[Abstract] Objective observation tetramethylpyrazine (TMP) in combination with fluorouracil (5  Fu) of gastric cancer multidrug resistant cells SGC7901/ADR cells in vitro killing effect. MTT assay was used to observe the the TMP joint 5  Fu of SGC7901/ADR cell killing effect, light microscopy and flow cytometry (FCM) to determine the cell cycle distribution of each drug group. 5  Fu half inhibition rate of SGC  7901/ADR cells (IC50) 13.001mg / L, 300mg / L TMP joint after the IC50 it down to 1.542mg / L, the reversal of a multiple of 8.43 times, and compared to the control group, the difference was statistically significant (P <0.01). Light microscopy showed 5  Fu + TMP (final concentration of 300mg / L) group than in the group of cancer cells to shrink smaller and round, apoptotic changes in 5  Fu. FCM analysis: the TMP joint 5  Fu with the control group compared to cell cycle arrest in DNA synthesis in the early and resting  G0/G1 (P <0.05). Conclusion TMP and 5  Fu joint MDR of gastric cancer cells SGC7901/ADR strong killing effect provides a new idea for the current treatment of gastric cancer.

Key words】 gastric cancer; multidrug resistance; SGC7901/ADR cells;  Fu; tetramethylpyrazine

Lethal Effect of Combination of Tetramethylpyrazine and Fluorouracil in Multidrug Resistance of Gastric Carcinoma Cell Lines SGC  7901/ADR

    YANG Ye, HOU Pei  zhen, ZHANG Juan

    The Emergency Department of First Affiliated Hospital, BaoTou Medical College, BaoTou 014010, China

    Corresponding Author: HOU Pei  zhen, E  mail: houpeizhen@126.comAbstract: Objective To investigate the lethal effect of combination of TMP and 5  Fu in the multidrug resistance of Gastric Carcinoma Cell Lines SGC  7901/ADR. Methods Gastric carcinoma cell lines SGC  7901 and the multidrug resistance cell lines SGC  7901/ADR were considered as target cells, MTT assay was used to investigate the lethal effect of combination of TMP and 5  Fu in vitro, which was observed by Light microscope. Flow cytometer (FCM) was employed to investigate the cell cycles. Results The half inhibition ratio (IC50) of 5  Fu against SGC  7901/ADR was 13.001 mg / L, which was reduced to 1.542mg / L by 300mg / L TMP, The reversal index was 8.43 (P <0.01). Malignant cell volume and morphous which exposed to 5  Fu + TMP became smaller and strinked rounder by Light microscope. FCM revealed that with the effect of 5  Fu + TMP, cell cycle was blocked at presynthetic phase and ambiguous phase  G0/G1 phase and The cells enter the period of DNA synthesis were reduced, inhibition mitosis of SGC  7901/ADR (P <0.05). Conclusion Combination of 5  Fu and TMP has stronger lethal effect against the multidrugs resistance Cell Lines SGC  7901/ADR in Gastric Carcinoma, which may provid a new therapeusis against Gastric Carcinoma.

    Key words: Gastric Carcinoma; MDR; SGC  7901/ADR; 5  Fu; TMP

Gastric cancer multidrug resistance (multidrug resistance, MDR) is one of the main reasons for the failure of chemotherapy [1]. Solve the main method of MDR by increasing the concentration of intracellular drug reversed MDR, but the majority of reversal agents toxic side effects limit their clinical application. The experimental observation of low toxicity, inexpensive new calcium channel blocker [2] tetramethylpyrazine (TMP) in combination with fluorouracil (5  Fu) of gastric cancer multidrug resistant cells SGC7901/ADR killing effect for the treatment of gastric cancer MDR new methods.

    1 Materials and methods

    1.1 cell line

    Human gastric cancer cell line SGC  7901 cells resistant to doxorubicin drug cell line SGC  7901/ADR cells by Xijing Hospital, Institute kindly.

    1.2 drugs, reagents and instrument

    TMP (Beijing Yongkang Pharmaceutical Co., Ltd.), 5-Fu (Nantong essence Pharmaceutical Co., Ltd.), adriamycin (ADM) (Shantou Special Economic Zone Meiji Pharmaceutical Co., Ltd.), RPMI 1640 (American GIBCO), newborn bovine serum (Hangzhou Evergreen Institute of Biology), MTT, DMSO, trypsin (American sigma), flow cytometry the kits (Exalpa products) inverted microscope (Chongqing Optical Instrument Factory products), microplate reader (U.S. Biotek company’s products), flow cytometry (U.S. BD).

    1.3 Methods

    1.3.1 Cell culture SGC  7901 cells in 10% fetal calf serum, 100u/ml penicillin, 100μg/ml streptomycin RPMI1640 medium. SGC  7901/ADR cells in the culture medium containing 1.0μg/ml ADM, 37 ℃, 5% CO2 saturated humidity incubator train the. SGC  7901/ADR cell experiment two weeks ago were cultured in a culture medium free of ADM.

    1.3.2 TMP non-cytotoxic dose MTT assay was used to take the logarithmic phase SGC  7901 cells and SGC  7901/ADR cells were seeded in 96-well cell culture plates, cultured 24h, 5 × 104/ml. Experimental holes with different concentrations of TMP (100,200,300,400,500 mg / L). Set five wells per concentration for 48h, 20μl (5 g / L) MTT hours after adding DMSO150μl shaker shake each well was measured, microplate reader 490nm wavelength absorbance A value. Calculate the survival rate of the cells under the different drug concentrations SR [3] (SR = experimental group A value / control group A value × 100%). When the cell viability SR ≥ 95% [4] to determine the non-cytotoxic dose of the present experiment.

    Ibid 1.3.3 TMP joint 5  Fu SGC  7901/ADR cell killing effect, adding in the SGC  7901 cells and SGC  7901/ADR cells ① different concentrations of 5  Fu (1 × 101 1 × 105μg / L), ② different concentrations of 5-Fu (1 × 101 to 1 × 105μg / L), and non-cytotoxic dose of TMP. SR, calculated cells under different drug concentrations 50% inhibitory concentration IC50, relatively resistant to RF [5] (RF = resistant cell line IC50 / gastric cancer cell lines IC50) and reversed multiples RI (RI = IC50 / joint reversal agent monotherapy After IC50).

    1.3.4 FCM analysis of cell cycle SGC  7901/ADR cells with various concentrations of drug treatment were collected, washed in PBS, 70% cold ethanol-fixed overnight, and centrifuged to remove the ethanol, adding 1% of the RNA enzyme solution and propidium iodide piperidine (PI) dye, dark dark 37 ° C water bath for 20min, specially designed nylon mesh filter (ice operations) in the test tube, on flow cytometry DNA content and cell cycle item with CELLQUEST software analysis.

    1.4 statistical methods

    SPSS11.5 statistical package, regression analysis and analysis of variance.

    2 Results

    A non-cytotoxic dose of 2.1 TMP

    TMP concentration of 300mg / L, SGC  7901/ADR cell survival rate of more than 95%, and the same dose of TMP-sensitive cells no significant toxic effects. Therefore TMP in a non-cytotoxic dose, the concentration of 300mg / L or less as shown in Table 1. Table 1 TMP at different concentrations on the tumor cell viability

    2.2 TMP joint 5  Fu SGC  7901/ADR cell killing effect

    SGC  7901/ADR resistant cells to 5  Fu, the relative resistance 97.49. 100 ~ 300mg / L TMP its resistance reversal effect and showed a dose-dependent manner (R = 0.972, P = 0.000) . 300mg / L TMP can significantly increase the SGC  7901/ADR cells 5  Fu sensitivity to the relative resistance dropped 12.89, the reversal fold to 8.43 times. 300mg / L TMP joint 5  Fu the cytotoxicity of SGC  7901/ADR strongest (P <0.01), are shown in Table 2. Light microscopy showed 300mg / L TMP strengthen the 5  Fu SGC  7901/ADR cell killing effect of cancer shrink smaller and round cell apoptosis change, as shown in Figure 1. Table 2 TMP SGC  7901/ADR cell resistance reversal effect

    2.3 cell cycle analysis

    FCM analysis confirmed TMP (final concentration of 300mg / L) after 5  Fu combined SGC  7901/ADR cell cycle arrest significantly higher than the percentage of cells in DNA synthesis in the early and resting (G0/G1 phase) 5  Fu group, enter the DNA synthesis phase (S phase) of DNA synthesis late and have wire split phase (G2 / M phase) cell percentage was significantly lower than the control group (P <0.05), which inhibit cancer cell DNA of synthesis and have wire division and induce cell apoptosis, as shown in Table 3. Table 3 TMP tumor cell cycle

    3 Discussion

    The main mechanism of tumor multidrug resistance (MDR) is the overexpression of P-glycoprotein (P glycoprotin of P  gp) and multidrug resistance associated protein (multidrug resistence associated protein, MRP) [6], to cause drug outflows and reduce intracellular drug savings. This study demonstrated that: SGC  7901/ADR cells resistant to 5  Fu relatively resistant to 97.49.

    TMP as the the Rhizoma Chuanxiong main active ingredient is the amide alkaloids. Discovered in recent years through a variety of channels, TMP play therapy and the role of adjuvant therapy of tumors. Calcium channel blocking activity, can be partially corrected mouse ascites cancer resistance to doxorubicin [7]. 100 ~ 320mg / L of a non-cytotoxic concentration range HL60/VCR cells, can increase the sensitivity of a variety of chemotherapeutic drugs [8]. The experimental results show that TMP 5  Fu compatibility does not occur any drug color change or precipitate formation, 100 ~ 300mg / L 5  Fu sensitivity can be improved by SGC  7901/ADR cells, 5  Fu resistant Reversal effect is dose-dependent manner, in which the role of 300mg / L TMP strongest (P <0.01), associated with TMP Ca2 + antagonism, large doses of TMP is more effective in reducing the cell membrane charge indirect effects of P  gp function [9], to improve 5  Fu drug concentration in the resistant cells exert pharmacological effects. FCM confirmed that the combination of two drugs arrest in the G0/G1 phase of the cell cycle, effectively inhibit cancer cell DNA synthesis and mitosis induced apoptosis (P <0.05). 5  Fu joint TMP specific mechanism of action, whether by reducing the multidrug resistance gene MDR1 high expression, affecting the function of P  gp, thereby increasing the intracellular concentration of 5  Fu enhance the effect of chemotherapy, the results need to be further in-depth study.