Jia-Ren Liu, Bai-Xiang Li, Bing-Qing Chen, Ying-ben Xue, Yan-Mei Yang, Yu-Mei Zheng, Department of Toxicological Health, Public Health College, Harbin Medical University, Harbin 150001, Heilongjiang Province, China
Xiao-Hui Han, ICU of Cardiological Surgery, The Second Hospital, Harbin Medical University,Harbin 150001,Heilongjiang Province,China
Rui-Hai Liu, Food Science and Toxicology, Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA
Supported by the National Natural Science Foundation of China, No. 39870661
Correspondence to: Dr. Jia-ren Liu, 199 Dongdazhi Street, Nangang District, Harbin 150001,Heilongjiang Province, China. jiarenliu@yahoo.com
Telephone: +86-451-3639459 Fax: +86-451-3641253
Received 2001-08-23 Accepted 2001-09-05

Abstract
AIM: To determine the effect of cis-9, trans -11-conjugated linoleic acid ( c 9, t 11-CLA) on the cell cycle of gastric cancer cells(SGC-7901) and its possible mechanism in inhibition cancer growth.

METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A,B₁,D₁,P16^ink4a and P21^cip/waf1 of SGC-7901 cells which were treated with various c 9, t 11-CLA concentrations (25,50,100 and 200μmol�L^-1)of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane).

RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c 9, t 11-CLA. SGC-7901 cells. Eight day after treatment with various concentrations of c 9, t 11-CLA mentioned above, the inhibition rates were 5.92%,20.15%,75.61% and 82.44%,respectively and inhibitory effect of c 9, t 11-CLA on DNA synthesis (except for 25μmol/L,24h) showed significantly less ³H-TdR incorporation than that in the negative controls (P<0.05 and P<0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c 9, t 11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%,24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B₁ (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D₁ (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B₁ and D₁ were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P<0.01), whereas the expressions of P16^ink4a and P21^cip/waf1, cyclin-dependent kinases inhibitors(CDKI), were increased.

CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c 9, t 11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B₁ and D₁ and enhanced expressions of CDKI(P16^ink4a and P21^cip/waf1).

Liu JR, Li BX, Chen BQ, Han XH, Xue YB, Yang YM, Zheng YM, Liu RH. Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line (SGC-7901).World J Gastroenterol 2002;8(2):224-229

INTRODUCTION
Gastric cancer is common in China^[1-12], and it is currently thought to be caused by environmental factors, with diet being an important modifying agent^[13-17]. Its mechanism of prevention and treatment still makes it become a hot spot in this area^[18-39]. Its anticancerous potential Dietary fat has been implicated as an enhancing agent in carcinogenesis by both epidemiological and animal studies. Consumption of meat, specifically animal fat, has been implicated in a number of disease processes^[40-42]. However, several epidemiological studies have suggested an association between increased consumption of meat and fat and decreased risk of stomach, mammary and esophageal cancers^[43,44]. Among the fatty acids, only the essential fatty acid, linoleic acid (LA), has been clearly shown to enhance mammary tumorigenesis^[43]. However, isomeric derivatives of cis -9, cis -12-octadecadienoic acid (linoleic acid, LA) containing a conjugated double-bond system (conjugated linoleic acid, CLA) showed inhibitory effect on carcinogenesis in animal studies^[45,46]. CLA has a mixture of positional (9/11 or 10/12 double bonds) and geometric (various cis/trans combinations) isomers of LA formed by rumen and colon bacteria. The ability of CLA to prevent mammary and other tumors in rodents has been identified and has been the subject of several reviews^[43]. There are eight potential isomers of CLA, but the cis -9, trans -11 and trans -9, cis -11 isomers are thought to be active as potential antioxidant and anticarcinogenic agents. Therefore, it is of interest to investigate more extensively the anticancer activities of CLA.
      In the present study, we investigated the effect of cis -9, trans -11-CLA( c 9, t 11-CLA) on the cell cycle of human gastric adenocarcinoma cells (SGC-7901).

MATERIALS AND METHODS
Materials
c 9, t 11-CLA, a monoisomer of c -9, t 11-octadecadienoic acid with 98% purity, was obtained from Dr. Rui-Hai Liu(Food Science and Toxicology, Department of Food Science, Cornell University, Ithaca, NY, USA). The c 9, t 11-CLA was dissolved in 96ml�L^-1 ethanol, and was then diluted to the following concentrations: 0,25,50,100 and 200μmol�L^-1 .

Methods
Cell culture Human gastric adenocarcinoma cells(SGC-7901), purchased from Cancer Research Institute of Beijing (China), were cultured in RPMI 1640 (Gibco) medium, supplemented with calf serum 100ml�L^-1 , penicillin (100�10³u�L^-1) and streptomycin (100mg�L^-1). The pH was maintained at 7.2-7.4, by equilibration with 5% CO₂.The temperature was maintained at 37℃.The cells were sub-cultured with a mixture Ethylenedinitrile tetraacetic acid (EDTA) and trypsin.
Cell growth curve The SGC-7901cells were seeded in six 24 well plates (Nuc,Co.); each well contained 2�10⁴ cells. After 24h, the medium of different plates was replaced with media supplemented with c 9, t 11-CLA at different concentrations. On the next day, the numbers of cells of 3 wells from each plate were determined daily by using the trypan blue staining. The means were obtained on each of eight days and were used to draw a cellular growth curve. The inhibitory rates(IR) on the 8^th day was calculated, as follows:

[³H]-Labeled precursor incorporation SGC-7901 cells(5�10⁴/well in 24 well plate) were cultured in appropriate medium for 24h prior to beginning the experiment. The medium was, then, replaced with different concentrations c 9, t 11-CLA. After 18 and 42h, the cells were incubated with [³H] thymidine (China Nucleus Institute, 0.5μci/mL, 1.0μci/well). After 6h the cells were harvested with trysin/EDTA. Cells were collected in an acetic fiber filter with cellular collector and washed three times with PBS. The filter was dried overnight at 37℃. The filter was transferred into liquid of scintillation(containing 1% po and 2% pop in xylene) and cpm value determined by liquid Scintillation Counter (LS6500, Beckmen Co.).

Cell samples
SGC-7901 cells were treated for 24 and 48h with various concentrations of c 9, t 11-CLA and collected by centrifugation. Specimens were fixed immediately in 40g�L^-1 formaldehydum polymerisatum and embedded in paraffin. Gastric cancer tissue from a patient served as a reference.

Primary antibodyies
To examine the proliferating cell nuclear antigen(PCNA) in cell proliferation and to determine cyclins (A,B₁ and D₁) and cyclin-dependent kinases inhibitors (P16^ink4a and P21^waf1) in cell cycle of SGC-7901, we used six primary antibodies: corresponding mouse monoclonal antibodies for cyclin B₁ and D₁, PCNA and P21^waf1 and corresponding rabbit polyclonal antibodies for cyclin A and P16^ink4a. PCNA,P16^ink4a, and cyclin D₁ were purchased from Calbiochem Co.USA; others from Zhongshan Co. China.

Immunocytochemistry
Immunocytochemical staining was performed on serial sections at room temperature, using the horseradish peroxidase method. The sections were deparaffinized in xylene and rehydrated through graded alcohol. The sections were incubated for 10min at 95℃ in 10mmol�L^-1 sodium citrate(pH 6.0) buffer for PCNA staining. Endogenous peroxidases were inactivated by immersing the sections in hydrogen peroxide for 10min, and then were incubated for 10min with 100ml�L^-1 normal goat serum in PBS to block non-specific binding. The sections were subsequently incubated overnight at 4℃ with relevant antibodies(1∶50 dilution) respectively. The next day, the sections were incubated with biotinylated anti-mouse or anti-rabbit IgG(Zhongshan Co. China) for 30min, followed by peroxidase-conjugated streptavidin(Zhongshan Co.China) for 30min. The chromogenic reaction was developed with DAB (diaminobenzidine) for 10min, and all sections were counterstained with hematoxylin. Controls consisted of omission of the primary antibody. The Positive Rate(PR) was calculated as follows:

Statistical analysis
Analysis of data was performed using the student's t test or χ² test. A value of P<0.05 is considered to be statistically significant.

RESULTS
Effect of c9,t11-CLA on SGC-7901 cell growth
As shown in Figure 1, Growth of the cells in various concentrations of c 9, t 11-CLA did not differ from the negative control within 3d. After 3d, SGC-7901 cells incubated in 25 and 50μmol�L^-1 of c 9, t 11-CLA grew at a lower rate than the negative control. While in 100 and 200μmol�L^-1 concentrations of c 9, t 11-CLA, cell proliferation was significantly inhibited. The inhibitory rate of various c 9, t 11-CLA concentrations were 5.9%, 20.2%, 75.6% and 82.4%, respectively.

Figure 1(PDF)Growth curve of SGC-7901 cells cultured in various concentration of c 9, t 11-CLA

Effect on DNA synthesis
The effect of CLA on isotope incorporation into SGC-7901 cells are presented in Table 1. SGC-7901 cells preincubated in media supplemented with various c 9, t 11-CLA concentration (except for 25μmol/L, 24h) incorporated significantly less 3H-TdR than did the negative control (P<0.05 and P<0.01, Table 1). The inhibitory rate (IR) displayed a dose-response relationship as the concentration of c 9, t 11-CLA increased.

Table 1 Inhibitory effect of c 9, t 11-CLA on DNA synthesis in SGC-7901 cells (n=6)

^aP<0.05, ^bP<0.01 vs negative control

Cell proliferation
As shown in Figure 2, expression raues of PCNA（Figure 3.1）on SGC-7901 cells gradually decreased after SGC-7901 cells were incubated with different concentrations of c 9, t 11-CLA at various times. Moreover, SGC-7901s cell expressed significantly less PCNA than did the negative control (P<0.01). The expression rate displayed a dose-response relationship as the concentrations of CLA increased.

Figure 2(PDF) Expression of PCNA on SGC-7901 cells treated with c 9, t 11-CLA

Expressions of cyclin A,B₁ and D₁ and P16 ink4a , p21^waf1
The the expression of rates cyclin A, B₁, and D₁ (Figure 3.2-4)on SGC-7901 cells was decreased (Table 2) after SGC-7901 cell were incubated with different concentrations of c 9, t 11-CLA for 24h and 48h while cyclin-dependent kinases inhibitors (P16^ink4a, and P21^waf1) increased(Table 3; Figure 3.5-6).

Figure 3 A: The expression of PCNA on SGC-7901 cells of the negative controls (immunocytochemistry staining SP method, original magnification �400); B: The expression of cyclin A on SGC-7901 cells of the negative controls (immunocytochemistry staining SP method, original magnification �400);C: The expression of cyclin B₁ on SGC-7901 cells of the negative controls (immunocytochemistry staining SP method, original magnification �400); D: The expression of cyclin D₁ on SGC-7901 cells of the negative controls (immunocytochemistry staining SP method, original magnification �400); E :The expression of P16^inf4a on SGC-7901 cells of c 9, t 11-CLA group(100μmol�L^-1) (immunocytochemistry staining SP method, original magnification �400);F:The expression of P21^cip/waf1 on SGC-7901 cells of c 9, t 11-CLA group(100μmol�L^-1) (immunocytochemistry staining SP method, original magnification �400)

Table 2 Positive rates of cyclin A, B₁, and D₁ on SGC-7901 cells treated with c 9, t 11-CLA(%)

^b P<0.01 vs negative control

Table 3 Positive rates of p16^ink4a and p21^waf1 on SGC-7901 cells treated with c 9, t 11-CLA(%)

^bP<0.01 vs negative control

DISCUSSION
CLA is a naturally occurring fatty acid in animal's food. Dietary sources of CLA include grilled beef, cheese, and related foods^[47]. Another source of CLA is its endogenous generation via the carbon centered free radical oxidation of linoleic acid^[45]. Over the past ten years, a number of research works of animal experiments have supported the observation that CLA is an effective chemopreventive agent of cancer, and that it can inhibit carcinogenesis of different tissues at different stages of induction by chemical agents ^[44,45]. Several investigators in our group have reported that c 9, t 11-CLA is an effective agent to prevent carcinogenesis ^[48,49] and cancer^[50-52]. Zhu's study^[48] demonstrated that c 9, t 11-CLA could significantly inhibit the mice forestomach neoplasia induced by B(a)P(50mg�kg^-1) in post-initiation in short term(23weeks).The incidences of tumors of mice in the B(a)P group, B(a)P with high dose CLA(5μL�g^-1) group and B(a)P with low dose CLA(2.5μL�g^-1) group were 100%, 60% and 69% respectively (P<0.05). Xue's research^[49] also indicated that the incidence of neoplasm in mouse forestomach in the B(a)P group,75% pure c 9, t 11-CLA group,98% pure c 9, t 11-CLA group and 98% pure t10,c12-CLA group were 100.0%, 75.0%, 69.2%, and 53.8%, respectively. This maybe due to an inhibition mitogen of activated protein kinase(MAPK)-a way to reduce carcinogenesis. The data from our research group suggested that c 9, t 11-CLA could inhibit proliferation of cancer cells, i.e. SGC-7901 cells^[50] and MCF-7 cells^[51,52], and induced cancer cell (SGC-7901) apoptosis^[53]. Moreover, the inhibiting effect of c 9, t 11-CLA on SGC-7901 cell proliferation may be related to cell cycle.
      As shown in Figure 1, c 9, t 11-CLA at various concentrations in 8 days reduced the proliferative activity of SGC-7901 cells and its inhibitory rates were from 5.92% to 82.44%, but the mechanism of such inhibition of c 9, t 11-CLA has not been clarified. However, we discovered that SGC-7901 cells supplemented with c 9, t 11-CLA incorporated significantly less [³H] thymidine than negative controls(shown in Table I). The inhibitory rates of, from 16.4% to 78.2% after incubating with c 9, t 11-CLA for 24h and from 14.0% to 97.5% after 48h displayed a dose-response relationship.In the meantime, we investigated further the expressions of PCNA and protein from cell cycle such as cyclins and cyclin-dependent kinase inhibitors (CDKI) on SGC-7901 cells treated with various concentrations of c 9, t 11-CLA. PCNA (proliferating cell nuclear antigen) plays an essential role in both the replication and repair of DNA, and is an essential component of the DNA replication machinery, acting as the processing factor for polymerases δandε.In addition to its role in replication, PCNA is not only required for base excision-repair of nucleotides, but also binds to cell cycle regulatory proteins such as P21 and Gadd45^[54]. In this study, we discovered that the expression of PCNA on SGC-7901 cells gradually decreased with increasing concentrations of c 9, t 11-CLA in comparison with negative controls(showed in Figure 2). In other words, DNA replication lessens, thereby resulting in slower on SGC-7901 cell proliferation.

Figure 4(PDF) The relationship between CDKI(P16 and P21) and cyclins in G₁/S transition

The fundamental task of the cell cycle is to ensure that DNA is faithfully replicated once during S phase and that identical chromosomal copies are distributed equally to two daughter cells during M phase. The cell cycle is a complex process, regulated by many factors, which can be divided into three groups: cyclins(A,B,D,E....H); cyclin-dependent kinases(CDK, including CDK₁-CDK 7); CDK inhibitors (CDKI, including P16 family and P21 family). They are balanced through mutual interactions. Uncontrolled cell proliferation is the hallmark of cancers which are the result of damage to genes that directly regulate their cell cycles. Using immunocytochemical technique to detect expressions of cyclins and CDKI, we demonstrated that the expressions of cyclin A,B₁ and D₁ on SGC-7901 cells treated with various concentrations c 9, t 11-CLA were reduced, whereas expressions of CDKI(P16^in4a and P21^waf1) increased, as compared with those of negative controls. Successive actions of CDKs promote cell-cycle progression in mammalian cells. Various cyclins bind and activate CDKs at specific times during the cell cycle.
      Mammalian cyclin A activates CDK₂^[55] in S-phase and CDK₁ (Cdc₂) in G₂- and M-phases. One important mechanism that enables sequential activation of cyclin-CDK complexes is the periodic synthesis and destruction of cyclins. Cyclin A expression starts late in G₁-phase and is increasing through S- and G₂-phase before the protein is degraded in M-phase. The cell cycle-dependent expression of cyclin B₁ is critical for the proper timing of a cell's entry into mitosis which is dependent both upon the binding of CDK₁ to cyclin B₁, as well as a series of phosphorylation and dephosphorylation events. The cyclin B₁ protein accumulates during interphase and peaks at the G₂-M phase transition^[56]. One of the crucial substrates of G₁ phase CDK, including CDK₄ in the complex with D-type cyclins(cyclin D₁,D₂ and D₃), is Rb protein (pRb), which is the product of the retinoblastoma susceptibility gene. Rb protein plays an important role in the regulation of the G₁ to S phase progression in normal cells and the function of pRb is regulated by phosphorylation. Thus, during the G₀ and G₁ phase, Rb protein is in an un- or underphosphorylated stage and binds to E₂F family transcription factors. Cylin Ds/CDK₄ becomes activated around the mid G₁ phase, resulting in the accumulation of increasingly phosphorylated, inactive forms pRb. This causes the release of E₂F family transcription factors which induce the expression of S-phase genes by positive regulation through E₂F-binding sites(see Figure 4)^[57]. It is also known that abrogation of the functions of Cylin A prevents entry into the S phase. From the beginning of the S phase Rb protein remains in the hyperphosphorylated inactive state until the end of M phase; such condition is thought to be due to both cylin A/CDK₂ and Cylin A,B₁/Cdc₂ in catalyzing the phosphorylation reaction^[58]. P16^ink4a is the founder member of a family of proteins with the ability to inhibit CDK₄ and the CDK₄-related kinase CDK₆.
      The INK4 family is composed of four members in mammalian organisms: P16^ink4a , P15^ink4b, P18^ink4c, and P19^ink4d. The four mammalian INK4 proteins have similar biochemical properties: all of them bind to CDK₄ and CDK₆ and inhibit the kinase activity of the CDK_4-6/Cylin D complexes(Figure 4)^[59]. The INK4 inhibitor causes G₁ arrest indicating that the phosphorylation of pRb on residues specific for CDK₄(and possibly CDK₆) is critical for G₁/S progression. While P21^CIP1/waf1 family, comprising P21^Cip1/waf1,P27^CIP1 and P57^CIP2, bind to a variety of CDKs and cyclins, preferentially to cyclin/CDK complexes rather than monomeric forms and also inhibit performed active cyclin/CDK complexes(see Figure 4)^[59]. In addition to its role as a CDKI, P21^Cip/waf1 has been shown to block DNA replication by direct interaction with PCNA mentioned above. However, P21^CIP1/waf1 does not inhibit the PCNA-dependent nucleotide excision-repair of DNA. In deed, DNA damage leads to an increase in the level of P53, and result in P21-mediated cell cycle arrest in the G₁ phase, which persists until DNA repair is completed^[60].Thus, it is proposed that P21^Cip/waf1 plays an important role under such conditions as terminal differentiation and cell senescence.
      In conclusion, c 9, t 11-CLA may inhibit cell growth and proliferation by a decrease in the expressions of cyclin A,B₁ and D₁ and an increase in that of CDKI(P16^ink4a and P21^Cip1/waf1) on SGC-7901 cells in comparison with the negative controls. This result suggested that the inhibition effect of c 9, t 11-CLA on SGC-7901 cell proliferation is related to the cell cycle. The whole mechanism of the action of c 9, t 11-CLA on SGC-7901 cell cycle further research.

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