Role of Phenolic Compounds in Nitric Oxide Synthase Activity in Colon and Breast Adenocarcinoma

Abstract

Cancer chemopreventive agents are designed to reduce the incidence of tumorigenesis by intervening at one or more stages of carcinogenesis. This study aimed to determine the effects of resveratrol (RES) and tannic acid (TA), which are chemopreventive agents, on the nitric oxide synthase (NOS) levels that are effective for development of cancer in colon and breast cancer cell lines. The CaCo-2 (human colon carcinoma cell line) and MCF-7 (Michigan Cancer Foundation-7; human breast adenocarcinoma cell line) cells were grown in the laboratory. RES and TA were used to treat CaCo-2 and MCF-7 cells. Nitric Oxide Synthase Assay Kit was used to determine the NOS enzyme activity of CaCo-2 and MCF-7. Statistical differences between control and RES- and TA-treated cells were calculated using the Student's t-test for double comparison. It was observed that NO activity was generally decreased in CaCo-2 and MCF-7 cells, in which RES and TA were applied. Results suggest that the phenolic compounds RES and TA have different effects on NOS enzyme activity of the colon and breast cancer cells.

Introduction

Carcinogenesis may arise as a result of chemical or biological insults to normal cells in a multistep process that involves changes at the genetic level (initiation) followed by promotion and progression that ultimately lead to malignancy.¹ In recent years, a considerable emphasis has been focused on the importance of the naturally available botanicals that can be consumed in an individual's everyday diet and that can also be useful as a chemopreventive or chemotherapeutic agent for certain diseases, including cancers. A wide variety of botanicals, mostly dietary flavonoids or polyphenolic substances, have been reported to possess substantial anticarcinogenic and antimutagenic activities because of their antioxidant and anti-inflammatory properties.²

Resveratrol (RES) and tannic acid (TA) are two of these chemopreventive agents.^1,3,4 RES (3,5,4′ trihydroxystilbene) is a phytoalexin phenolic compound that is known to regulate the metabolism of lipid, inhibit eicosanoid synthesis, and suppress platelet aggregation, apart from functioning as an antioxidant, and is known as anticarcinogenic, anti-inflammatory, antimutagenic, antiproliferative, antiviral, antibacterial, estrogenic, and vasodilator agent.^1,5 TA is a polyphenolic compound and can function as anticarcinogenic, antioxidant, antimutagenic, antimicrobial, antiallergic, and anti-inflammatory agent.⁴

Nitric oxide (NO) is a multifunctional inorganic gas synthesized by three different isoforms of nitric oxide synthase (NOS) and is generated in neuron, glia, endothelial cells, and macrophages.^6
–8 As an endothelium-derived relaxing factor, a mediator of immune responses, a neurotransmitter, a cytotoxic free radical, and a signaling molecule, NO plays crucial roles in virtually every cellular and organ function in the body. The discovery of NO synthesis has unified traditionally diverse research areas in nutrition, physiology, immunology, pathology, and neuroscience. Increasing evidence over the past decade shows that many dietary factors, including protein, amino acids, glucose, fructose, cholesterol, fatty acids, vitamins, minerals, phytoestrogens, ethanol, and polyphenols, either are beneficial to health or contribute to the pathogenesis of chronic diseases partially through modulation of NO production by inducible NOS (iNOS) or constitutive NOS (cNOS).⁹ In addition to reactive oxygen species, reactive nitrogen in the form of NO has been also implicated in the regulation of cellular proliferation.¹⁰ The highly reactive free radicals, produced by NOS, have been implicated in the modulation of carcinogenesis. Overexpression of iNOS, a common phenomenon during chronic inflammatory conditions, generates sustainable amounts of NO; the fact that its reactive intermediates are mutagenic, causing DNA damage or impairment of DNA repair, has been well established in carcinogenesis. Recent studies also implicate NO as having a key signaling molecule that regulates processes of tumorigenesis. Increased expression of iNOS has been observed in tumors of the colon, lung, oropharynx, reproductive organs, breast, and central nervous system besides its occurrence in chronic inflammatory diseases. Thus, developing selective inhibitors of iNOS and NO-releasing agents may lead to important strategies for chemoprevention of cancer.¹¹ Although most published studies have focused on only a single nutrient and have generated new and exciting knowledge, future studies are necessary to investigate the interactions of dietary factors on NO synthesis and to define the underlying molecular mechanisms.⁹

The aim of this study was to investigate the effects of RES and TA on NOS activity in colon (human colon carcinoma cell line: CaCo-2) and breast (human breast adenocarcinoma cell line: Michigan Cancer Foundation-7 [MCF-7]) cancer cell lines.

Materials and Methods

Cell culture

The MCF-7 and CaCo-2 cells were grown in the Laboratory of Medical Biology, Eskisehir Osmangazi University, Eskisehir, Turkey. The MCF-7 breast cancer cells were cultured in the flasks using RPMI 1640 (Roswell Park Memorial Institute 1640, Biological Industries) supplemented with 10% fetal calf serum (Sigma-Aldrich) and penicillin–streptomycin (Sigma-Aldrich). The CaCo-2 colon cancer cells were cultured in the flasks using Eagle's minimum essential medium (Sigma-Aldrich) supplemented with 10% fetal calf serum and penicillin–streptomycin. Cells were maintained in a 5% CO₂ atmosphere at 37°C.

Cell treatment

RES (Sigma-Aldrich) and TA (Acros Organics) were used to treat CaCo-2 and MCF-7 cells. The cells were plated in the flasks and in a medium containing different concentrations of RES and TA (0, 25, 50, and 100 μM in dimethyl sulfoxide) for 24, 48, and 72 hours.

NOS activity measurement

Required number of cells were prepared for each measurement by counting them on a Neubauer slide by trypan blue staining. Nitric Oxide Synthase Assay Kit (EMD Chemicals) was used to determine the NOS enzyme activity of CaCo-2 and MCF-7 before and after the RES and TA treatments. The kit was designed for measuring total nitrate and nitrite contents, which are the stable end products of the reaction of NO in biological fluids and are derived from NO produced by NOS under controlled assay conditions.

Statistical analysis

Data were reported as means ± standard error. Statistical differences between control and RES- or TA-treated cells were calculated using the Student's t-test for double comparison. A p-value of <0.05 was considered to indicate a significant difference.

Results

In CaCo-2 cell line

NOS activity according to RES and TA in different doses and hours is shown in Table 1. It was observed that NOS activity was gradually decreased in all groups at the 24, 48, and 72nd hours in the CaCo-2 cell line. It was determined that NOS activity was also gradually decreased in the cells administered with RES of 25, 50, and 100 μM at the 24th hour and 50 and 100 μM at the 48th hour. Nevertheless, it was observed that application of 25 μM RES at the 48th hour and 25, 50, and 100 μM RES at the 72nd hour increased NOS activity when compared with the control. It was determined that NOS activity was gradually decreased in the cells administered with TA at the 24th and 48th hours as a result of different dose applications. However, it was observed that application of 50 μM TA at the 48th hour and 25, 50, and 100 μM TA at the 72nd hour increased NOS activity when compared with the control.

Table 1.

Nitric Oxide Synthase Enzyme Activity in CaCo-2 Cell Line

			NOS activity (total nitrate and nitrite content in μM)
Cell line	Groups	Dose (μM)	24th hour	48th hour	72nd hour
CaCo-2	Control	0	2.7033 ± 0.007	1.223 ± 0.019	0.430 ± 0.017
	Resveratrol	25	1.797 ± 0.024^**	1.433 ± 0.024^**	0.917 ± 0.012^**
		50	1.440 ± 0.026^**	0.847 ± 0.020^**	0.830 ± 0.035^**
		100	1.553 ± 0.030^**	1.027 ± 0.032^**	0.497 ± 0.029^a
	Tannic acid	25	1.517 ± 0.032^**	0.650 ± 0.025^**	0.507 ± 0.092^a
		50	1.330 ± 0.000^**	1.303 ± 0.003^*	0.723 ± 0.026^**
		100	1.727 ± 0.018^**	0.980 ± 0.006^**	0.607 ± 0.023^**

NOS enzyme activity was analyzed using Nitric Oxide Synthase Assay Kit in CaCo-2 cell line. NOS enzyme activity in resveratrol and tannic acid treatments was calculated according to the control. Mean ± standard error values are shown for three experiments (n = 3).

Not significant, *p < 0.05, **p < 0.001.

NOS, nitric oxide synthase.

In MCF-7 cell line

NOS activity according to RES and TA in different doses and hours is shown in Table 2. It was observed that NOS activity was increased in the control group of MCF-7 cell line at the 48th and 72nd hours when compared with the 24th hour. It was determined that NOS activity was decreased in the cells administered with RES at the 48th and 72nd hours as a result of different dose applications. Nevertheless, it was observed that application of 25, 50, and 100 μM RES at the 24th hour significantly increased NOS activity when compared with the control. Although no significant difference was determined in NOS activity for all RES doses at the 48th hour when compared with the control group, it was specified that all RES doses significantly decreased NOS activity at the 72nd hour when compared with the control. It was determined that application of 25, 50, and 100 μM TA at the 24th hour significantly increased NOS activity when compared with the control group. However, it was observed that NOS activity was decreased at the 48th hour for all TA doses when compared with the control group. It was determined that application of 25 and 50 μM TA at the 72nd hour significantly decreased NOS activity when compared with the control, whereas application of 100 μM TA at the 72nd hour significantly increased NOS activity when compared with the control.

Table 2.

Nitric Oxide Synthase Enzyme Activity in MCF-7 Cell Line

			NOS activity (total nitrate and nitrite content in μM)
Cell line	Groups	Dose (μM)	24th hour	48th hour	72nd hour
MCF-7	Control	0	0.967 ± 0.012	1.280 ± 0.021	1.040 ± 0.015
	Resveratrol	25	1.631 ± 0.021^***	1.223 ± 0.019^a	0.547 ± 0.027^***
		50	1.661 ± 0.026^***	1.260 ± 0.017^a	0.597 ± 0.042^***
		100	1.801 ± 0.026^***	1.210 ± 0.012^a	0.640 ± 0.025^***
	Tannic acid	25	2.263 ± 0.032^***	1.133 ± 0.019^***	0.810 ± 0.021^***
		50	1.487 ± 0.007^***	1.171 ± 0.015^**	0.430 ± 0.017^***
		100	2.333 ± 0.023^***	1.190 ± 0.045^*	2.827 ± 0.035^***

NOS enzyme activity was analyzed using Nitric Oxide Synthase Assay Kit in MCF-7 cell line. NOS enzyme activity in resveratrol and tannic acid treatments was calculated according to the control. Mean ± standard error values are shown for three experiments (n = 3).

Not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

NOS, nitric oxide synthase.

Discussion

This study aimed to determine the effects of NOS activity, whose effects on different cancer types had been studied, and to determine the effects of RES and TA, which were thought to be potential chemopreventive agents in colon and breast adenocarcinoma cells and other cancer types. Reactive oxygen species, reactive nitrogen in the form of NO, have been also implicated in the regulation of cellular proliferation, but its role as a proliferative signal is not well defined, because it appears to depend on the cell type responsible for its release and the NOS isoforms within that cell, as well as on the concentration of released NO and the composition of the intracellular milieu.¹⁰ There are ongoing studies about NOS and various chemopreventive agents. In a study performed on RES, Tsai et al. reported that RES significantly inhibited NO production in active macrophages in RAW 264.7 murine cell activated with bacterial lipopolysaccharide and decreased systolic iNOS protein quantities at the level of mRNA.³ In the present study, it was observed that this activity was decreased in CaCo-2 cells through RES in all doses of the 24th hour and in all doses of the 48th hour except for the dose of 25 μM. It was determined that the activity was decreased in MCF-7 cells at the 72nd hour depending on the increasing time. In the study of Hsieh et al. on BPAE cells isolated from the intrapulmonary artery of calf lungs, it was specified that application of 10 μM RES had no effect on endothelial cell NOS (ecNOS) activity, whereas 50–100 μM doses increased ecNOS activity three times.⁵ Besides, in a study in which the antiproliferative effect of RES was indicated on the gastric adenocarcinoma cells (SNU-1) and the regulatory effect of reactive nitrogen and oxygen production was specified, it was suggested that NO was produced at low quantities through stimulation of NOS.¹⁰ In the present study, it was observed that this activity was significantly increased in CaCo-2 cells through RES in 25 μM dose at the 48th hour and all doses at the 72nd hour and in MCF-7 cells through RES in all doses of the 24th hour. The present study specifies that this effect varied depending on time rather than the dose, because RES was effective at the early hours (the 24th hour), whereas a decrease was observed with increasing time. Holian et al. reported that NOS activity was increased three times as a result of applying RES for 16 hours, although SNU-1 cells did not express any measurable NOS activity under basal conditions. However, a measurement that was performed at a later hour was not reported. Similarly, in the same study, it was reported that NO induced apoptosis in colonic epithelial cells and that there was evident apoptotic response in SNU-1 cells that were subjected to high concentrations (100 μM) of RES, resulting in maximal NOS activation; however, RES applied in low concentrations (10 μM) at similar times did not induce apoptosis.¹⁰ In a previous study, CaCo-2 cells were analyzed in terms of RES and apoptosis.¹² On analyzing the results of the present study in terms of this aspect, it was observed that apoptosis was increased depending on the increasing dose, whereas there was no parallelism between NOS increase and apoptosis increase. In another study, it was stated that NO may both stimulate and suppress apoptosis. In this study, it was shown that ecNOS was expressed in MCF-7 cells; however, other NOS isoforms were not expressed.¹³

There are limited studies on TA, another agent used in the present study, as it is a more recent chemopreventive agent when compared with RES.

A previous study reported that TA and green tea polyphenols were potential inhibitors of iNOS activity.¹⁴ In another study, it was reported that epigallocatechin gallate, which is a green tea polyphenol, decreased iNOS activity and protein levels through a decrease in expression of iNOS mRNA.¹⁵ In another study, it was observed that on incubation with epigallocatechin gallate, iNOS gene expression was inhibited when murine peritoneal macrophages were activated.¹⁶ In the present study, it was observed that this activity was generally decreased in CaCo-2 and MCF-7 cells administered with TA.

Conclusions

Results suggest that the phenolic compounds RES and TA have different effects on colon and breast cancer cells. The present study suggests that dose arrangements and processes are important in using chemopreventive agents for stopping or slowing down the cancer process. This shows that chemopreventive agents to be used in cancer treatment have different effects on different cancer types. In the future, further studies are needed with respect to these chemopreventive agents.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Bhat

, Pezzuto

. Cancer chemopreventive activity of resveratrol. Ann NY Acad Sci, 2002; 957:210.

Nandakumar

, Singh

, Katiyar

. Multi-targeted prevention and therapy of cancer by proanthocyanidins. Cancer Lett, 2008; 269:378.

Tsai

, Lin-Shiau

, Lin

. Suppression of nitric oxide synthase and the down-regulation of the activation of NFkappaB in macrophages by resveratrol. Br J Pharmacol, 1999; 126:673.

Lopes

, Schulman

, Hermes-Lima

. Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions. Biochim Biophys Acta, 1999; 1472:142.

Hsieh

, Juan

, Darzynkiewicz

et al. Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21^WAF1/CIP1, and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G₂ . Cancer Res, 1999; 59:2596.

Cai

, Li

, Goethe

et al. Downregulation of endocardial nitric oxide synthase expression and nitric oxide production in atrial fibrillation: Potential mechanisms for atrial thrombosis and stroke. Circulation, 2002; 106:2854.

Castillo

, Rama

, Dávalos

. Nitric oxide–related brain damage in acute ıschemic. Stroke, 2000; 31:852.

Tsumori

, Tanaka

, Yoshimura

et al. Cytotoxic effect of nitric oxide on human hematological malignant cells. Acta Biochim Pol, 2002; 49:139.

, Meininger

. Regulation of nitric oxide synthesis by dietary factors. Ann Rev Nutr, 2002; 22:61.

10.

Holian

, Wahid

, Atten

et al. Inhibition of gastric cancer cell proliferation by resveratrol: Role of nitric oxide. Am J Physiol Gastrointest Liver Physiol, 2002; 282:G809.

11.

Rao

. Nitric oxide signaling in colon cancer chemoprevention. Mutat Res, 2004; 555:107.

12.

Cosan

, Soyocak

, Basaran

et al. The effects of resveratrol and tannic acid on apoptosis in colon adenocarcinoma cell line Saudi. Med J, 2009; 30:191.

13.

Mortensen

, Skouv

, Hougaard

et al. Endogenous endothelial cell nitric-oxide synthase modulates apoptosis in cultured breast cancer cells and ıs transcriptionally regulated by p53. J Biol Chem, 1999; 274:37679.

14.

Srivastava

, Husain

, Hasan

et al. Green tea polyphenols and tannic acid act as potent inhibitors of phorbol ester-induced nitric oxide generation in rat hepatocytes independent of their antioxidant properties. Cancer Lett, 2000; 153:1.

15.

Lin

, Lin

. (−) Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-ınduced activity of transcription factor nuclear factor-κB. Mol Pharmacol, 1997; 52:465.

16.

Ying Chan

, Fong

, Hot

C-H

et al. Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea. Biochem Pharmacol, 1997; 54:1281.