Chinese-German Journal of Clinical Oncology February 2012, Vol. 11, No. 2, P104–P108DOI 10.1007/s10330-011-0906-4

Bcl-2 is an important gene for depressing apoptosis, which involves some biological functions. It plays an im-portant role in the initiation and progression of malignan-cies by inhibiting apoptosis of carcinoma cells. A number of studies have been showed that to block or decrease the related protein expression of Bcl-2 can be induce the apoptosis of carcinoma cells, and increase the sensitivity of carcinoma cells to radiotherapy and chemotherapy, and can improve therapeutic efficacy. The development technology of RNA interference is a specific gene silence in recent years, which transfers target genes into cells by carriers to cause post-transcriptional related gene silenc-ing, thereby, block or decrease the related protein expres-sion specifically.

In this study, objective to through the effect on SGC-7901 by synergism between a siRNA targeted to Bcl-2 and celecoxib to explore whether it can provide the tradition-al treatment of gastric cancer a new therapeutic approach [1, 2].

Materials and methods

Main regentsRPMI 1640 medium, fetal bovine serum, were purchased

from Gibco, LipofectamineTM2000 were purchased from Invitrogene (USA), and multiply-table tournament (MTT)

was from Sigama. ELISA kit was obtained from Beyotime Institute of Biotechnology. A siRNA [3] targeted to Bcl-2 was designed affirmatively by our research team and syn-thesized by Shanghai GenePharma Co., Ltd (China), sence: 5’-CACCTGACGCCCTTCTTCAAGAGAGAAGGGCGTCAGGTGCAGCTTTTTTG-3’, antisence: 5’-GATCCAAAAAAGCTGCACCTGACGCCCTTCTCTCTTGAAGAAGGGCGTCAGGTGCAGC-3’.

Cell cultureThe SGC-7901 human gastric cancer cells were cul-

tured in RPMI-1640 medium, supplemented with 10% fetal bovine serum and 100 kU/L penicillin and strepto-mycin at 37 ℃ in humidified atmosphere of 5% CO2.

TransfectionDivided into three groups: the control group, Lipo-

fectamine group, small hairpin RNA (shRNA) group. Cells were seeded at a density of 1 × 105 cells/cm2 in 24-well plates, corresponding to a density of 70%–80% at the time of transfection, 0.5 μg of plasmid DNA, 1.5 μL of Lipofectamine were added into each well, all the steps were carried out strictly according to the specifications of LipofectamineTM 2000. Cells were harvested at 24 h, 48 h, 72 h and 96 h.

Test of transfection efficiencyTwenty-four h, 48 h, 72 h and 96 h after transfection,

the expression of protein could be observed under a fluo-

Effects of transfecting Bcl-2 shRNA and celecoxb on the SGC-7901 cell line Weiwei Liu, Aiqin Wang, Wenjing Xiao, Fangzhen Shen, Minggang Lin, Ruyong Yao

Department of Oncology, The Affiliated Hospital of Medical College Qingdao University, Qingdao 266003, China

Received: 20 September 2011 / Revised: 20 October 2011 / Accepted: 25 November 2011© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012

Abstract Objective: The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA (shRNA) targeted Bcl-2 and celecoxib in vitro. Methods: To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000, the rate of cell growth inhibition of all groups was detected by multiply-table tournament (MTT) method, apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa. Results: After siRNA was transfected to SGC-7901 cells, the rate of cell growth inhibition was increased, the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa. Conclusion: The growth of SGC-7901 cells which was transfected siRNA has been inhibited, and the sensitivity to celecoxib has been increased.

Key words gastric cancer; Bcl-2; RNA interference (RNAi); small hairpin RNA (shRNA); gene transfected ; celecoxib

Correspondence to: Fangzhen Shen. Email: shenfangzhen@163.com

105Chinese-German J Clin Oncol, February 2012, Vol. 11, No. 2

fluorescent cells in the whole cells under fluorescence microscope, the transfection efficiency was defined as av-erage of 3 visions. 24 h after transfection, we found that the transfection efficiency was about 41.5 ± 1.5 % and few green fluorescence was observed in the cells. The expres-sion of green fluorescence was on the advance with time increased in 48 h, the maximum value reached 89.6 ± 2.3 %, the expression of green fluorescence become decreased for about 35.8 ± 2.1 % in 72 h, and the lowest in 96 h for about 21.5 ± 1.9 %, the transfection efficiency of control group was almost 0. Cell morphology was roughly normal and endochylema was roughly clear in 48 h. The growth of cells were inhibited and apoptotic cells were observed in 72 h: smaller cell size, nuclear chromatin condensa-tion, cytoplasm appeared coarse particles, part of the cell membrane appears shrunken (Fig. 1).

Synergism between a siRNA targeted to Bcl-2 and celecoxib in inhibiting SGC-7901 cell proliferation

After different doses of celecoxib were treated to dif-ferent groups for 48 h, according to IC50 formula, IC50 of the group celecoxib alone was 51.89 ± 0.77 μmoL/L, the rate of cell growth inhibition for groups of shRNA joined to celecoxib were increased obviously. The IC50 was 18.18 ± 4.12 μmoL/L (Fig. 2). Celecoxib group, the joined group compared with the control group, the differ-ence was statistically significant (P < 0.05).

Effects of transfection on apoptosisApoptosis rate of all groups had appearance of Fig. 3,

and had a positive correlation with different doses of ce-lecoxib, which increased with the increase of doses of ce-lecoxib, compared with control group, all the groups had statistical difference (P < 0.05). Compared with control group, celecoxib group and the Joined group group had statistical difference (P < 0.05).

Influence of synergism between a siRNA and celecoxib on the expression of Bcl-2 in SGC-7901

The quantitative changes of expression of Bcl-2 in in SGC-7901 of all groups had appearance of Fig. 4, com-pared with control group, Bcl-2 level was much lower in group of treated with celecoxib, and had a negative corre-lation with different doses of celecoxib, which decreased with the increase of doses of celecoxib, however Bcl-2 level in joined groups went further down, and had a sig-nificant difference (P < 0.05).

Discussion

Gastric cancer is one of the most common malignant tumors, and the 5-year survival rate of it is only about

rescence microscope, transfection efficiency = (number of protein expression cells / total number of cells) × 100%. 48 h after transfection was used as follows: one as con-trol group cultured in RPMI-1640 medium, group shRNA SGC-7901 and group SGC-7901 were cultured in differ-ent concentrations of 5 μmoL/L, 10 μmoL/L, 20 μmoL/L, 40 μmoL/L, 80 μmoL/L celecoxib, respectively.

Cell proliferation by MTTCells were added at a density of 1 × 103 cells/cm2 into

96-well plates, all the transfection steps were carried out strictly according to the specifications of LipofectamineTM 2000. 6 h later, the culture medium was exchanged for the same range of concentrations of 5 μmoL/L, 10 μmoL/L, 20 μmoL/L, 40 μmoL/L, 80 μmoL/L celecoxib, after 48 h, cultured 100 μg MTT was added into all the wells for 4 h, then the supernatant was removed and 200 μL of DMSO was added to. After 10 min at room temperature, the ab-sorbance was read at a wavelength of 570 nm determined with a BioRad reader. The survival rate and inhibitory rate were calculated in the following formulas: survival rate (%) = OD values of experimental wells / OD values of control wells × 100%, inhibitory rate (%) = (1－OD values of experimental wells / OD values of control wells) × 100%. Ratios of all groups were analyzed by statistical software.

Apoptosis analysis by flow cytometrySGC-7901 cells of all the groups were collected, and

re-suspended with PBS and adjusted to a density of 1 × 105 cells/mL and carried out strictly according to the specifications. Data were got and analyzed by Cell Quest software.

Expression of Bcl-2 by ELISASGC-7901 cells of logarithmic growth phase were col-

lected and digested by trypsin (25%), washed twice with PBS, added to cell lysis solution for 30 min, the specific methods were carried out strictly according to the speci-fications.

Statistical analysis All the data were analyzed using software SPSS 14.0

for windows. Data were expressed as χ ± s and Student’s t test was used to compare the means of two groups. A dif-ference of P < 0.05 was considered to be statistically sig-nificant, statistical analysis was done using the one way ANOVA for multiple samples.

Results

Transfection efficiencyTwenty-four h, 48 h, 72 h and 96 h after transfection,

viewed from each vision and counted the percentage of

106 www.springerlink.com/content/1613-9089

35%, the mortality rate up to the 2nd malignant tumors in the world. Thus combined treatment are served as major group to cure advanced carcinoma of stomach, available evidence suggest [4] that preoperative radiotherapy com-bined with surgery can improve 3-, 5-, 10-year survival rate. But it has low sensitivity to chemotherapy, people who suffer cancer experience ill effects from chemother-apy treatments and lower their life quality. To find more effective and more harmfulless therapeutic method and to enhance the holistic treatment level of gastric cancer are the problems that must be solved in no time at present in the world.

There are significant relationship between the oc-currence, development and metastasis of gastric cancer and gene, such as Ras, C-myc, C-met, Erb-2, P16, P53, Bcl-2, and so on. Among these genes, Bcl-2 is essential to the occurance, development and metastasis of gastric cancer [5]. Bcl-2 is an important anti-apoptosis gene, not only does it regulate and control apoptosis, but also may participate in regulation of telomerase activity [6, 7], which plays an important role in the occurrence, development and prognosis of tumor [8–10]. Research indicates [11] that high expression of Bcl-2 in human gastric carcinoma, a siRNA targeted to Bcl-2 can lower the expression of Bcl-2 obviously, and suppress the growth of SGC-7901 in vi-tro and in vivo. It should be considered as an important method in human gene therapy gradually.

Celecoxib is a part of specific cyclooxygenase-2 (COX-2) inhibitor, and a member of NSAIDs. The recent studies showed that COX-2 performed a very useful role in diges-tive system carcinoma especially in the occurrence, de-velopment, differentiation and metastasis of gastric can-cer. COX-2 is a inducible enzyme, it can not be detected

in most tissues under normal physiological condition, and begin to synthesise, express when irritated by inflamma-tory factor or Hp. Extensive research suggest that COX-2, aside from its obvious role in inflammatory reaction, serves some important functions in most digestive system carcinoma especially in gastric cancer [12]. The positive rate of COX-2 expression is significantly higher than con-trast group in gastric cancer [13]. The incidence of COX-2 in cardia cancer is significant higher than that in the

Fig. 1 The expression of SGC-7901 in 24 h, 48 h, 72 h and 96 h after transfection by fluorescence microscope (10 × 10). (a) 24 h; (b) 48h; (c) 72 h; (d) 96 h

Fig. 2 Different doses of celecoxib influenced on the cell growth inhibi-tion for SGC-7901 before and after transfection

Fig. 3 Different doses of celecoxib influenced on the changes of apop-tosis rates

Fig. 4 The expression of Bcl-2 in joined group

107Chinese-German J Clin Oncol, February 2012, Vol. 11, No. 2

endmost tissues. High COX-2 expression in gastric cancer tissues accompanies high Bcl-2 expression. The expres-sion of COX-2 is closely associated with VEGF in gastric cancer, the more malignant the gastric cancer, the more coordinated expression of COX-2 and VEGF. The positive of Hp in gastric cancer tissues, increase the expression of COX-2, especially in the mucosa of the gastric antrum. The expression rate of COX-2 and VEGF in gastric cancer with lymph node metastasis are higher than those with-out lymph node metastasis. This suggests that the overex-pression of COX-2 may accelerate the metastasis of gastric cancer, mechanisms of COX-2 carcinogenesis as follows: (1) preventing apoptosis, the increase activity of COX-2 can increase the expression level of anti-apoptosis gene Bcl-2, decrease the expression level of Bax and BlcXL and decrease the level of TGF-β2 mediated cell apoptosis and the activity of E-cadherin to promote the proliferation of tumor cells; (2) stimulating tumor angiogenesis through the up-regulation of angiogenic growth factor expression, such as VEGF; (3) immunological suppression and so on. It has been reported that [14] COX-2 inhibitor can enhance the effect of chemoradiotherapy, inhibit angiogenesis and the expression of VEGF, suppress the tumor invasion and metastasis, lower the expression of cancer gene and upper the expression of cancer suppressor gene. Many litera-tures have reported [15] that inhibiting the expression of COX-2 in gastric cancer tissues through the technology of RNAi in order to inhibit tumor cell proliferation and induce tumor cell apoptosis. Does COX-2 inhibitor regu-late Bcl-2? Does it cause the inhibition of gastric cancer cells through synergism between a siRNA targeted to Bcl-2 and celecoxib, but these issues are at present unsettled.

This study was therefore to synergize a siRNA tar-geted to Bcl-2 and celecoxib to act on SGC7901, the re-sults showed that the transfection was most efficient after 24 h of transfection, about 89.6 ± 2.3 %. the groups of RNAi and celecoxib could suppress the proliferation of SCG-7901. The IC50 of the celecoxib alone was 51.89 ± 0.77 μmoL/L, and the group of shRNA combined with ce-lecoxib was 18.18 ± 4.12 μmoL/L, moreover the growth inhibition rates of all the groups were positively corre-lated with the concentration of celecoxib. Flow cytom-etry analysis showed that the apoptosis rates of all groups increased with the increase of celecoxib concentration. ELISA showed that the expression of Bcl-2 were posi-tively correlated with the concentration of celecoxib, and the lowest level of expression of Bcl-2 could be detected in the concentration of 80 μmoL/L, which was 0.212 ± 0.04 g/L.

The results of the experiment indicated that there would be a relation between Bcl-2 and COX-2 inhibitor: maybe COX-2 inhibitor can decrease the expression of Bcl-2 through a channel. Studies showed [16] that COX-2 inhibitor could induce apoptosis of tumor cells through

changing signal transduction pathway of apoptotic cell related protein, such as Bcl-2/Bax, etc. After being treated with COX-2 inhibitor, the levels of Bcl-2 began to drop slowly, at the same time the apoptosis biomarkers began to increase, so the effect of RNAi combined with celecox-ib was considered as synergy. This provided a new tool for treatment of gastric cancer. After the expression of COX-2 inhibitor, the production of PGE2 were running down, in addition PGE2 can induce cell proliferation and stimulate the expression of Bcl-2. that is decreased PGE2 production can reduce the expression of Bcl-2, combining with RNAi to increase cell apoptosis and cyclin D1, to ar-rest the cell cycle G1 at gene level so as to curtail life cycle of damaged DNA, thereby reducing the tumor. Though gene transfection are carried out only in animal experi-ments and cellular level, have not been used in clinical trials. There are so many problems to be resolved about gene therapy [17], such as security of vector, the carcino-genic problem of random-insert and integrating of vector, and the deficiency of transgene in vivo and so on. With continuing development of molecular biotechnology, the above problem will be solved step by step, the true dint-ing will evenly come true, and solve the difficult problem of gene therapy fundamentally.

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