Download Advances in Environmental Biology Patients

Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
The Effects of Omega 3, 6, 9 Fatty Acids on the Caspase-3 in Gastric Cancer
Patients
1Behzad
Ordoubadi, 2Manuchehr Norazariyan, 3Mohhamad Hoseyn Somei
1
Department. of Biology, Ahar Branch, Islamic Azad University, Ahar, Iran
Assistant Professor in Clinical Biochemistry, Dept. of Laboratories Sciences, Faculty of Para-Medicine, Tabriz University of Medical
Sciences, Tabriz, Iran.
3
Professor in Liver and Gastrointestinal Disease, Tabriz Liver and Gastrointestinal Disease Research Center, Tabriz, Iran
2
ARTICLE INFO
Article history:
Received 11 June 2014
Received in revised form 21 August
2014
Accepted 25 September 2014
Available online 25 November 2014
Keywords:
Gastric Cancer, Chemoresistance,
Omega 3, 6, 9 fatty acids, Apoptosis,
Caspase3
ABSTRACT
Introduction and objects: Where of Omega 3, 6, 9 fatty acid have role in
inflammatory procedure and inflammatory is one of the main elements of Apoptosis
activation, so, if using of PUFAs fatty acids beside chemotherapy cause to the
activation of Apoptosis. This procedure makes gradation of chemotherapy effectiveness
in patients with gastric cancer. Caspase3 is one of the main caspases in upholding of
process start that measuring active enzyme scale in cell and its gene recitation is good
assessment for Apoptosis scale. So the purpose of this study is to investigate PUFAs
fatty acids effect on the Caspase3 enzyme in patients with gastric cancer. Materials
and Methods: This study is a Clinical Trial that the target group of this research is
patients with gastric cancer who were recognized for the first time and were cured
under chemotherapy. Thirty-four patients were chosen in two groups of 17 subjects.
Three biopsy samples from tumor were taken these patients. In the next stage,
chemotherapy of these patients started under inquest of Oncologist. In two selective
groups, at first group, chemotherapy started with Cis-Platin without supplementary
medication, and in the second group, chemotherapy started with Omega 3, 6, 9 fatty
acids supplement in the scale of 3600 mg daily and in length of 3 courses. Once again,
biopsy sampled from these patients. Quantitative scale of caspase3 enzyme's protein
upon biopsy samples was surveyed by Frozen Section and Immunohistochemistry
method and gene expression measure was investigated by RT-PCR and Real-Time PCR
method. Results: The means of caspase3 enzyme's protein in gastric tumor after 3
course chemotherapy in 2 groups of study , shows increase , but this increase was more
greater in second group and it is meaningful from the statistic point of view ( p= 0.0065
) . In order to certify this growth in the next stage, derivation and making quantitative of
caspase3 enzyme's gene was accomplished. And they gained results confirm this
increase. Discussion: Due to the results of this study and different accomplished studies
it can be seen likewise that PUFAs fatty acids effect of medication and also usual drugs
of chemotherapy, which were used for controlling of gastric cancer, can be beneficial.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Behzad Ordoubadi, Manuchehr Norazariyan, Mohhamad Hoseyn Somei, The Effects of Omega 3, 6, 9 Fatty Acids on
the Caspase-3 in Gastric Cancer Patients. Adv. Environ. Biol., 8(23), 57-63, 2014
INTRODUCTION
Gastric cancer is fourth prevalent cancer and second cause of death in the world. According to international
estimations and statistics, 930000 cancers are diagnosed annually among which 700000 result in death. North
and North West of Iran are the most vulnerable areas in the country to such disease. Aden carcinoma is the most
common form of gastric cancer. Infection caused by helicobacter pylori (HP), salty diet, smoking and not
receiving sufficient anti oxidants are the causes of this disease in Iran [1]. Tumor location of 75% of patients
with gastric disease is distal third. Lymphatic drainage of these tumors is toward lymphatic groups beneath the
pylori, liver navel and both of them have been extended toward large and small curve of the gastric. In addition
to attacking lymphatic glands, adjourning buildings i.e. lower end of esophagus, Gastric carcinoma is developed
by sub mucosa, pancreas, Transverse colon, peritoneum and rarely duodenum release. Tumor blood release
results in renal, liver, cerebral and bone metastases [1, 2].
Normally, most cases of gastric cancer are diagnosed when the patient is in his developed stages and
meanwhile surgery would play a limited role in treatment procedure. Hence systematic chemotherapy is the only
Corresponding Author: Behzad Ordoubadi, Department. of Biology, Ahar Branch, Islamic Azad University, Ahar, Iran
This article is extracted from a senior thesis skins
58
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
efficient way of treatment [1]. By the term chemotherapy, it is meant: using different drugs for treatment and in
proper sense it means medical treatment of the tumor. Using anti cancer drugs or chemotherapy is one of several
treatment ways of such tumors and it can be combined with other treatment methods such as surgery and
radiotherapy [2].
Chemotherapy is widely efficient in tumor treatment and nowadays with exploration of new anti tumor
drugs, this effect is growing considerably. According to studies it was identified that gastric cancer is sensitive
to chemotherapy to some extent., resulting in tumor growth reduction in 50-70% of patients. This is while
reduce in duration of tumor growth was short and increase in the life of patients was not successful and
resistance against chemotherapy was observed. Considering that molecular mechanism of drug resistance has
not been yet identified properly, so better understanding of tumor generation and overcoming cytotoxic effects
of chemotherapy and increasing efficiency of chemotherapy in patients with gastric cancer with change in food
diet, is considered as effective step taken to develop treatment methods and overcoming drug resistance (2).
Necessary fatty acids such as omegas are fatty acids which can not be made in the body and they should be
provided by food. Inappropriate diets with omitted or limited use of some fats have caused us to confront with
lack of such fatty acids in the body, lack of which faces us with different problems through depriving the body
of their crucial role in different crucial issues [1].
Apoptosis is one kind of planned cell death which plays an important role in unnatural cells growth,
evolution, immune response and clearing. Moreover this procedure is an important way of maintaining number
of cells in living body and it s activation is crucial in inhibition of cancer development [3]. Understanding of
apoptosis mechanism is of great importance and it helps considerably in understanding pathogens of conditions
created due to apoptosis disorder, development in using diet in medical chemistry and medicals targeting genes
and apoptosis pathways [1-3]. Apoptosis reduce or resistance to it plays an important role in cancer growth and
development and cancer cells can inhibit or decrease apoptosis in different ways. Apoptosis induction requires
massaging molecules cooperation, receptors and gene inhibitor proteins [3]. Among these molecules Caspase
messaging water fall system which is controlled by different molecules such as apoptosis inhibitor proteins,
Calpain and Bcl-2 families, are crucial in doing apoptosis process [3]. In fact caspases are CysteineAsparginase protease which can be activated in different ways, though 2 pathways have been studied with more
details. First way is carried out by Fas/TNF receptor which carries out Caspase activation by messaging
mechanism. It is so that, related ligand is connected to membrane receptor and activates apoptosis. Second way
is apoptosis activation by Caspase activator proteins released from mitochondria to cytosol. First pathway is
known as external path relying on death receptors and the second path is known as internal pathway. Studies
have indicated that Caspase-8 in external pathway and Caspase-9 in internal pathway act as crucial caspases. In
external pathway of apoptosis, death signals connection such as TNF and Fas-L to their receptors in membrane
surface of cells result in receptors activation cell death receptors. TNF and Fas activate Caspase-8 through
connecting to FADD and TRADD, respectively. In internal pathway, also, release of Cytochrome-C from
mitochondria activates Caspase-9 through connecting to apoptosis-1 activator factor and in the rest of pathway
these two caspases influence Caspase-3 and complete apoptosis process [1-3].
Hence Caspase-3 in apoptotic cell is activated during both external method (death ligand) and internal
method (mitochondria). As a result Caspase-3 is considered as one of the main caspases in confirming apoptosis
process start. Synthesis rate measurement of this Caspase in cells and its gene expression indicates apoptosis
activation or deactivation. Since fatty acids of Omega 3, 6, 9 plays a role in inflammation and inflammation is
among the main factors of apoptosis activation, so using a food diet containing fatty acids of Omega 3, 6, 9
along with chemotherapy will increase the efficiency of chemotherapy in patients with gastric cancer, if it
activates apoptosis [4].
MATERIALS AND METHODS
Research method: present study is of blind clinical trial (intervention). Neither oncologist medical nor the
patient is aware of treatment to prevent probable errors in this way.
Selecting patients and sampling method:
Among patients referred to endoscopy clinic of Tabriz University of Medical Sciences which were
suspected to have gastric cancer and considering present study conditions and objectives, 34 patients were
selected and divided into two groups. Patients' selection was carried out with diagnosing gastric cancer based on
physical symptoms and checkups by design colleague who was digestion specialist. Definitive diagnosis was
carried out after clinical examinations of digestion specialist through doing endoscopy and biopsy taking from
gastric and pathologic investigation of these samples and observing cancer cells. After definitive diagnosis of
gastric cancer, 3 biopsy samples were taken from gastric tumor tissue and distal third and transferred to nitrogen
tank. Patients were selected with written consent and complete knowledge of study subject and these patients
were referred to oncologist in each stage and the patients' chemotherapy started under oncologist supervision. In
59
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
the two selected groups, treatment group 1 with Cis-Platin along with placebo without supplement and treatment
group2 with Cis-Platin along with 1200 mg Natural Factors Ultimate-Omega Factors tablet and fatty acids
supplementation of Omega 3, 6, 9 with formulation of Fish Oil Blend 400mg, Flaxseed Oil 400mg and Borage
Oil 400mg started experiment daily with 3600 g per a day (three 1200 mg tablets) for three courses (each course
taking three weeks). After this time along with treatment mandatory following they received endoscopy and
with gastric biopsy taking, tumor treatment was pursued.
Once again samples taken from patients were transferred to nitrogen tank and then they were sent to
training groups to do required experiments.
Frozen Section Method and Immuno-histochemical staining:
Semi quantitative amount of Caspase-3 enzyme proteins was carried out with frozen section and Immunohistochemical staining methods. To do so one biopsy sample was transferred to nitrogen tank immediately. After
dewatering in pathology laboratory, frozen tissue samples were cut and then they were stained with Immunohistochemical staining method. It was so that, first 4 micron sections were prepared from tissues and then
samples were put into citrate buffer for 15 minutes. In order to prevent unprofessional binding, the samples were
blocked using skim milk and then the samples were incubated with initial monoclonal antibodies against
enzyme protein of Caspase-3 at 4 centigrade for one night. Secondary antibody was added after three times of
washing with TBST buffer and then it was incubated for 1 hour. For enzyme protein expression of Caspase-3,
DAB was used as substrata and hematoxylen was used as counter stain.
RNA Extraction:
The extraction was carried out by Exigon company kit. The basis of RNA extraction in this kit is solid
phase. First, RLT buffer which involves thiocyanate guanidine for laysis of cell through protein denaturation
was used. It is, of course, worth mentioning that thiocyanate plays a role in RNAase inhibition in addition to cell
laysis inhibition. It is necessary to add Mercapto-ethanol (2ME) to buffer 2 RLT. 2-ME causes restoration of
disulfide connections. Hence 2-ME causes riboneuclease omission. RNA dilution phase was carried out by
column, based on solid phase. There are some polytimin roots on columns surface which causes RNA isolation
from cell body and other components with higher molecular weight through connecting to a bridge tail. And
finally RW1 solution which involves ethanol and thiocyanate to some extent was used for RNA concentration
and sedimentation. In fact ethanol isolates nucleic acid from solution. After obtaining RNA to do next stages,
this RNA was confirmed both qualitatively and quantitatively to reduce probability of error and erroneous
results at next phase. Nano-drop machine was used to determine quality or value of RNA. Electrophoresis was
carried out on agarose 1% to determine no failure or intact in RNA molecule and investigation of DNA
contamination to confirm RNA quality. If RNA was healthy, two distinguished bands should be seen, one of
which is related to 18 sRNA and the other is related to 28 sRNA which are considered as the main component of
RNA in Eukaryotic cells.
cDNA synthesis: Fermantas kit and related manual were used for cDNA synthesis. This kit works based on
reverse transcription. I other words with transcription of RNA, it makes its supplement DNA.
Real-time RT-PCR:
Real-time PCR was carried out using SYBR green color. Its basis is so that, SYBR green color emits
Fluorescence light through connecting to small duplicated DNA thread. Hence as reaction goes on, since cDNA
is propagated, more SYBR Green will be able to connect to cDNA and consequently fluorescence increases
accordingly. In this study, beta Glucuronide gene was used as reference to compare gene expression level of
Enzyme Caspase-3 in studied groups and it is due to consistent gene expression in the cell.
Primer design and selection: using available software of Primer 3 and Primer Express, related frequency
investigation was selected and designed in primer Gene Bank and specified probe for enzyme gene of Caspase3, in a way that it was able to show maximum efficiency. Common PCR methods were used to confirm
designed primers' efficiency as follows:
Caspase-3:
Sense, 5′-TTCAGAGGGGATCGTTGTAGAAGTC-3′;
Antisense, 5′-CAAGCTTGTCGGCATACTGTTTCAG-3′
To prepare primer, 1ml of sterile distilled water was added to vials specific for maintaining primer which
have liyoflization form and after mixing they were distributed in 50ul micro tubes and micro tubes were kept in
-200 centigrade.
Statistical Analysis:
Obtained data were analyzed by SPSS version 22 and using ANOVA statistical tests. Differences were
considered significant when P<0.05. Data were displayed in the form of standard deviation (mean-+SE).
60
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
Results:
Results of Frozen Section and Immuno-histochemical staining:
Results obtained from Staining obtained from frozen section and Immuno-histochemical staining from
gastric biopsy is explained as follows:
Fig. 1: Immuno-histochemical staining by antibody of anti-Caspase-3 in gastric cancer tissues, before and after
chemotherapy in studied groups.
As it can be seen in Figure-1(section a of this figure), gastric biopsy samples were taken from patients at
first day, when they referred to endoscopy unit for diagnosis and they were studied by frozen section and
Immuno-histochemical staining. Section b of the figure shows gastric cancer tissue biopsy taken and studied
after three courses of chemotherapy, treated by Cis-Platin in patients of group 1. The section c of Figur-1 show
gastric cancer tissues biopsy in gastric cancer patients which have been taken and studied from patients of group
2 after three courses of chemotherapy by Cis-Platin along with fatty acids of omega 3, 6 and 9. As it has been
identified with black arrows in the figure, active enzyme protein of Caspase-3 indicated significant increase in
patients of group 2 after three courses of chemotherapy along with using omega fatty acids supplement.
Extraction and quantification of enzyme gene of Caspase-3 was carried out to confirm such increase in next
phase.
Fig. 1: Omega fatty acids effect on protein level of Caspase-3 in gastric cancer tissue after three courses of
chemotherapy in patients of group 1 and 2. (*p=0.0065)
As it can be seen in diagram 1, enzyme protein level of caspase 3 indicated increase in gastric cancer tissue
of both groups after three curses of chemotherapy with Cis-Platin. But this increase was significant among
patients of group 2 i.e. cancer patients who received fatty acids of omega 3,6 and 9 as supplement along with
61
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
chemotherapy and It was significant statistically (p=0.0065) the explanation for this is that, values obtained
from frozen section method and Immuno-histochemical staining were obtained as semi quantitative.
Results of Real-time RT-PCR:
Synthesized cDNA was extracted from RNA to be used in doing Real-time and RT-PCR: caspase 3 gene
expression level was compared in studied samples. GUSB gene was used as housekeeping gene for normalizer.
Standard curve was used for semi quantitative analysis of propagated products which was carried out with
∆∆CT method. In this method, first some parts of synthesized cDNA were entered to real time PCR reaction in
duplicate form and standard curve along with propagation curve were drawn for caspase-3 enzyme gene using
BIO-RAD iQ5 software version 2. Closeness of efficiency to 1 indicated efficiency of 100% and consequently
optimal propagation conditions. Moreover considering that efficiency level of caspase-3 enzyme gene
(considered gene) was close to GUSB gene (reference gene), data analysis became possible in ∆∆CT method.
Fig. 2: Gene expression level curve of caspase-3 in studied groups compared to standard gene.
As it can be seen in Fig. 2 caspase-3 gene expression level has been shown for studied groups compared to
standard gene. Red line indicates standard gene or reference gene and blue line shows caspase-3 enzyme gene
expression before starting treatment and green line indicates capase-3 enzyme gene expression level after three
courses of chemotherapy with cisplatin (group 1) and purple line indicates caspase-3 enzyme gene expression in
group 2 i.e. gastric cancer patients after three courses of chemotherapy along with consuming fatty acid
supplements of omega 3, 6, 9.
Discussion:
Chemotherapy is one of standard treatment methods for treating most cancers. Although this treatment
method is notable for cell death induction or apoptosis in cancer cells, it is observed in most cases, it is along
with cancer recurrence, finally resulting in patient's death. Gastric cancer treatment is also one of most important
challenges of medical sciences. The aim of present investigation is to investigate effects of prescribing fatty acid
food of omega 3, 6, 9 on gastric cancer development in patients with gastric Adenocarcinoma and reduce of
medical resistance through investigation of activity level of caspase-3 enzyme. Prior to our study, Mad Havi et
al (1994), Lu et al (2010) and Die et al (2013) have indicated that unsaturated fatty acids have toxic effect on
cancer cell through double bond (PUFA). But still the exact mechanism of how these fatty acids influence
cancer cells isn’t determined. However different studies have indicated different mechanisms for anti-cancer
effects of these fatty acids.
Cursta et al (2011), Shiruta et al (2005), Liu et al (2009, Manjazeb et al (2006) and Nariyan et al (2001)
indicated that anti cancer mechanisms of omega unsaturated fatty acids are accompanied with increase of cell
death level in cancer cells. These findings are in balance with findings of our study.
Moreover Nasar et al ( 1992), Chilik et al (2007), Liang et al (2009) indicated that omega 3,6 fatty acids
have toxic effect on cancer cells, resulting in oxidative stress induction and decrease in cancer cells growth. All
of these findings were obtained from above mentioned studies and mechanisms obtained from cell medium and
studies on experimental animals. Hence evidences mentioned in these studies can not mention the exact
mechanism of anti-cancer effects of omega fatty acids and they cannot be generalized for clinical samples and to
findings of our study. So it can be claimed that our study is the first to investigate anti-cancer effects of omega
fatty acids on human samples and it can be said that we are just at the beginning of the way.
62
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
As it was shown in our study, omega 3, 6, 9 fatty acids prescription as oral supplements along with CisPlatin drug increased active protein of caspase-3 enzyme in patients with gastric Adenocarcinoma and also it
increased gene expression level of these enzymes in gastric cancer tissue of patients participated in this study.
These have been displayed in diagram (1) and Fig.(2).
An ambiguous point in such studies is that whether toxic effects of omega fatty acids are the same on
healthy cells or not? In a study carried out by Die et.al(2013), Begin et.al(1985) and Dus et al (1991) it has been
mentioned that toxic effects of fatty acids are on cancer cells selectively and they have no effect on healthy cells
or its effects are inconsiderable on them and if used dosage of such fatty acids are more than 40 micro gram per
each ml in ratio of every 105 cells, they can be toxic in healthy cells, also. Hence considering results of present
investigation, dosage for selected patients of this study was common and normal (i.e. 3600 mg per a day or in
better words 1200 mg per a day for each fatty acid). Doing so, we were able to study and observe toxic effects
on cancer cells. One weak point of our study was that this study should be carried out on healthy tissue of the
gastric. This should be taken into consideration in future studies.
However since no similar study on gastric cancer sample has been reported, so we cannot reach an overall
conclusion with findings of present study.
Conclusion:
Considering results of present investigation and different studies carried out in the field it seems that the
effects of omega 3,6,9 fatty acids supplement along with ordinary drugs used in chemotherapy can be efficient
to control gastric cancer. This was shown in present investigation with increase in caspase-3 enzyme activity
protein level and increase of this enzyme gene expression can be promising point in reducing medical resistance
in patients with gastric Adenocarcinoma.
Suggestions:
It is proposed that the effect of fatty acids to be studied simultaneously on gastric healthy tissue in patients
with gastric Adenocarcinoma. Moreover it is proposed that other anti-cancer mechanisms of omega 3, 6, 9 fatty
acids, mentioned in discussion section, to be evaluated and investigated.
REFERENCES
[1]
Malekzadeh, R., M. Derakhshan, Z. Malekzadeh, 2009. Gastric Cancer in Iran: Epidemiology and Risk
Factors. Arch Iran Med, 12(6): 576–583.
[2] Huei, K., 1998. High Expression of Thymidylate Synthase Is Associated with the Drug resistance of
Gastric Carcinoma to High Dose 5-Fluorouracil–Based Systemic Chemotherapy. American Cancer
Society, 6: 1626-1631.
[3] Ting-Jun, F., H. Li-Hui, C. Ri-Shan, L. Jin, 2005. Caspase Family Proteases and Apoptosis. Int J Cancer,
37(11) :719-727.
[4] John, CR., 2000. Mechanisms of Apoptosis. American Journal of Pathology, 157(5): 1415-1430.
[5] Li, J., S. Qin, J. Xu, W. Guo, J. Xiong, Y. Bai, G. Sun, Y. Yang, L. Wang, N. Xu, Y. Cheng, Z. Wang, L.
Zheng, M. Tao, X. Zhu, D. Ji, X. Liu, H. Yu, 2013. Apatinib for chemotherapy-refractory advanced
metastatic gastric cancer: results from a randomized placebo-controlled parallel-arm phase II trial. J Clin
Oncol, (31): 3219-3225.
[6] Bang, YJ., YW. Kim, HK. Yang, HC. Chung, YK. Park, KH. Lee, KW. Lee, YH. Kim, SI. Noh, JY. Cho,
YJ. Mok, YH. Kim, J. Ji, TS. Yeh, P. Button, F. Sirzén, SH. Noh, 2012. Adjuvant capecitabine and
oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): a phase 3 open-label randomised
controlled trial. Lancet, 379: 315-321.
[7] Kim, HS., HJ. Kim, SY. Kim, TY. Kim, KW. Lee, SK. Baek, TY. Kim, MH. Ryu, BH. Nam, DY. Zang,
2013. Second-line chemotherapy versus supportive cancer treatment in advanced gastric cancer: a metaanalysis. Ann Oncol, 24: 2850-2854.
[8] Madhavi, N., UN. Das, 1994. Effect of n-6 and n-3 fatty acids on the survival of Vincristine sensitive and
resistant human cervical carcinoma cells in vitro. Cancer Lett, 84: 31-41.
[9] Lu, X., H. Yu, M. Qi, SR. Shen, UN. Das, 2010. Linoleic acid suppresses colorectal cancer cell growth by
inducing oxidant stress and mitochondrial dysfunction. Lipids Health Dis, 9: 106.
[10] Dai, J., J. Shen, W. Pan, S. Shen, UN. Das, 2013. Effects of polyunsaturated fatty acids on the growth of
gastric cancer cells in vitro. Lipids in Health and Disease, 12: 71.
[11] Corsetto, PA., G. Montorfano, S. Zava, IE. Jovenitti, A. Cremona, B. Berra, AM. Rizzo, 2011. Effects of
n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane Lipids Health Dis, 10:
73.
[12] Shirota, T., S. Haji, M. Yamasaki, T. Iwasaki, T. Hidaka, Y. Takeyama, H. Shiozaki, H. Ohyanagi, 2005.
Apoptosis in human pancreatic cancer cells induced by eicosapentaenoic acid. Nutrition, 21: 1010 –1017.
63
Behzad Ordoubadi et al., 2014
Advances in Environmental Biology, 8(23) Special 2014, Pages: 57-63
[13] Liu, WH., LS. Chang, 2009. Arachidonic acid induces Fas and FasL upregulation in human leukemia
U937 cells via Ca2+/ROS-mediated suppression of ERK/ c-Fos pathway and activation of p38 APK/ATF2 pathway.Toxicol Lett, 19: 1140–148.
[14] Monjazeb, AM., KP. High, A. Connoy, LS. Hart, C. Koumenis, FH. Chilton, 2006. Arachidonic acidinduced gene expression in colon cancer cells. Carcinogenesis. 27: 1950–1960.
[15] Narayanan, BA., NK. Narayanan, BS. Reddy, 2001. Docosahexaenoic acid regulated genes and
transcription factors inducing apoptosis in human colon cancer cells. Int J Oncol, 19: 1255–1262.
[16] Nassar, BA., UN. Das, YS. Huang, G. Ells, DF. Horrobin, 1992. The effect of chemical
hepatocarcinogenesis on liver phospholipid composition in rats fed n-6 and n-3 fatty acid- Supplemented
diets. Proc So Exp Biol Med, 199: 365–368.
[17] Celik, GE., FO. Erkekol, Z. Mgil, M. Melliw, 2007. Lipoxin A4 levels in asthma: relation with disease
everity and aspirin sensitivity. Clin Exp Allergy, 37: 494–1501.
[18] Liang, G., Y. Pu, L. Yin, R. Liu, B. Ye, Y. Su, Y. Li, 2009. Influence of different sizes of titanium dioxide
nanoparticles on hepatic and renal functions in rats with correlation to oxidative stress. J Toxicol Inviron
Health (A), 72: 740–745.
[19] Begin, ME., UN. Das, G. Ells, DF. Horrobin, 1985. Selective killing of tumor cells by polyunsaturated
fatty acids. Prostaglandins Leukot, Med, 19: 177–186.
[20] Das, UN., 1991. Tumoricidal action of cis-unsaturated fatty acids and its relationship to free radicals and
lipid peroxidation. Cancer Lett, 56: 235–243.