DOI:http://dx.doi.org/10.7314/APJCP.2015.16.8.

3457
Morinda Citrifolia (Noni) Effects on DNA Repair Genes in SiHa Cervical Cancer Cells

RESEARCH ARTICLE

Influence of Morinda citrifolia (Noni) on Expression of DNA

Repair Genes in Cervical Cancer Cells
Rakesh Kumar Gupta, Deepti Bajpai, Neeta Singh*
Abstract
Background: Previous studies have suggested that Morinda citrifolia (Noni) has potential to reduce cancer
risk. Objective: The purpose of this study was to investigate the effect of Noni, cisplatin, and their combination
on DNA repair genes in the SiHa cervical cancer cell line. Materials and Methods: SiHa cells were cultured and
treated with 10% Noni, 10 µg/dl cisplatin or their combination for 24 hours. Post culturing, the cells were pelleted,
RNA extracted, and processed for investigating DNA repair genes by real time PCR. Results: The expression
of nucleotide excision repair genes ERCC1, ERCC2, and ERCC4 and base excision repair gene XRCC1 was
increased 4 fold, 8.9 fold, 4 fold, and 5.5 fold, respectively, on treatment with Noni as compared to untreated
controls (p<0.05). In contrast, expression was found to be decreased 22 fold, 13 fold, 16 fold, and 23 fold on
treatment with cisplatin (p<0.05). However, the combination of Noni and cisplatin led to an increase of 2 fold,
1.6 fold, 3 fold, 1.2 fold, respectively (p<0.05). Conclusions: Noni enhanced the expression of DNA repair genes
by itself and in combination with cisplatin. However, high expression of DNA repair genes at mRNA level only
signifies efficient DNA transcription of the above mentioned genes; further investigations are needed to evaluate
the DNA repair protein expression.
Keywords: Morinda citrifolia (Noni) - DNA repair genes - cervical cancer - SiHa cells

Asian Pac J Cancer Prev, 16 (8), 3457-3461

Introduction in malignant cells, the suppression of DNA repair would,

presumably, increase the effectiveness of chemotherapy
Cervical cancer is the second most common through damage accumulation and consequent apoptosis
malignancy among women worldwide (Gupta et al., (Slyskova et al., 2012).
2013). Human papillomavirus (HPV) has been established The nucleotide excision repair (NER) genes include
as an important etiological factor for the development of ERCC1, ERCC2, and ERCC4; whereas, base excision
cervical cancer. However, only a small fraction of women repair (BER) gene includes XRCC1. The nucleotide
infected with HPV develop cervical cancer, implying the excision repair (NER) pathway mainly removes bulky
involvement of environmental and genetic cofactors in DNA adducts and maintains genomic stability (Min and
cervical carcinogenesis (Rosa et al., 2009). Pavletich, 2007); whereas the base excision repair (BER)
Cisplatin (CP), an antitumor drug, is employed as pathway is responsible for removal of oxidized DNA bases
a first-line chemotherapeutic agent in the treatment of that may arise from endogenous or exogenous agents (Zhi
epithelial malignancies, including cancer of cervix, lung, et al., 2012).
ovarian, testicular, and others (Gupta and Singh, 2013). We selected XRCC1 because deregulation of base
The destruction of the cancer cells by cisplatin requires excision repair gene expression (XRCC1) has been shown
binding of the drug to DNA and the formation of platinum- to enhance proliferation in head and neck squamous
DNA adducts, which may establish inter- and intra-strand cell carcinoma (Mahjebeen et al., 2014). Evidence has
DNA crosslinks, thereby inhibiting DNA replication (Su revealed that expression levels of ERCC4 correlated with
et al., 2014). risk, progression, response to cisplatin chemotherapy,
DNA repair is a defensive mechanism that copes with and clinical outcome of multiple human cancers,
ubiquitous DNA damage occurring as a consequence of including head and neck cancer (Yu et al., 2012), thereby
cellular metabolism or from exogenous exposure (EI-Zein suggesting that altered ERCC4 expression may lead to
et al., 2010). Moreover, a large number of antineoplastic altered DNA repair capacity (DRC), thereby modulating
drugs impart their effect by DNA damage. Therefore, cancer susceptibility. Earlier studies have linked cisplatin
an effective DNA damage response is essential for the resistance to the expression of ERCC1 mRNA in cell
maintenance of genome stability in normal cells, whereas lines of cervical cancers (Bai et al., 2012). Reduced

Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India *For correspondence:
singh_neeta26@rediffmail.com

Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 3457

Rakesh Kumar Gupta et al
expression of ERCC2 has been reported in squamous cell humidified atmosphere. Post treatment, the cells were
carcinoma of head and neck (Kumar et al., 2012). These pelleted and stored at -700C for various assays (Gupta
reports suggest that DNA repair genes could be involved and Singh, 2013).
in cervical cancer, especially as HPV is necessary but
not sufficient for cervical cancer formation; and genomic RNA extraction
instability/host genetic background often participate in Total cellular RNA was harvested from the cell pellet
its development and progression as they influence HPV using Triazol reagent (Sigma Aldrich, St.Louis USA)
acquisition and persistence (Bajpai et al., 2013). according to the manufacturer’s instructions.
Morinda citrifolia, commonly known as Noni, is
considered to be one of the most important medicinal Reverse Transcription Assay
plants in the Hawaiian Islands (Gupta et al., 2013). Synthesis of cDNA was carried out by using cDNA
Several reports have described health benefits of Noni fruit synthesis kit (Fermentas Life Sciences, New Delhi, India)
including immune modulation and antioxidant activities as per manufacturer’s protocol (Kumar et al., 2012).
in vitro and in vivo (Gupta and Singh, 2013). Noni has
also been suggested to mitigate oxidative damage (Wang Real time-polymerase chain reaction
et al., 2008); reduce the cancer risk in heavy cigarette Real Time PCR was done using gene specific primers
smokers (Wang et al., 2009). It does not have a genotoxic (Table 1). The PCR was carried out in a volume of 20 μl
potential and that genotoxic anthraquinones do not exist in ABI 7500 Thermocycler using Fast Taq Polymerase
in Noni juice (Westendorf et al., 2007). It also prevents (Chromous Biotech, Bengaluru, India ) under the
mammary breast cancer at the initiation stage of chemical following conditions: denaturation at 94°C for 10 sec;
carcinogenesis (Clafshenkel et al., 2012). annealing (as per primer) for 20 sec; extension at 72°C
As accumulation of DNA damage and increased for 5 sec. The mRNA expression of DNA repair genes
genomic instability have been proposed to play a critical was normalized to the housekeeping gene 18S. The
role in several pathophysiological conditions associated comparative CT method was used in order to evaluate the
with cervical cancer; manipulation of DNA repair differential DNA repair gene expression in treated cervical
mechanisms by Noni/Cisplatin may provide a strategy to cancer SiHa cells with respect to untreatedcontrols. For
prevent cellular dysfunction in the normal cell process. each target gene, we performed three replicates of qRT-
Hence, the aim of this study was to determine the relative PCR. The relative amount of each target gene to 18S was
expression of DNA repair genes (XRCC1, ERCC1, determined using the following equation:
ERCC2, and ERCC4) at mRNA level by real time PCR,
and to examine if these DNA repair genes are altered in 2-ΔΔCt, where ΔΔCtgene= [Ctgene - Ct18S] Patient
HPV-16 positive cervical cancer SiHa cells in response to - [Ctgene - Ct18S] Control.
either Noni, Cisplatin, and their combination, to provide
potential therapeutic targets. Statistical analysis
Results of each experiment represent the mean±standard
Materials and Methods deviation (SD) of three independent experiments carried
out in triplicate. All the data were analysed using Student’s
cell culture and treatments t-test. Statistical analysis showing a value of p<0.05 was
SiHa (HPV16+) cervical cancer cell line was used in considered significant.
this study and cultured in DMEM media, supplemented
with 10% fetal bovine serum (FBS), 100 U/ml Penicillin, Results
and 100 µg/ml Streptomycin.
Noni juice was obtained from Health India Laboratories Effect of Noni on the expression level of NER genes and
(a unit of Noni BioTech Pvt. Ltd., Chennai, India). BER gene
Cisplatin was purchased from Sigma, USA. The fruit juice of Morinda citrifolia (Noni) is in
SiHa cells were treated with 10% Noni (v/v), 10 µg/ high demand in alternative medicine for different kinds
dl Cisplatin, and their combination i.e. 10% Noni and 10 for illnesses. Several studies have also demonstrated
µg/dl Cisplatin for 24 hours at 370C in 5% CO2 and 95% anti-inflammatory, antioxidant, and apoptosis-inducing

Table 1. Primer Sequences of DNA Repair Genes in the Present Study

Gene name Primers Sequences Amplicon size(bp)
XRCC1 Forward 5’-CAGCCCTACAGCAAGGACTC-3’ 209
Reverse 5’-GCTGTGACTGGGGATGTCTT-3’
ERCC2 Forward 5’-GACCTGGTGTCCAAGGAACTG-3’ 165
Reverse 5’-GATCCTGAGCACCGTCTTCTG-3’
ERCC4 Forward 5’-TGACTCCAGCACCTCGATG-3’ 237
Reverse 5’-AGTACTTGACCTGGACCACC-3’
ERCC1 Forward 5’-TTGTCCAGGTGGATGTGAAAGATC-3’ 151
Reverse 5’-GCTGGTTTCTGCTCATAGGC-3’
18S Forward 5’-GTAACCCGTTGAACCCCATT-3’ 145
Reverse 5’-CCATCCAATCGGTAGTAGCG-3’

3458 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015

 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.8.3457
Morinda Citrifolia (Noni) Effects on DNA Repair Genes in SiHa Cervical Cancer Cells
effects of Noni in various cancers. Our results showed folds respectivelyl (p<0.05) (Figure 2).
increased expression level of nucleotide excision repair
genes (ERCC1, ERCC2, and ERCC4) and base excision Effect of Noni and Cisplatin on NER genes and BER gene
repair gene (XRCC1) with Noni treatment as compared The combination of Noni and Cisplatin has been found
to control; and the increase was found to be statistically to induce apoptosis through the mitochondrial pathway,
significant. The expression of ERCC1, ERCC2, ERCC4, decrease lipid peroxidation, and enhance Catalase activity
and XRCC1 increased by 4 folds, 8.9 folds, 4 folds, and in human cervical cancer cell lines. Our results showed
5.5 folds respectivelyl (p<0.05) (Figure 1). increased expression level of nucleotide excision repair
Effect of Cisplatin on the expression level of NER genes genes (ERCC1, ERCC2, and ERCC4) and base excision
and BER gene repair gene (XRCC1) on treatment with the combination
Cisplatin is among the most effective and widely of both Noni and Cisplatin, as compared to control. The
used chemotherapeutic agent employed in the treatment expression of ERCC1, ERCC2, ERCC4, and XRCC1
of solid tumors. It is a potent inducer of cell cycle arrest, increased by 2 folds, 1.6 folds, 3 folds, and 1.24 folds
and apoptosis in most cancer cell types including human respectively (p<0.05) (Figure 3).
cervical cancer cell lines. Our results showed decreased
expression level of nucleotide excision repair genes Discussion
(ERCC1, ERCC2, and ERCC4) and base excision repair
gene (XRCC1) on Cisplatin treatment as compared to Exposure to different endogenous and exogenous
control. The expression of ERCC1, ERCC2, ERCC4, and mutagens and carcinogens can result in various types of
XRCC1 decreased by 22 folds, 13 folds, 16 folds, and 23 DNA damage. These alterations, if not repaired, can cause
genetic instability, mutagenesis and cancer. Importantly,
5 Control to counteract the deleterious consequences of the DNA-
0 CP damaging agents, evolution has moulded a number of
DNA repair systems that as a whole take care of most of
Fold  Change  

-5 ERCC1 ERCC2 ERCC4 XRCC1

the insults inflicted on a cell’s vital genetic information.
-10 The repair of different types of DNA damage is important
-15 for safeguarding genomic integrity. Among the main DNA
* maintenance mechanisms operating in humans, the NER
-20 * and BER are the primary defence against DNA damage
-25 * (Li et al., 2012).
*
Given that cervical carcinogenesis is usually a
DNA Repair Genes (NER and BER) multistep, multigenic process, it is unlikely that one
Figure 1. Effect of Cisplatin on the Expression Level individual DNA repair gene would have a significant effect
of ERCC1, ERCC2, ERCC4, and XRCC1 on cancer risk. Therefore, single-gene studies are likely to
provide limited value in evaluating cervical cancer risk.
10 * Control Evaluation of the combined effect of a panel of DNA repair
8
Noni genes that interact in different DNA repair pathways may
amplify the effects of individual variation in expression
Fold  Change  

*
6 level and enhance predictive power. Although reduced
* *
4 expression of DNA repair genes has been shown to be
associated with squamous cell carcinoma of head and
2 neck (Kumar et al., 2012) and smoking-related cancers
0 such as lung cancer (Wei et al., 1996); few studies have
ERCC1 ERCC2 ERCC4 XRCC1 evaluated the expression level of DNA repair genes in
the etiology of cervical cancer. Hence, we measured
DNA Repair Genes (NER and BER)
the relative expression levels of NER genes (ERCC1,
Figure 2. Effect of Noni on the Expression Level of
ERCC2, and ERCC4) and BER gene (XRCC1) in SiHa
ERCC1, ERCC2, ERCC4, and XRCC1
cells on treatment with Noni alone, Cisplatin alone, and
3.5 Control combination of both Noni and Cisplatin for 24 hours.
3 *
N + CP We found that Noni enhanced the expression level of
ERCC1, ERCC2, ERCC4, and XRCC1 by itself and in
Fold  Change  

2.5
2 * combination with Cisplatin, suggesting that Noni might
1.5 *
*
influence and enhance the expression level of these DNA
1 repair genes. However, their expression was found to
0.5 be decreased with Cisplatin as compared to control. On
0 Cisplatin treatment, the mRNA expression level of both
ERCC1 ERCC2 ERCC4 XRCC1 NER and BER decreased significantly as compared to
control. This is due to the fact that SiHa cells are Cisplatin
DNA Repair Genes (NER and BER)
sensitive cells (Bergs et al., 2007); and hence Cisplatin
Figure 3. Effect of Noni and Cisplatin on the Expression forms DNA-adducts, primarily intra-and inter strand
Level of ERCC1, ERCC2, ERCC4, and XRCC1 DNA adducts (Tanida et al., 2012), and activates several
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 3459
Rakesh Kumar Gupta et al
signaling pathways such as p53, leading to activation Based on the above results, present findings also
of apoptosis (Gupta et al., 2013), and thereby adversely demonstrates the presence of possible interrelationship
affecting DNA repair genes (Rabik and Dolan, 2007). and crosstalk between Noni and Cisplatin which may be
Some studies have suggested that low ERCC1 related, at least, partly to the expression of DNA repair
expression is associated with increased chemotherapeutic genes in cervical cancer SiHa cell line.
sensitivity and thus considered a predictive marker for In conclusion, these results showed the ability of Noni
patients with colorectal cancer (Ni et al., 2014). Our by itself and in combination with Cisplatin to increase
findings also corroborates with earlier studies which have the expression level of ERCC1, ERCC2, ERCC4, and
linked cisplatin resistance to the expression of ERCC1 XRCC1; and this might assist in treatment of cervical
mRNA in cell lines of cervical cancers (Bai et al., 2012). cancer patients. However, high mRNA expression of
Notably, recent clinical evidence suggests that ERCC1 above genes only signifies efficient DNA transcription of
levels predict response to platinum-based therapies in non- above mentioned genes; and does not necessarily reflect
small cells lung cancer (Olaussen et al., 2007) and bladder the production of their functional protein. The protein
cancer (Bellmunt et al., 2007). Reduced expression of expression of above mentioned DNA repair genes needs
ERCC2 has been reported in squamous cell carcinoma of to be evaluated to determine whether these findings can be
head and neck (Kumar et al., 2012). Chinese hamster cell used for selection of cervical cancer patients for treatment.
lines defective in ERCC4 were found to be hypersensitive
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