Abstract Objective To investigate the gastric cancer cell lines MGC803 cisplatin (CDDP)-resistant generated mechanisms. The fluorescence staining cells observed by different concentrations of CDDP (0.1, 1.0, 10.0 mg / L) of MGC803 cell proliferation, apoptosis and anti-apoptosis gene, survivin, bcl  2 mRNA expression in cell growth inhibition rate was detected by MTT; The rate of apoptosis; measured by flow cytometry cell cycle changes; RT  PCR was used to detect the expression of survivin, bcl  2 mRNA expression. Results of 10mg / L the CDDP of MGC803 cells inhibit proliferation and promote apoptosis, 0.1,1.0 mg / L the CDDP intervention 24h, the inhibition of cell proliferation and promote apoptosis gradually disappear; CDDP role cells in S phase cells 48h after increased G2 / M phase decreased; MGC803 cells 1.0mg / L CDDP role, survivin mRNA expression increased gradually after 24h, bcl  2 mRNA expression decreased (P <0.05). Conclusion CDDP to interference MGC803 cell cycle, but the easy to produce drug-resistant cells to low concentrations of CDDP of survivin mRNA expression increased may be resistant to one of the reasons MGC803 cells to CDDP.

Key words gastric cancer cell line cisplatin-resistant cell apoptosis survivin bcl  2

Gastric cancer is a common malignant tumors in China, and its mortality rate ranks first in various malignancies, one of the efficacy of chemotherapy as an important treatment of gastric cancer is still not satisfactory, mainly due to cancer resistant. To this end, the experiment according to the literature [1], gastric commonly used chemotherapy drug cisplatin (Cisplatin, CDDP) is set to three different role concentration, the role of cisplatin in the cell cycle by detecting MGC803 cells, cell proliferation, apoptosis and apoptosis-related gene expression of survivin, bcl  2 mRNA expression changes in cisplatin-resistant gastric cancer cells to cytotoxic drugs generate and possible mechanisms.

1 Materials and methods

1.1 Drugs and reagents

RPMI 1640 medium, Trizol (GibcoBRL company); calf serum (Hangzhou Evergreen), four thiazole blue (MTT), bromine has given (EB), acridine orange (AO), dimethyl sulfoxide (DMSO) (Sigma), survivin, bcl  2 primers [2] (Shanghai Sangon synthetic),; β  actin (Ruike); two-step RT  PCR reagents: Taq enzyme oligo  dT dNTP, DEPC ( the Sangon.), M  NLV Rnasin the (Promeg company), CDDP (Shandong Qilu Pharmaceutical Factory, saline solution, the culture was diluted).

1.2 Experimental Methods

1.2.1 Cell culture and subculture

People poorly differentiated gastric adenocarcinoma cell line MGC803 purchased from Xiangya School of Medicine Cell Center. Cells containing penicillin 100 U / L, streptomycin 100 mg / L, 10% inactivated fetal calf serum in RPMI 1640 culture medium, set to 37 ° C, 5% CO2 incubator culture.

1.2.2 MTT colorimetric determination of the impact of drugs on cell proliferation

5 × 103 / well of vaccination MGC803 cells in 96-well plates, set of three wells plus CDDP treatment of the experimental group, so that the final concentration of 0.1mg / L, 1.0mg / L, 10mg / L, control group plus physiological saline, incubated together 12,24,48,72 h, plus 20μl of MTT, culture was continued 4h, supernatant was removed, added 150μl of DMSO, enzyme immunoassay analyzer the 570nm absorbance at value A570nm and calculate the concentration Inhibition rate = [(control group A570nm – test group A570nm) / control group A570nm] × 100%.

1.2.3 fluorescence microscopy to detect groups of drugs on the role of apoptosis

Plus CDDP treatment MGC803 cells to a final concentration of 0.1 mg / L and 1.0 mg / L, 10.0 mg / L, the role of 12, 24, 48 and 72 h, located three wells, the cells were collected by centrifugation, and the supernatant was removed, PBS washed 3 times to prepare a cell suspension was added to a final concentration are 100μg/ml AO, EB, mixing, in the ratio of apoptosis in at least 200 cells is counted under a microscope. Early apoptotic nuclei staining quality green was pyknotic-like or bead-like; the late apoptotic nuclear chromatin nacarat, was the pyknosis shape or rupture like; necrosis nucleus chromatin orange red was normal cells structure; live Chromatin from green, was the structure of the normal cells [3].

1.2.4 groups of drugs on the cell cycle by flow cytometry

MGC803 cells plus CDDP treatment, so that the final concentration of 0.1 mg / L and 1.0 mg / L, 10.0 mg / L, the control group plus saline, co-48h, the conventional digestion cells, centrifugation, PBS washed, each tube by adding 70 % of the ice ethanol 1 ~ 1.5ml, so that each sample containing cells of at least 1 × 106, -20 ℃ fixed inspection flow cytometry.

1.2.5 RT  PCR assay CDDP before and after the intervention survivin, bcl  2 mRNA expression

Total RNA preparation: MGC803 cells were seeded in 6-well plates, test groups plus CDDP processing, so that the final concentration of 1.0mg / L, control group plus saline, located three wells using Trizol method before drug intervention and intervention 12 , 24,48,72 h of total cellular RNA.

RNA reverse: M  MLV manual, cDNA set stored at -20 ℃.

: PCR amplification reaction system including 10 × Taq Buffer5μl 25 mM / MgCl23μl, of 10 mM DNTP1μl, 10 pmol of target genes, the downstream primer (survivin upstream primer: 5 ‘ CAG ATT TGA ATC the GCG GGA CCC  3’, downstream primers: 5 ‘ the CCA AGT CTG GCT CGT TCT CAG  3’; expression of bcl  2 upstream primer material: 5 ‘ GTG GAG GAG CTC the TTC AGG GA  3’, downstream primer material: 5 ‘ AGG CAC the CCA GGG TGA TGC AA  3 ‘), β  actin on each primer, 2μl cDNA templates 4μl, 2.5U/μl Taq enzyme 1 μl of complement DEPC water to 50 μl. Amplified set PCR amplification, 95 ° C denaturation 5min, denaturation, annealing, elongation at 94 ° C for 30, X ° C (of survivin to 61 ° C, the expression of bcl  2 to 59 ° C) 1min, 72 ° C 1min, 30 cycles after 72 ℃ final extension at 10min. The PCR product was purified by agarose gel electrophoresis containing 0.5mg/Lde1 the EB gel imaging analyzer results, calculation of the ratio of the target gene and of β  actin.

1.3 Statistical

Quantitative and semi-quantitative data, expressed as mean ± standard deviation, SKN  q test was used for statistical analysis.

2 Results

2.1 MTT colorimetric determination of the impact of various concentrations of CDDP for gastric cancer cell proliferation

The mean absorbance value for each multiple holes findings group extended over time, high concentrations of CDDP (10.0mg / L), cell proliferation were significantly lower (P <0.05); low concentration (0.1mg / L, 1.0mg / L) CDDP group decreased after 24h, no longer easily tolerance MGC803 cell proliferation inhibition by low concentrations of CDDP, as shown in Table 1. Table 1 CDDP MGC803 cell proliferation in the experimental group with CDDP processing comparison, * P <0.05, ** P <0.012.2 fluorescent staining groups CDDP on apoptosis of gastric cancer

Staining counting apoptotic cells found that the effect of high concentrations of CDDP (10.0mg / L), the rate of apoptosis with action time to increase (P <0.05), see Figure 1, while the low concentration of CDDP (0.1mg / L, 1.0mg / L) role MGC803 cells after 24h, the apoptosis rate almost no change in Table 2. Expression Table 2CDDP for MGC803 apoptosis (bcl  2 mRNA 304bp, β  actin 500bp)

2.3 Flow cytometry groups CDDP gastric cancer cell cycle

MGC803 cells were treated with different concentrations of CDDP for 48h, cells in S phase ratio gradual increase in the G2 / M phase decreased. CDDP cell cycle has a significant dose-dependent relationship, as shown in Table 3. Table 3 CDDP on MGC803 cell cycle

2.4RT  PCR detection CDDP gastric cancer cells of survivin, bcl  2 mRNA expression

RT  PCR to detect the expression of survivin, bcl  2 mRNA expression changes (2,3), gel imaging analyzer results (Table 4) of MGC803 cells at low concentrations (1.0mg / L) CDDP role, survivin mRNA 24,48,72 h expression continued to improve after 72 hours, the expression of close to three times that of the control group. Bcl  2 mRNA in the intervention 12,24,84,72 h expression decreased gradually. Table 4 semi-quantitative RT  PCR detection CDDP MGC803 cells

3 Discussion

Clinical drug side effects, patients can not tolerate small doses can easily become resistant due to high-dose chemotherapy, which greatly affect its efficacy. Actively explore the mechanism of resistance of tumor cells to chemotherapeutic agents, to overcome the resistance of tumor cells produce, is one of the urgent problems of the non-surgical treatment of gastric cancer.

the widely expressed survivin embryo and malignant tissues in the body [4,5], it is expressed in gastric cancer histopathology as high as 82% [6], while negative expression in normal tissues, terminally differentiated tissues, survivin expression and tumor tissue metastasis, invasion, and chemical resistance are closely related to the malignant behavior [7], its mechanism of action and the IAP family, similar to other proteins, thereby inhibiting apoptosis by inhibiting caspase  3 and 7 zymogen activity, in addition to survivin is also involved in cell mitosis, promotes cell proliferation, the study found that the application survivin antisense oligonucleotide inhibition of survivin expression in normal and tumor cells results in increased apoptosis, polyploid produce increased soft agar medium colony reduce [8] [9], accelerate the proliferation rate of 293 cells transfected with survivin gene expression of the phosphorylation defects survivin mutant increased apoptosis in human melanoma cells, enhanced cisplatin-induced cell death [10 ]. bcl  2 is detected earlier, it is important apoptosis suppressor gene is overexpressed in a variety of malignant tissue by preventing calcium from the endoplasmic reticulum to release to the cytoplasm, the endonuclease activity of calcium-dependent nucleic acid reduce, inhibit the generation of oxidative free radicals blocking apoptosis, increased invasion ability of tumor spread. Found in this experiment, the presence of the inhibitor of apoptosis gene, survivin, and bcl  2 mRNA expression MGC803 cells.

CDDP tumor cell cycle can be change cycle specific cells, thereby inducing apoptosis [11, 12], our study found that of MGC803 cells after CDDP treatment, the increase in the proportion of cells in S phase and G2 / M phase decreased, indicating that the cells blocked S phase, can not enter the G2 / M phase, which is consistent with the study reported on esophageal [13]. However, its clinical efficacy is not very optimistic, the main reason for the resistance of tumor cells, the results show that the large dose of CDDP (10.0mg / L) can inhibit gastric cancer cell the strains MGC803 proliferation, induction of apoptosis, and has good aging , but this concentration has been far greater than the clinical dose, the maximum dose of clinical serum peak concentration of 2 mg / L, [14], while the low concentration group (0.1 mg / L, close to this concentration of 1.0mg / L) this weak role of gastric cancer cell lines, and 24 hours after drug intervention, almost no change of the rate of apoptosis inhibitory effect on cell proliferation waning to show that MGC803 cells become resistant to the low concentration of CDDP easily, its mechanism not very clear. Over-expression of the mdrl encoding P-glycoprotein, in addition to tumor cells can increase the drug discharged, but also in that the tumor cells resistant to drug-induced apoptosis. Contact with tumor cells, chemotherapy drugs can induce tumor cell COX  2 expression increased, and thus induce the anti-apoptotic gene bcl  2 expression [15] In addition, recent studies also found that, CDDP induced apoptosis inhibitory gene family (IAP), a new member of survivin expression [16,17]. The study found that, of MGC803 cells after low concentration (1mg / L) CDDP role of survivin mRNA expression increased to intervene 24,48,72 h, 72h close to three times that of the control group, Ikeguchi reported with a small dose of CDDP ( 0.1mg / L, 1mg / L) the role of early (1h) significantly increased apoptosis in gastric cancer cell lines, MKN  45 intervention late (48h) no longer changes the percentage of apoptotic cells, cell survivin mRNA and protein expression significantly increased, consistent with its reported the results of this study. CDDP-induced apoptosis mechanism Caspases  3 activation, survivin is by inhibiting the activation of Caspases  3 inhibition of apoptosis, and thus that the increased expression of survivin resistant to CDDP-induced apoptosis, resulting in resistance drugs [16  18], bcl  2 mRNA expression in the after CDDP intervention, gradually decline, indicating that the inhibition of its expression by CDDP, this result is inconsistent with some literature reports, the reason may be related to the use of tumor cells, the literature suggests that bcl  2 expression increased tumor cells in the CDDP effect due to COX  2 receptor-mediated negative expression of COX  2 protein gastric cancer MGC803 cell lines.

The results confirm that low concentrations of CDDP-induced gastric cancer cell lines MGC803 survivin mRNA expression increased, this change of apoptosis-related genes, probably one of the reasons leading to gastric cancer cell resistance to chemotherapeutic drugs, and in the process bcl  2 The role seems obvious.