Phospholipase A2 Isoforms in Benzo (α) Pyrene-Induced Molecular Changes in Colon Cancer Cells: A Plausible Therapeutic Target

Background: Colorectal cancer (CRC) is the third most common cancer prevalent in men, and the second in women worldwide. The exact cause and pathogenesis of this disease remains unknown. In every cell multiple phospholipase A 2 (PLA 2 ) exists, but their contribution to the cell function has yet not been determined. The increased activity of these isoforms may be important in the generation of inflammatory mediators which finally are involved in the initiation of carcinogenesis. This study has explored the pathways involved in the differential response of HCT-15 and HT-29 colon cell lines during benzo(α)pyrene [B(α)P]-induced molecular changes. Methods: HT-29 and HCT-15 cells were used for the present study. Benzo(α)pyrene [B(α)P]-induced molecular changes were evaluated through cell viability using MTT assay, reactive oxygen species (ROS) measurement using 2,7 dichloro-dihydro-fluorescin diacetate (DCFH-DA), The gene expressions of PLA2 isoforms were estimated by RT- PCR. Further, knockdown of PLA 2 isoform in transfected cells was done with siRNA. Results: The cell viability decreased significantly after exposure with B(α)P in both cell lines. Reactive oxygen species (ROS) production after exposure with B(α)P was significantly induced in both types of colon cell lines. The gene expressions of PLA2 isoforms were also estimated. Three PLA 2 isoforms such as IB and, IID, IVA were induced after exposure with B(α)P respectively in HT -29 and HCT-15 cell lines. Further, a significant knockdown of PLA 2 isoform in transfected cells with siRNA, and B(α)P exposure was observed in HCT-15 and HT-29 cells. There was a significant induction of ROS in IVA transfected HCT-15 cells exposure with B(α)P. Conclusion: It is concluded that each cell line behaved differently, specific PLA 2 siRNA are involved in controlling the cascades related ROS, depending upon the origin of the cells. 2 ) (CSC) and B(α)P (a major carcinogen of cigarette smoke) in vitro . This study aims to explore the pathways involved in the differential response of HCT-15 and HT-29 colon cell lines during [B(α)P]-induced molecular changes. DHE and mitochondrial ROS using DHR123. Our results clearly show that PLA2 IVA is identified in the intracellular H2O2 mediated oxidative stress. In this study, we have found that B(α)P induced intracellular ROS in both the colon cell lines (HCT-15 and HT-29). However, the extent of ROS production was more in HT-29 cells than in HCT-15 cells. Interestingly the induction of ROS, in HT-29


Introduction
Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide [1]. The incidence of CRC has been reported to increase 2-4 times in the past few decades in many countries of the Asia-Pacific region. Presently the most common treatment option is surgery, and that also depends on various factors like the site of the cancer and the presence and extent of metastasis.
The risk of colorectal cancer begins with the formation of abnormal polyp growth that later grows to cancer. The major causes include the over-eating, alcohol consumption and smoking. Studies also suggest that a proper life-style maintenance including the controlled diet could reduce the abnormal poly formation and further cancer. Ursolic acid is a phytochemical present in most plants and has the ability to reduce the cell proliferation of breast and colon cancer cells through various pathways [2].
The Benzo(α)pyrene [B(α)P], a known carcinogen, presents in diet and environment such as cigarette smoke (CS), grilled meats and byproducts of industrial incineration [3]. The oxidative stress caused by the constituents of CS such as B(α)P in normal as well as in the mutated cells may lead to the remodeling of membrane lipids through upregulation of various signaling events such as activation of lipid specific enzymes especially phospholipases. Almost in every cell type, multiple isoforms of phospholipase A 2 (PLA 2 ) but their contribution to the cell functions has not yet been clearly determined.
Phospholipase A 2 superfamily is composed of many lipolytic enzymes whose common feature is to hydrolyze the fatty acid present in the sn-2 position of glycerophospholipids. It is reported that PLA 2 can act synergistically with reactive oxygen species (ROS) to cause cellular injury due to enhanced susceptibility of peroxidized membrane to the action of PLA 2 . Phospholipases type A 2 is involved in the generation of a wide range of biomediators, including lysophospholipids, arachidonic acid, and metabolites of arachidonic acid that can enhance tumor cell growth, invasion, and metastasis [4]. However, it is not known whether PLA 2 isoforms are expressed in human colon cancer and whether PLA 2 isoforms influence the malignant potential of colon cancer cells. Besides that, PLA 2 isoenzymes may behave differentially to cigarette smoke condensate (CSC) and B(α)P (a major carcinogen of cigarette smoke) in vitro.
This study aims to explore the pathways involved in the differential response of HCT-15 and HT-29 colon cell lines during [B(α)P]-induced molecular changes.

Cell lines:
Colon adenocarcinoma cancer cell lines (HCT-15 and HT-29) were procured from National Centre for Cell Science, Pune, India.
[b(α)p] Preparation: B(α)P were procured from sigma USA and dissolved in DMSO, the working concentration (0.025%) was prepared in a sterile medium.

Ros And Superoxide Radicals (sor):
Levels of intracellular ROS, mitochondrial ROS and SOR were measured by the shift in fluorescent intensity resulting from oxidation of DCFH-DA (for ROS), DHR123 (for Mitochondrial ROS) and DHE (for Superoxide radical) fluorescence dye [6]. Briefly, 0.5 × 10 5 cells/well were seeded into 12-well plates and allowed to grow overnight in complete media. After 24 hr of incubation, the medium was replaced with a fresh medium and the cells were treated with varying concentrations of B(α)P for a specific time interval. Cells were incubated with 5 µM DCFH-DA, 10 µM DHR123 or 10 µM DHE fluorescence dye for 30 min. Cells were challenged with B(α)P for 24 hr and 48 hr. Thereafter, cells were washed, harvested and resuspended in chilled phosphate buffer saline (PBS). Finally, cells were subjected to fluorescence-activated cell sorting (FACS; Beckton Dickinson FACSCan). Levels of ROS and SOR were represented in terms of mean fluorescent intensity (MFI).

Rna Extraction And Cdna Synthesis:
Total RNA was isolated by using Trizol reagent according to the manufacturer's instructions. Briefly, 1 ml Trizol reagent was added to the cells followed by chloroform. The mixture was incubated at room temperature for 15 min and centrifuged at 12000 x g for 15 min at 4ºC resulting in the separation of an upper aqueous phase and lower organic phase. The upper aqueous phase was carefully transferred to a fresh eppendorf tube. After adding and shaking with isopropanol mixture was incubated for 10 min and then centrifuged at 12000 x g for 10 min. The pellet thus obtained was washed with 75% ethanol followed by centrifugation for 5 min at 7,500 x g. The RNA pellet was air-dried and dissolved in an appropriate volume of DEPC water and stored at -80ºC. Spectrophotometric measurement of RNA was done to quantify its purity. Further, one microgram RNA was used for cDNA synthesis. The cDNA synthesis was carried out from the purified and intact total RNA by using the Revert AidTM first strand cDNA synthesis kit (Fementas) according to the manufacturer's instructions.
Reverse Transcription-polymerase Chain Reaction (rt-pcr): Effect of B(α)P on mRNA expression of PLA 2 isoforms was conducted through RT-PCR for the analysis of mRNA levels of PLA 2 groups (IB, IID, III, IVA, IVB, IVC, VI, X, acid iPLA 2 , iPLA 2 ). In RT-PCR reactions (25 µl) 100 ng cDNA was used as template DNA. The primer sequences were designed from the mRNA sequence of genes available in the Genebank or from previously published literature. The details of primer sequences and RT-PCR conditions used for the study are given in Table 1. The expression levels of different genes were calculated by relative quantification using b-actin transcript levels for normalization. The fold change in mRNA expressions was calculated by the comparative ΔΔCt method [7]. PLA 2 siRNA (IB, IID and IVA from Sigma Aldrich, USA) and scrambled siRNA were transfected into HCT-15 cells using transfection lipofectamine™ 2000 reagent as described previously [8]. The sequences of all siRNAs used in this study are provided in Table 2. Briefly, in HT-29 (IB) and HCT-15 (IID and IVA) cells the PLA 2 gene was silenced by using specific siRNA against the group IB, IID and IVA PLA 2 gene.
The cells (1 × 10 5 per well) were seeded in 2 ml antibiotic-free medium supplemented with 10% FBS in a six-well culture plate. After 60-80% confluency the cells were siRNA (IB, IID and IVA from Sigma Aldrich, USA) and scrambled siRNA were transfected into HCT-15 cells using transfection lipofectamine™ 2000 reagent. Briefly, in HT-29 (IB) and HCT-15 (IID and IVA) cells the PLA 2 gene was silenced by using specific siRNA against the group IB, IID and IVA PLA 2 gene. The cells (1 × 10 5 per well) were seeded in 2 ml antibiotic free medium supplemented with 10% FBS in a six well culture plate. After 60-80% confluency the cells were transfected with transfection reagents for 6-8 hr at 37 0 C in a CO 2 incubator following the recommended protocol provided with siRNA oligos. Before transfection, the medium was replaced with fresh medium without antibiotics. After transfection, additional media was added with 20% FBS so that the final concentration of FBS becomes 10% in the final mixture followed by the treatment with a specific concentration of B(α)P for 48 hr. After completion of the incubation time, total RNA was isolated as mentioned above transfected with transfection reagents for 6-8 hr at 37 0 C in a CO 2 incubator following the recommended protocol provided with siRNA oligos. Before transfection, the medium was replaced with fresh medium without antibiotics. After transfection, additional media was added with 20% FBS so that, the final concentration of FBS becomes 10% in the final mixture followed by the treatment with a specific concentration of B(α)P for 48 hr. After completion of the incubation time, total RNA was isolated as mentioned above. Similarly, one microgram of RNA was converted into cDNA and expression of group IB, IID and IVA PLA 2 was checked by qRT-PCR (Lightcycler 480, Roche diagnostics, Germany). Table 2 Sequences of siRNAs S. No.
Name of target gene Sequence of siRNAs 1.
PLA2 IB Statistical analysis: The data were analyzed using the SPSS 16.0 software. All results were expressed as mean ± SD. The differences in all data were assessed by one-way analysis of variance (ANOVA) by the Bonferroni test and nonparametric method such as Kurskali Wallice wherever required. The difference was taken as statistically significant at p < 0.05.

Intracellular Ros Production:
Total ROS production in HCT-15 and HT-29 cells by B(α)P concentrations at different time intervals is shown in Fig. 2. In HCT-15 cells, ROS production was found to be significantly increased 48 hr after treatment of cells with varying concentrations of B(α)P (0.1, 1.0, 10, 50, 100 and 200 µM) respectively as compared to control (100%) (p=) 0.036) ( Fig. 2A)  Effect of group IB, IID, and IVA PLA 2 gene silencing 11 Cell Viability: Effects of B(α)P on cell viability after silencing the specific PLA 2 genes are shown in Fig. 7 at 48 hr.  (Fig. 8D). Whereas ROS production in the presence of group IID PLA 2 siRNA in HCT-15 cells was 121 ± 2.8%, 97 ± 10.3% and 92 ± 8.6% in scrambled siRNA control and siRNA (IID) without and with 0.1 µM of B(α)P respectively (Fig. 8E). On the other hand, ROS production in the presence of group IVA siRNA with exposure to 0.1 µM B(α)P was 104 ± 12.5%, 99 ± 5.2%, and 96 ± 4.2% in scrambled siRNA control and siRNA (IVA) without and with 0.1 µM of B(α)P respectively in HCT-15 cells (Fig. 8F). However, all the values in the experimental groups mentioned above were statistically non-significant to scrambled control.
Sor Production: Relative SOR production in the presence of group IB, IID and IVA siRNA in both cell lines at 48 hr is shown in Fig. 9. Relative SOR production in HT-29 cells transfected with group IB PLA 2 gene siRNA treated with 0.1 µM B(α)P in scrambled siRNA control and siRNA (IB) without and with 0.1 µM of B(α)P were found to be 92 ± 4.9%, 103 ± 9.6% and 116 ± 3.5% respectively (Fig. 9A). Whereas, SOR production in scrambled siRNA control and siRNA (IID) without and with 0.1 µM of B(α)P was 91 ± 8.0%, 86 ± 6.7%, and 90 ± 3.6% respectively in HCT-15 cells (Fig. 9B). However, the SOR production in HCT-15 cells in the presence of siRNA (IVA) was 96 ± 12.0%, 73 ± 10.7% and 69 ± 13.0% in scrambled siRNA control and siRNA (IVA) without and with 0.1 µM of B(α)P respectively (Fig. 9C). Like other parameters such as cell viability, ROS, mitochondrial ROS, etc in this case also the changes were non-significant to scrambled control.

Discussion
CS continues to remain one of the most important health hazards and it contributes extensively to many diseases including respiratory disorders characterized by inflammatory changes in the lungs [9,10]. It is involved in many other cancers like pharynx, larynx, esophagus, etc. various studies also suggested the relationship between smoking and stomach, liver and colon cancer [11,12]. However, the molecular and cellular mechanism involved in the pathogenesis of smoking-related colon cancer remains unknown.
CS is a very complex, multi-factorial stimulus with over 5300 identified [13]. Approximately 100 carcinogens, co-carcinogens, mutagens, and/or tumor promoters are present in CS [14]. One of the most prominent carcinogens in burned as well as, unburned tobacco, is benzo(α) [15]. The complex changes in cellular functions, morphology and gene expression caused by CS constituents that are initiators as well as promoters of carcinogenesis [16], involve a combination of direct and indirect effects on cells and their components. We found that treatment with B(α)P resulted in a significant change in cell shape and size in both the cell lines (HCT-15 and HT-29 cells). There was prominent fragmentation, loss of cohesiveness, irregular shape and the cells appeared in clumps. Our present study is in check this word with an earlier report, on MCF-10A and MCF-12A cell lines which also showed these cellular changes after CSC treatment. These morphological changes may be due to multiples factors like altered cell membrane integrity caused by increased formation of ROS leading to changes in cytotoxicity, proliferation and apoptosis.
Cytotoxicity, an important factor in understanding the mechanism of action of chemicals on cells and tissues, may modulate the activity of other agents, including free radicals, oxidants, irritants and genotoxins [17]. These intracellular moieties have been reported to induce adverse effects in the body [18]. In this study, we observed that no cytotoxic effects of B(α)P at concentrations below 1 µM in HCT-15 and below 50 µM in HT-29 cells, but at higher concentrations, the cell viability decreased significantly. Similar observations have been reported earlier on the effects of B(α)P in other cell lines [19].
Cellular levels of ROS reflect a delicate balance between ROS production and detoxification. Despite the existence of such well-coordinated cellular ROS detoxification systems, the uncontrolled ROS production may overwhelm these defense mechanisms. Many previous studies have shown that aqueous cigarette tar extracts increase cellular production of ROS [20,21] leading to increased proinflammatory gene expressions [22].  Another pathway of regulating the superoxide radicals is through ubiquinol (QH 2 ) which has been shown to act as a reducing agent in the elimination of various peroxides in the presence of succinate [24]. Though like total ROS, SOR production by B(α)P was more in HT Many previous studies documented the PLA 2 s such as cPLA 2 , sPLA 2 group IIA, sPLA 2 group IID and sPLA 2 group X has been involved in colon tumor development [28,29]. Our findings are similar to that of Valentine et al., 2000 [30] to some extent, wherein they found over-expression of cPLA 2 in 49% of colon carcinomas patients as well as in four colon cancer cell lines. These results coincide with the elevated expression of cPLA 2 α in human colon adenocarcinoma [31]. On the other hand, cPLA 2 expression has been reported to be was diminished in azoxymethane-induced mouse colon tumors and knock-out of cPLA 2 enhanced colon tumor development [32].

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