer in Polish population. Similar results have been presented by Petrova who has not found relation between SNPs ABCB1 3435C>T, ABCB1 2677G>T/A and the risk of sporadic colorectal cancer development in Bulgarian population [30].

Earlier researches indicate that TT3435 genotype develops more frequently among ulcerative colitis patients but not among Crohn disease patients. Both diseases are considered to be factors predisposing to colorectal cancer development [35, 36]. Furthermore, it was noticed that earlier-diagnosed colorectal cancer patients (below the age of 50) have TT3435 genotype and T3435 allele more frequently (2.7-fold and 1.7-fold higher risk) [21]. However, other researches indicate rather a protective role associated with the presence of T3435 allele. Gaikovitch indicated a higher risk (1.65-fold) of colorectal cancer development among CC3435 genotype carriers than among T3435 allele (p = 0.01) carriers. Similarly, the presence of T2677 allele decreases the risk of colorectal cancer development in relation to G2677 allele (OR = 0.65, p = 0.02) [37]. Protective role of T3435 and T2677 alleles (possibly also T1236 allele) may be associated with the function of P-gp protein, which influences functions of c-Myc and cyclin D1 and contributes to unblocking of cell death pathways suppression.

Impact of ABCB1 1236C>T and ABCB1 3435C>T polymorphisms on the function of P-gp can be explained by importance of LD of ABCB1 1236C>T, ABCB1 2677G>T/A and ABCB1 3435C>T. The ABCB1 1236C>T and ABCB1 3435C>T are “silent” SNPs, and therefore it may be linked with the causal polymorphisms. We detected a strong LD between ABCB1 1236C>T and ABCB1 2677G>T/A SNPs (the LD value D′ = 0.621, r 2 = 0.318) but also observed the remains of pair-wise LD between ABCB1 1236C>T and ABCB1 3435C>T (D′ = 0.394, r 2 = 0.104) as well as ABCB1 2677G>T/A and ABCB1 3435C>T SNPs (D′ = 0.384, r 2 = 0.120). Also, a strong association between ABCB1 1236C>T, ABCB1 2677G>T/A and ABCB1 3435C>T alleles was found by Tanabe [27]. Strong linkage disequilibrium of SNPs: ABCB1 2677G>T/A and ABCB1 3435C>T (D′ = 0.739, r 2 = 0.428) was also described by Petrova [30].

These results suggested that three studied SNPs of ABCB1 were located in one haplotype block. ABCB1 1236C>T and ABCB1 3435C>T are in linkage disequilibrium with other common functional non-synonymous polymorphisms such as ABCB1 2677G>T/A. In fact, these SNPs are a part of a common haplotype [15]. It is very probable that other potential SNPs, especially in regulatory region of ABCB1 gene, influence P-gp expression and function, e.g. ABCB1 -2410 T>C, ABCB1 -1910 T>C, ABCB1 -692 T>C and ABCB1 -129 T>C [38, 39]. Rund proved that mutations in the promoter region of ABCB1 gene were associated with haematological malignancies, so screening for SNPs and other types of polymorphism in the promoter region of this gene is important for studying human cancers [40].

Haplotype may often provide more useful information than genotype about interindividual and interethnic differences [41]. Kroetz defined 32 haplotypes and their subtypes (64 distinct haplotypes obtained for 28 variant sites) [10]. Estimates in our laboratory of ABCB1 haplotype frequencies showed that T1236–T2677–T3435 (24.0% vs 3.0%, OR = 9.61) and C1236–C2677–C3435 (39.0% vs 19.0%, OR = 2.77) of eight haplotypes found in colorectal cancer patients were significantly higher than in a control group. Moreover, haplotype analysis showed that haplotypes C1236–T2677–T3435 and T1236–G2677–C3435 were found higher in healthy population compared to colorectal patients (26.0% vs 8.0%, OR = 0.25 and 23.0% vs 3%, OR = 0.11). In addition, statistically significant differences were found in haplotype distributions between investigated populations. Potocnik identified relationship between haplotypes of ABCB1 gene (1236C>T–rs2235035–2677G>T/A–3435C>T) and the presence of high microsatellite instability. It also suggests that changes of ABCB1 gene function may contribute to the initiation and development of MSI-H tumours. Most frequently noticed haplotype T1236–Crs2235035–T2677–T3435 is associated with significant risk of MSI-H occurrence (p = 0.004, OR = 0,48) [42]. A transcription factor complex TCF4/β catenin responsive element was identified in the ABCB1 promoter region pointing to a direct link between the ABCB1 gene and the Wnt signalling pathway, the most important pathway altered in colorectal cancers [43]. Therefore, somatic mutations and functional polymorphisms within the ABCB1 gene in colorectal tumours and corresponding normal mucosa were previously characterised for microsatellite instability and lymphoid infiltration [44].

Several haplotypes of ABCB1 gene have clinical relevance, e.g. the C1236–C2677–C3435 haplotype has been associated with pharmacoresistance [45]. This haplotype correlated with treatment failure indicated that the degree of pharmacoresistance may be modulated by ABCB1 gene, for example drug resistance in temporal lobe epilepsy [46]. On the other hand, the T1236–T2677–T3435 haplotype was shown to be associated with high risk of developing refractory Crohn's disease [44].

These results suggested that ABCB1 haplotypes based on three or more sites may be useful for colorectal cancer patient characterization. In the future, large-scale studies are warranted to appropriately investigate this possibility.

Tumour cells could become resistant to anticancer drugs by a variety of mechanisms. Several factors affect ABCB1 gene expression or the MDR (multidrug resistance) phenotype such as: inhibitors of P-gp, calmodulin inhibitors, X-ray, cytotoxic agents, proapoptotic agents and others. Allelic variants in ABCB1 gene have attracted attention as a possible explanation of inter-individual differences in drug response. Also, in this study, the influence of ABCB1 genotypes on outcome of colorectal cancer therapy was estimated. Survival probability of patients with wild-type C3435 allele was higher than among patients without this allele present. Because 5-FU is probably not the substrate for P-gp, some indirect mechanisms than transports via membrane could have impact on efficiency of chemotherapy in colorectal cancer. Moreover, silent mutation in a haplotype, mammalian membrane transport protein alters the substrate specificity [47]. P-gp was implicated in the system regulating cell differentiation, proliferation and survival [4]. On the other hand, P-gp may play a role in regulating some caspase-dependent apoptotic pathways, a function completely independent of its drug transport properties [48]. The combined actions of 5-FU metabolites are associated with inhibition of DNA biosynthesis, altered DNA stability, production of DNA damages and interference with DNA repair. The genotoxic stress resulting from 5-FU administration may activate apoptosis in susceptible cells via activated P53 or inactivated cyclin-dependent kinases [49]. On the other hand, protection function of P-gp against carcinogens may reduce the degree of accumulation of mutations in genes (e.g. P53 gene) of cell, thus increasing sensitivity of cancer cell to action of anticancer agents.

The incidence of colorectal cancer is increasing in Poland, and the results of treatment are some of the worst among other western European countries [50]. The reason of that is late diagnosis, which in consequence hinders treatment. A few coexisting SNPs may influence the therapeutic outcome and the risk of cancer disease development. Larger number of samples must be examined to reach more reliable conclusions. Therefore, further studies are needed to determine whether environmental factors, unexamined ABCB1 genotypes and genes other than ABCB1 were more predominant for the development of colorectal cancer.