Abstract Objective To investigate the PI3K/Akt signal transduction pathway inhibitor LY294002 lung adenocarcinoma cell line A549 and xenograft tumor chemotherapy sensitizing effect. Methods MTT assay and flow cytometry LY294002, paclitaxel, LY294002 combined with paclitaxel on proliferation and apoptosis of A549 cells; nude mouse model of detection LY294002, paclitaxel, LY294002, in combination with paclitaxel tumorigenicity of A549 cells. Results LY294002 enhanced paclitaxel inhibition of A549 cells, and can increase the rate of apoptosis. Nude mice experiments showed that LY294002 and paclitaxel inhibited tumor growth associated with inhibition rate of increase. Conclusion LY294002 enhanced paclitaxel chemosensitivity of the A549 cell xenografts in nude mice, inhibition of the PI3K/Akt signaling pathway may improve the effect of chemotherapy of lung adenocarcinoma.

Key words lung cancer; apoptosis; LY294002; paclitaxel

Improving Chemotherapeutic Effect of Lung Adenocarcinoma by Inhibiting PI3K/Akt Signal Pathway

    WU Qiu  ge1, WANG Jing2, ZHANG Mao  lin3, MIAO Li  jun2

    1.Department of Gerontology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China, 2.Department of Respiratory; 3. Department of Chemistry, Zhengzhou UniversityAbstract: Objective To explore the effects of specific inhibitor LY294002 of the PI3K/Akt signaling pathway in enhancing sensitivity to chemotherapeutic agent of lung adenocarcinoma cell line A549 and implanted tumor of nude mice. Methods The effects of LY294002, paclitaxel liposome, LY294002 combined with paclitaxel liposome on proliferation and apoptosis of human lung cancer cell line A549 were evaluated by MTT reduction assay and flow cytometry respectively; and the effects of that on neoplasia were verified by modeling subcutaneous implanted tumor of nude mice. Results LY294002 could increase the inhibitory effect of the paclitaxel liposome and increase the apoptosis ratio on cell line A549 in vitro. LY294002 and paclitaxel liposome could inhibit the growth of subcutaneous implanted tumor of nude mice and the inhibitory rate of LY294002 combined with paclitaxel liposome was higher significantly than that of LY294002 and paclitaxel liposome alone (P <0.01). Conclusion The LY294002 can enhance sensitivity to chemotherapeutic agent of lung adenocarcinoma cell line A549 and subcutaneous implanted tumor of nude mice. Inhibiting the PI3K/Akt signaling pathway can increase the chemotherapeutic sensitivity of lung adenocarcinoma.

    Key words: Lung carcinoma; Apoptosis; LY294002; Paclitaxel liposome
Lung cancer is a common malignancy world today, systemic chemotherapy is the main treatment for the majority of lung cancer patients, the resistance of lung cancer cells is a major cause of failure of chemotherapy. In recent years, the study found that the activation of the PI3K/Akt that are resistant to radiotherapy and chemotherapy-induced apoptosis, the PI3K/Akt inhibitor can enhance tumor of radiotherapy and chemotherapy sensitivity [1  2]. But its mechanism is not fully understood. Joint application PI3K/Akt inhibitor LY294002 and chemotherapy drug paclitaxel liposomes in vitro and in vivo lung adenocarcinoma cells to explore the impact of the the PI3K/Akt inhibitor of the effect of chemotherapy in lung adenocarcinoma.

    1 Materials and methods

    1.1 Materials

    Cell lines A549 (Shanghai Institute of Cell Biology, Research Institute); F12K medium, LY294002 and thiazolyl tetrazolium (MTT) (Sigma); fetal bovine serum (Chinese Academy of Medical Sciences, Institute of Biological Engineering); paclitaxel liposome (trade name: force the paclitaxel, Nanjing Cisco Pharmaceutical Co., Ltd., Zhunzi H20030357); nude mice (Peking University School of Medicine).

    1.2 Experimental Methods

    1.2.1 Cell culture

    A549 cells were treated with F12K medium at 37 ° C, 5% CO2 incubator culture, 0.25% trypsin digestion and passage.

    1.2.2 MTT colorimetric assay detected of LY294002 on A549 cell growth inhibition

    Logarithmic growth phase of the A549 cells were inoculated at 1 × 104 / well in 96-well culture plate, each hole 200 μL. 24h culture medium was changed to set up the blank zero Groups (not inoculated cells), and the control group (only containing equal amounts of solvent) and the experimental group (plus Taxol, paclitaxel concentration followed by 0.05, 0.1, 0.2, 0.4 and 0.8 μg / ml, Each LY294002 final concentration were 20μmol / L), and each group of six wells. MTT (5g / L) 20μl, for 48h culture for 4h plus DMSO 150μl / hole in enzyme-linked immunoassay instrument wavelength of 490 nm, the absorbance A value of each well was measured. Calculate the inhibition rate of cell growth, the inhibition rate = (1 – the average experimental group A / control group A mean value) × 100%.

    1.2.3 flow cytometry cell cycle and apoptosis rate

    Taking the logarithmic growth phase of A549 cells were seeded in culture flasks, adding the different concentrations of the drug (LY294002 final concentration of 20μmol / L paclitaxel final concentration of 0.4μg/ml) to obtain cells for 48h, 4 ° C, 70% cold ethanol fixed. On the machine to detect the cell cycle and apoptosis rate.

    1.2.4 Western blot assay p  Akt protein expression

    The same 1.2.3 Methods Cell Lysates Cells were lysed by adding p  Akt monoclonal antibody, an image analysis system for determination of the absorbance of each band A value of p  Akt protein quantitative analysis.

    1.2.5 nude mouse model to establish and Intervention

    24 nude mice were female mice aged 3-4 weeks, weighing about 18 ~~ 20g. Randomly divided into four groups of six, in the upper right back of nude mice inoculated subcutaneously with A549 cells. Until a week after the tumor to grow. (1) LY294002 group: LY294002 25mg/kg, intraperitoneal injection of 2 times per week; (2) control group: normal saline was injected intraperitoneally; (3) paclitaxel: paclitaxel 20mg/kg intraperitoneal injection; (4) LY294002 + paclitaxel: paclitaxel 20mg/kg + LY294002 25mg/kg, intraperitoneal injection; medication for three weeks. Weighing tumor nodules end of the experiment, the inhibitory rate was calculated: tumor inhibition rate = (control group tumor nodule weight-LY294002 group tumor nodules weight) / control group tumor nodules weight × 100%. Groups of nude mice liver, stomach and intestinal tissue HE staining.

    1.3 statistical methods

    Measurement data ± s, SPSS13.0 statistical software univariate analysis of variance and Pearson correlation analysis was used to compare rates χ2 test, P <0.05 for the difference was statistically significant.

    2 Results

    2.1 LY294002 combined with DDP on A549 cell growth inhibitory effect

    The MTT colorimetric results show: LY294002 in A549 cell proliferation inhibition, and the dose and time-dependent manner [3], as shown in Table 1. According to the concentration of the inhibitory effect of selection for the concentration of 20μmol / L LY294002 as with DDP joint intervention.

    Paclitaxel alone in A549 cells As the concentration increased, the inhibition rate and then increases when the concentration of 0.4μg/ml, and the inhibition rate reached 56.08%; its inhibition rate does not increase when the concentration is further increased, and no dose 0.05)。">effect relationship (r = 0.620, P> 0.05). When LY294002 + paclitaxel concentration group with drugs alone compared inhibition rate of A549 cells was an increasing trend, as shown in Table 2.

    2.2 LY294002 by A549 cells p-Akt protein expression

    Western blot the protein quantitative results show: and LY294002 inhibits p  Akt protein expression, and the concentration of LY294002 intervention dose-dependent manner (r = -0.913, P <0.05), as shown in Table 3.

    2.3 LY294002 and paclitaxel A549 cell cycle and apoptosis rate

    LY294002, paclitaxel and LY294002 with paclitaxel combined with intervention in A549 cells, the apoptosis rate of each drug group compared with the control group, the differences were statistically significant (P <0.01); paclitaxel + LY294002 group rate of apoptosis with LY294002 group and the paclitaxel group phase Table 1 LY294002 A549 cell growth inhibition effect Compared with the corresponding control group, all P <0.01 than the differences were statistically significant (P <0.05). The cell cycle distribution compared with the control group changes, LY294002 + paclitaxel with paclitaxel alone than in A549 cells, G0 / G1 phase was increased, the difference was statistically significant (P <0.05), as shown in Table 4.

    2.4 LY294002 and DDP nude mice growth

    Nude mice inoculated subcutaneously with A549 cells after about a week or so, see subcutaneously transplanted tumor, were round or oval-shaped nodules. 0.05)。">During the experiment, nude mice were not significantly spirit, eating, abnormal bowel movements, and death, the weight of nude mice in each group had no significant difference (P> 0.05). LY294002, paclitaxel and LY294002 + paclitaxel tumor nodule weight were lower than the control group (P <0.01). LY294002 + paclitaxel tumor nodules weight below the LY294002 groups and paclitaxel Table 4 LY294002 plus paclitaxel in A549 cell cycle P <0.05 group, the difference was statistically significant (P <0.01). Prompted LY294002 and paclitaxel inhibited the growth of xenografts in nude mice, the combination of the two inhibitory effect is stronger than the separate application, as shown in Table 5.

    3 Discussion

    The incidence of lung cancer increased year by year, including non-small cell lung cancer (non  small cell lung cancer, NSCLC) accounts for about 85% of the incidence of lung cancer, 55% of patients with NSCLC treatment Ⅲ B ~ Ⅳ, lost the opportunity of surgery . Chemotherapy is the main treatment of such patients, but the tumor cells through a series of adaptive responses to avoid its killing effect of chemotherapy drugs, which do not lead to the effect of chemotherapy. Caused by the tumor cells resistant to many mechanisms, including the PI3K/Akt pathway regulates cell proliferation, survival and differentiation of Table 5 LY294002 combined paclitaxel liposome tumor growth inhibitory effect on nude mice: P <0.01, PI3K can raised the anti-apoptotic gene expression, inhibition of c  myc-induced apoptosis [4]. Therefore speculate P13K signal transduction pathway may play a role in a multi-cell drug. The tumor molecular targeted therapy study recently found that the signal transduction pathways of tumor progression and is closely related to radiotherapy and chemotherapy resistance, PI3K/Akt inhibitors can inhibit the growth of various tumor cells in vitro, leading to tumor cell apoptosis [5  6 ].

    This study found that paclitaxel and LY294002 in combination improve paclitaxel trend inhibitory effect on lung adenocarcinoma cell line A549, can increase the rate of apoptosis, the combination can significantly inhibit tumor growth in lung cancer, the experiment paclitaxel LY294002 in combination with paclitaxel alone increased inhibition rate of lung cancer cells, comparing the two was not statistically significant, significant tumor inhibitory analysis of the reasons may LY294002 in vitro and in vivo effects , may also be related to the cell lines, but overall inhibit the PI3K/Akt signal transduction pathways can enhance the killing effect of paclitaxel on human lung adenocarcinoma A549 cells PI3K/Akt signal transduction pathway activation may lead to tumor cell resistance occurred An important reason. Akt inhibition mechanism of apoptosis may have the following: (1) through phosphorylation of the forkhead family transcription factor, thereby preventing it from play in regulating apoptosis-related gene transcription function [7  8]; (2) activation of Akt IKK mediated I  κB mediated degradation and NF  κB activation [9], to induce anti-apoptotic gene Bcl  2, Bcl  XL expression; (3) activation of Akt directly phosphorylated cAMP response element binding protein (CREB) Ser133 sites the CREB connected to the promoter region induced CAMP response element, Bcl  2 of c  fos and other genes; (4) PI3K-dependent activation of Akt, Bad, Ser136 sites phosphorylation, Bad and Bcl  2 or Bcl  XL can not form a heterodimer, the free Bcl  2 or Bcl  XL restore the anti-apoptotic effects, effectively blocking Bad-induced apoptosis. Another study showed that one of the PI3K/Akt substrate P70s6k also able to phosphorylation of Bad [10]. This study also found that the joint application of LY294002 can significantly increase the rate of lung adenocarcinoma cell apoptosis, indicating that inhibition of the PI3K/Akt signal transduction pathways can effectively improve the drug-induced apoptosis of tumor cells.

    Promote apoptosis through inhibition of the PI3K/Akt signal transduction pathway, to enhance the effect of chemotherapy for lung cancer cells, providing a new idea for us to further improve the quality of life and survival of lung cancer patients, the initiation of apoptosis is a complex process involving multi-gene process, LY294002 induced apoptosis of A549 cells regulate gene activity, expression and transcriptional changes mechanism, will be the direction of future research, which will LY294002 provide a theoretical basis for a possible drug for treatment of cancer.