DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3761 CYP450 and GSTM1 Polymorphisms and CRC Risk in Saudi Arabia
$VLDQ3DFLÀF-&DQFHU3UHY 14 (6), 3761-3768
Introduction
Colorectal cancer (CRC) is one of the most frequent causes of cancer death in industrialized countries with a yearly incidence of 50 new cases for every 100,000 people in the population (Boyle and Ferlay, 2005). CRC is the third most common cancer in the world and its prevalence has been steadily increasing over the last century, while mortality rates have declined as a result of improved WUHDWPHQWDQGHIÀFLHQWVFUHHQLQJDQGVXUYHLOODQFH +HDYH\
et al., 2004; Parkin et al., 2005). CRC is traditionally FODVVLÀHGLQWRVSRUDGLFDQGIDPLOLDORUKHUHGLWDU\IRUPV
and represents a complex disease which development is mediated by genetic and environmental factors (Potter,
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reported the association between polymorphisms for gene encoding enzymes involved in biotransformation of xenobiotics and susceptibility to cancers (Induski and Lutz, 2000; Chang et al., 2003; Terry et al., 2003). The human body is exposed daily to a number of xenobiotics
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Abstract
Background: The Saudi population has experienced a sharp increase in colorectal and gastric cancer incidences within the last few years. The relationship between gene polymorphisms of xenobiotic metabolizing enzymes and colorectal cancer (CRC) incidence has not previously investigated among the Saudi population. The aim of the present study was to investigate contributions of CYP1A1, CYP2E1, and GSTM1 gene polymorphisms.
Materials and Methods: Blood samples were collected from CRC patients and healthy controls and genotypes were determined by polymerase chain reaction restriction fragment length polymorphism and sequencing.
Results and Conclusions:&<3( ZDVQRWVLJQLÀFDQWO\DVVRFLDWHGZLWK&5&GHYHORSPHQW RGGUDWLR
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considered polymorphisms. Investigation of genetic risk factors and susceptibility gene polymorphisms in our Saudi population should be helpful for better understanding of CRC etiology.
Keywords: Cytochrome P450 - xenobiotic - colorectal cancer - single nucleotide polymorphism
5(6($5&+$57,&/(
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and Susceptibility to Colorectal Cancer in the Saudi Population
Hesham Mahmoud Saeed
1,2*, Mohammad Saud Alanazi
1, Howaida Attia 1RXQRX
3, Manal Ali Shalaby
4, Abdelhabib Semlali
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, Abdeulrahan Aljebreen
, Othman Alharby
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1, Jilani Shaik
1, Maha
including drugs, dietary compounds and environmental carcinogens which are metabolized by a variety of enzymes through phase I and phase II reactions (Bozina et al., 2009). These enzymes participate in the conversion of xenobiotics to more water soluble metabolites which are readily excreted from the body. During metabolism of some of these xenobiotics a variety of unstable and reactive intermediates can be formed which could attack '1$ FDXVLQJ FHOO WR[LFLW\ DQG WUDQVIRUPDWLRQ %R]LQD
et al., 2009). The key enzyme system involved in such activation process are phase I xenobiotic metabolizing enzymes (XMEs) such as cytochrome P450s (CYPs) DQGGHWR[LÀFDWLRQE\SKDVH,,;0(VVXFKDVJOXWDWKLRQH
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xenobiotics, including environmental carcinogens, chemotherapeutic agents and reactive oxygen species (Taspinar et al., 2008). Mutations in the genes that code for these enzymes can affect the metabolism of chemical carcinogens and which alters susceptibility to different
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long arm of chromosome 15q22-qter (Corchero et DO $ SKHQRW\SLF SRO\PRUSKLVP LQ &<3$
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after exposure to inducer than the non exposed group (Kellerman et al., 1973). In genotyping studies, two FORVHO\OLQNHGSRO\PRUSKLVPVRIWKH&<3$JHQHKDYH
been well studied in Caucasian and Oriental populations, WKH ·ÁDQNLQJ UHJLRQ 0VS, VLWH WKDW DVVRFLDWHG ZLWK
increased sensitivity to inducer in some studies and the exon 7 Ile-Val substitution that appears to result in higher HQ]\PHDFWLYLW\LQYLWUR +D\DVKLHWDO ,QGLYLGXDOV
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higher rates of carcinogen activation than individuals with the wild-type allele. The CYP2E1 enzyme, a member of the cytochrome P450 superfamily, is a natural ethanol- inducible enzyme that is involved in the metabolic oxidation of low molecular weight carcinogens such as N-nitrosoamines, benzene and vinyl chloride (Liu et al., 2009). CYP2E1 gene is located on the 10q24.3-qter. It is 18,754 bp long consisting of nine exons and eight introns, which encodes a 493 amino acid protein. CYP2E1 gene contains six restriction fragment length polymorphisms, RIZKLFKWKH5VD,3VW,SRO\PRUSKLVPLQLWVÁDQNLQJ
region has been shown to affect its transcriptional level.
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plasma ratios in healthy subjects possessing at least one
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theoretically at a high risk of cancer development. Case control studies have shown that some of the mentioned JHQH SRO\PRUSKLVPV DUH DVVRFLDWHG ZLWK D VLJQLÀFDQW
increase in the risk of cancer including CRC in some populations (Chang and Yang, 2000).
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cancer patients (65 males and 27 females, age range, 26-80 years; mean age, 58.4 years) and 79 healthy controls matched for age and sex. The control samples were collected from subjects referred to the hospital for general medical checkups. Colon cancer tissue samples were also collected from 12 unrelated colorectal cancer patients 8 males and 4 females and histologically normal tissues in the distant margin to the tumour were collected at the time of surgery from the patient who undergoing resection of colorectal tumours. The diagnosis of cancer was based on standard clinical, endoscopic, radiological, and histological criteria. Clinical and demographic characteristics were recorded, including age at diagnosis, gender, family history, smoking habits, disease behaviour, disease location, and need for surgery. Tissue samples to EHXVHGIRU51$DQDO\VLVZHUHLPPHGLDWHO\VXEPHUJHG
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DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3761 CYP450 and GSTM1 Polymorphisms and CRC Risk in Saudi Arabia expression levels.
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Results
It should be noted that the molecular tools used for the genotyping do not determine whether other polymorphisms in the same gene are also present in the tested subject. The wild type (wt) denotation thus refers to the wild type allele at the investigated polymorphic site only, regardless of other unstudied polymorphisms for that particular gene. Furthermore, in this study, there were no VLJQLÀFDQW GLIIHUHQFHV LQ WKH GLVWULEXWLRQ RI JHQRW\SHV
for the three investigated genes when male groups were compared with females groups (data not shown).
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tested subjects and statistical analysis of the obtained data are detailed in Table 1. The frequency of the
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different from the control healthy individuals (OR=4.17;
&, Ƶ2=9.63 and p=0.0019).
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and B. The distribution of CYP2E1 genotypes among the tested subjects and the statistical analysis of the obtained data are detailed in Table 1. The frequency RI WKH &<3( DOOHOH ZDV LQ &5& SDWLHQWV
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of the CRC patients but not in the control group. The GLVWULEXWLRQRI&<3(ZW JHQRW\SHLQ&5&SDWLHQWV
was not statistically different from that of healthy controls )LVKHU·V H[DFW S WKH 25 EHLQJ &,
0.81-3.05). Moreover, the results remained statistically QRQVLJQLÀFDQWZKHQERWK&<3( DQGZW ZHUH
considered together as one group (OR=1.29 at CI 0.68-
Table 1. 6WDWLVWLFDO$QDO\VLVDQG'LVWULEXWLRQRI&<3$ $ 0VS,7!&UV DQG&<3( 'UD,
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DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3761 CYP450 and GSTM1 Polymorphisms and CRC Risk in Saudi Arabia
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polymorphic sites in the three investigated genes was conducted (data not shown). None of the tested subjects carried the variant allele for the three gene polymorphisms VLPXOWDQHRXVO\$OO RWKHU FRPELQDWLRQV EHWZHHQ WKH
genotypes of the three genes showed no significant variation from the results of the analysis of each gene separately.
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highly expressed in colon cancer tissues as compared with normal control adjacent tissues and their expression levels were found to be 4, 4.2 and 4.8 times as compared to the FRQWUROUHVSHFWLYHO\*HQHH[SUHVVLRQOHYHOVRI&<3$
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Figure 1. $ $JDURVHJHO (OHFWURSKRUHVLVIRU
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Cancer Patients (Lanes 1-21).The amplicon is subjected to digestion with MspI prior to electrophoresis. The 340 bp uncut DPSOLFRQ XSSHU EDQG UHYHDOV &<3$ ZWZW KRPR]\JRXV
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two fragments (200 and 140 bp), and the heterozygous genotype
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fragment and two restriction fragments of 200 and 140 bp).
Lanes 1-3 represent control uncut PCR products. Lanes 4 and 21 UHSUHVHQWES'1$ODGGHUPROHFXODUZHLJKWPDUNHUV
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Figure 3. (A) Real Time PCR Analysis of CYP1A1,
&<3( DQG *670 P51$ LQ 1RUPDO FRQWURO and Colon Cancer Tumor Tissues. (B) Expression of CYP1A1, 2E1 and GSTM1 in Different Cancer Cell Lines as Compared to GAPDH
2.45) as shown in Table 1.
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lines (Figure 3B). On the other hand, detectable levels of
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under investigation (Figure 3B).
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immunohistochemistry on colon cancer tumour samples DQGQRUPDOFRQWUROV&\WRFKURPH3$ZDVIRXQGWR
be highly expressed in colon cancer tissues as indicated by strong staining (Figure 4C) when compared with control WLVVXHV ZHDN VWDLQLQJ )LJXUH $ DQG SUHPDOLJQDQW
tissues (polyp) (weak staining) (Figure 4B). Over- H[SUHVVLRQRI&<3$LQWXPRXUWLVVXHVFRUUHODWHGZLWK
tumour progression indicating a potential role in tumour development.
Discussion
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factorial disease involving inherited and environmental factors, the present study focused on the genetic basis RI FDQFHU VXVFHSWLELOLW\ HVSHFLDOO\ WKH 613V 6LQJOH
nucleotide polymorphism are the most abundant forms among variations in human genomes, and it has been GHPRQVWUDWHG WKDW VRPH 613V DUH VWURQJO\ DVVRFLDWHG
with pathophysiological properties of individuals such as responses to medications and mortalities of hereditary GLVHDVHV %URRNHV 5RVHV 6DFKLGDQDQGDPHW
DO +HQFHJHQRW\SLQJWKHVHELDOOHOLFPDUNHUVPD\
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of markers for individual medications (Carlson et al., 2001). Moreover, susceptibility to cancer is determined by the activation of enzymes involved in carcinogens DFWLYDWLRQ RU GHDFWLYDWLRQ *HQHWLF YDULDWLRQV LQ WKHVH
genes encoding the enzymes, possibly by altering their expression levels and functions, may increase or decrease FDUFLQRJHQDFWLYDWLRQGHWR[LÀFDWLRQDQGPRGXODWH'1$
UHSDLU5HFHQWO\DFRPPRQO\VWXGLHG613LQWKH&<3$
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at nucleotide 3801 in the 3’ non-coding region containing a single T to C substitution that results in a polymorphic
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polymorphism, rs4646903). The MspI restriction site polymorphism results in three genotypes: a predominant KRPR]\JRXV P DOOHOH ZLWKRXW WKH 0VS, VLWH W\SH$
TT), the heterozygote (type B, TC) and a homozygous rare m2 allele with the MspI site (type C, CC) (Zhou et DO 7KH &<3$ $ SRO\PRUSKLVP KDV EHHQ
associated experimentally with increased catalytic activity DQG LQGLYLGXDOV SRVVHVVLQJ &<3$ $ DUH H[SHFWHG
to exhibit high rates of carcinogen activation (Landi et al., 1994). The present study showed that the variant
&<3$ $DOOHOHLVVLJQLÀFDQWO\DVVRFLDWHGZLWK&5&
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agreement with studies in other populations, for example VWDWLVWLFDOO\VLJQLÀFDQWHLJKWIROGLQFUHDVHGLQULVNIRU&5&
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shown in Italy (Boccia et al., 2007), the United Kingdom <HDQG3DUU\ DQGLQ/HEDQRQ 6ODWWHU\HWDO
It has been reported that the detection of an association EHWZHHQ&<3$ $DQGFDQFHUFRXOGQRWEHFRQÀUPHG
in Caucasian populations because of the low frequency RIWKLVDOOHOHDPRQJWKHVHSRSXODWLRQ *DUWHHWDO
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been associated experimentally with increased catalytic DFWLYLW\ DQG LQGLYLGXDOV SRVVHVVLQJ &<3$ $ DUH
expected to exhibit high rates of carcinogen activation (Lindi et al., 1994). The present study showed that the
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CRC compared to control normal tissues as measured by T3&5 )LJXUH$ LQGLFDWHGWKDWWKHKLJKP51$OHYHO
IRU&<3$ZDVGHWHFWHGLQWXPRXUWLVVXHVDVFRPSDUHG
with normal tissues. This finding was confirmed by immunohistochemistry study using anti-cytochrome P450
$DQWLERG\ )LJXUH ,WZDVFOHDUO\REVHUYHGWKDWFRORQ
FDQFHUWXPRXUWLVVXHVH[SUHVVHGPRUH&<3$SURWHLQ
than normal adjacent control tissues.
Many studies have investigated the relationship between CYP2E1 gene variation and the risk of CRC. The PRVWH[WHQVLYHO\VWXGLHG613VRI&<3(DUH5VD,3VW,
VLWHLQWKH·ÁDQNLQJUHJLRQDQGWKH'UD,VLWHLQLQWURQ
+D\DVKLHWDO /LXHWDO 7KHSUHVHQWVWXG\
indicated that the polymorphism caused by the presence or absence of a DraI site in CYP2E1 intron 6 has no VLJQLÀFDQW DVVRFLDWLRQ ZLWK &5& LQ 6DXGL SRSXODWLRQV
This is in agreement with a previous study about CRC in a Taiwanese population (Dil LIio et al., 1987) and Lebanese population (Darazy et al., 2011). The effect of this mutation on CYP2E1 enzyme activity is still not well established; however, an increased risk for CRC was detected among Italian population ever drinkers carrying
&<3( %RFFLDHWDO ,WVHHPVWKDWWKHORZ
frequency of this allele and the low number of samples ZRXOGPDNHLWGLIÀFXOWWRGHWHFWDQDVVRFLDWLRQZLWK&5&
LQ 6DXGL SRSXODWLRQ 6WXG\ RI &<3( H[SUHVVLRQ E\
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F i g u r e 4 . M i c r o p h o t o g r a p h o f C Y P 1 A 1 Immunohistochemistry in Colorectal Adenocarcinoma & 1RUPDO 1RQ1HRSODVWLF &RORQ $ DQG 3RO\
SUHPDOLJQDQW 1RUPDO &RORQ % &RORUHFWDO
Adenocarcinoma Overexpressed CYP1A1 Protein and Showed a Strong Staining in a Granular Cytoplasmic 3DWWHUQ1RQ1HRSODVWLF1RUPDO&RORQ(SLWKHOLDO&HOOV
were CYP1A1 Weak Staining (A, B) in Comparison with Adenocarcinoma (C)
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.6.3761 CYP450 and GSTM1 Polymorphisms and CRC Risk in Saudi Arabia ZDVEDVHGRQ3&5DVVD\WKDWLGHQWLÀHGWKH*670DQG
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individuals. The present study demonstrated that the
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times higher in CRC patients (Figure 3C) compared ZLWKQRUPDOFRQWUROKHDOWK\DGMDFHQWWLVVXHV*670LV
generally considered as a protective enzyme because it GHWR[LÀHVDQXPEHURIWR[LFDQGFDUFLQRJHQLFVXEVWDQFHV
VXFKDVQLWURVDPLQHVDQG3$+VLQFOXGLQJ%3'( .HWWHUHU
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in larger tumour could comply with an adaptive cellular response to disease progression and this response was OHVV HYLGHQW LQ WKH GHOHWLRQ YDULDQWV *670 JHQH
expression varies widely inter-individually and this may EH GXH WR WXPRXUVSHFLÀF H[SUHVVLRQ RU WR PHWDEROLF
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linked and may affect an individual’s susceptibility for cancer (Katoh et al., 1996; Rawal et al., 1999; Martinez HW DO *670 JHQRW\SH ZDV OLQNHG WR DQ
elevated risk of CRC in several populations such as the 6SDQLVK $WHVHWDO 7XUNLVK .LVVHWDO
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an elevated risk of gastric cancer in Turkish and Iranian SRSXODWLRQV 6DDGDWDQG6DDGDW 7DPHUHWDO
Other studies investigating the association between the
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al., 1999; Piao et al., 2009). Meta analysis of 49 published case-control and cohort studies showed that there was D VLJQLILFDQW DVVRFLDWLRQ EHWZHHQ WKH *670
JHQRW\SHDQGJDVWULFFDQFHUDPRQJ$VLDQVEXWQRWDPRQJ
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with CRC in Caucasians but not in other ethnic groups *DRHWDO 7KHSRSXODWLRQGHSHQGHQWGLIIHUHQFH
in term of association might be due to a difference in the frequency of this polymorphism among these populations as well as to other factors related to the diet and the environment. In addition, the presence of other genetic SRO\PRUSKLVPV DIIHFWLQJ ;0(V VLPLODU WR *670
might also affect the strength of the association. For LQVWDQFH*677ZKLFKLVLQYROYHGLQWKHGHWR[LÀFDWLRQ
of chemical carcinogens, also exhibits a gene deletion PXWDWLRQ$FDVH²FRQWUROVWXG\LQD6SDQLVKSRSXODWLRQ
REVHUYHG WKDW WKH *670 JHQRW\SH DORQH ZDV
associated with a 1.91-fold increase in the risk of CRC, but WKHFRPELQDWLRQRIERWK*670 DQG*677
genotypes was associated with an even higher OR of 4.98 $WHVHWDO
In conclusion, the work undertaken in this study raises a number of interesting observations. First, the
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factors in the analyzed population. Third, all genes under investigation were found to be highly expressed in CRC patients which is likely to be an important determinant in predicting the outcome of cancer chemotherapy. Finally, large-scale studies with more CRC patients are required to draw a clearer picture about the genetic factors of CRC HWLRORJ\LQ6DXGLSRSXODWLRQ
Acknowledgements
The authors extend their appreciation to the Deanship RI6FLHQWLÀF5HVHDUFKDW.LQJ6DXG8QLYHUVLW\IRUIXQGLQJ
the work through the research group project number 5*3933
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enzymes and susceptibility to colorectal cancer. $QWLFDQFHU
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