| Background and ObjectiveThe first morbidity and mortality of malignant tumors is lung cancer in the world, it is a serious threat to people’s health. Non-small cell lung cancer (NSCLC) is the most important pathological type of lung cancer, accounting for 80-85% incidence. Lung adenocarcinoma is the highest grade malignancy of NSCLC, distant metastasis by blood way could occur in early stage. An effective inspection method is lack in the early stage lung cancer, and patients have no symptoms in the early stage, most of them was found by health examination. Most patients with symptoms were in the middle and advanced stage. Early stage lung cancer can be removed by surgery, patients with advanced stage rely mainly on radiotherapy and chemotherapy.Taxol (paclitaxel) is the first-line treatmen currently in NSCLC chemotherapy. Paclitaxel is the main active ingredient in Taxus brevifolia, it is a microtubule stabilizing agent. Effect on the tumor cells, paclitaxel can promote tubulin polymerized to form microtubules, stabilize microtubules, interfering cells spindle formation, thus interfering mitosis leading to cell death. Although paclitaxel has an efficient killing effect in the treatment of NSCLC, but the overall survival rate is still relatively low (30-50%), which is due to the primary or secondary resistance of tumor cells to paclitaxel, the majority of patients become resistance to paclitaxel at the beginning of treatment or therapy in the process. Resistance is an important factor affecting the paclitaxel chemotherapy.Under the premise of no next generation of more effective chemotherapy drugs appears, to improve sensitivity of existing chemotherapy drugs or reverse drug resistance of chemotherapy is the important strategy to improve the treatment of NSCLC.The main mechanism of paclitaxel drug resistance according to their role in the process are various:(1) The overexpression of membrane efflux transport protein P-glycoprotein (P-gp). Paclitaxel is’pumped’ out of celluar by the P-gp membrane efflux transport protein when it enter the cell. The result is reducing the concentration of the drug, and then the threshold of killer cells by paclitaxel can’t be reached. P-gp is an important member of ATP-binding cassette (ABC) family, and was coded by ABCB1 genes. (2) The mutations or abnormalities of paclitaxel binding site in the microtubule network including a-tubulin and β-tubulin. Paclitaxel can not bind to tubulins of the mutations. Tubulins lost the polymerization function. (3) Down-regulation of cell death pathways, including Bcl-2, P53, etc, cell death signaling was terminated.In the study of paclitaxel resistance reversal, there is no breakthrough, and the drugs could not be applied to clinical practice due to drug toxicity. Chinese medicine has now become a breakthrough study of antitumor mechanism because of its multi-targeted anti-tumor effect of high efficacy. Triptolide (TPL) is the main active ingredient of a traditional Chinese medicine Tripterygium wilfordii, as a double terpenoids. A variety of biological activity have been confirmed in studies, including immunosuppressive, anti-inflammatory and anti-tumor effects, particularly in the field of anti-tumor it has a good prospect. TPL has entered 1 clinical trial stage in the treatment of leukemia. Currently a large number of studies have confirmed that a variety of tumor cells could be inhibited by triptolide, such as breast cancer, gastrointestinal cancer, prostate cancer and nervous system tumors.Since 1972 triptolide was discovered, the molecular mechanism of action was conducted in-depth research by scholars, such as TPL exert immunomodulatory effects, mainly through inhibition of nuclear transcription factor -κB (NF-κB) reducing inflammatory factors generate weakened immune response; In the aspect of anti-tumor effect, TPL can induce a variety of tumor cell apoptosis by inhibiting HSF1 (heat shock factor 1), API (activator protein 1), NF-κB and other transcription regulatory factors play an anti-tumor effect. This shows that NF-κB may be a important basic that TPL play biological activity. Denis found that inhibition of tumor cell RNA polymerase II transcription is an important underlying mechanism of anti-tumor effect of triptolide.NF-κB is a group of important proteins of transcripting, it is a nuclear transcription factor that having variety ability of regulatory function. As early as 1986 Sen found the most important biological transcriptional activator from B lymphocytes nucleus extract of mouse, it was found in a wide variety of eukaryotic cells. NF-κB involved in various physiological processes, the regulation of various cytokines and play a role in a variety of cellular processes, including stress, gene expression of cytokines, free radicals, cell adhesion, cell cycle activation, apoptosis and tumor formation. It is an important nuclear transcription factors. In normal cells, most NF-κB combined with its inhibitor IκBα, IκBβ, IκBε in a combination in the cytoplasm, and formed an inactive NF-κB/I-κB complex in the cytoplasm. TNF-a transfer signal through the cell membrane receptor, and gradually activate TNF-a receptor-associated factor TRAF, activate NF-κB inducing kinase NIK and I-κB activation kinase IKK. I-κB was phosphorylated by IKK and degraded by the proteasome. NF-κB/I-κB complex was disintegrated, and the free NF-κB was released and transferred into the nucleus to regulate the transcription of target genes, which are regulated genes include genes associated with drug resistance, such as anti-apoptotic gene Bcl-2, XIAP etc.In addition, studies show that multidrug resistance gene ABCB1 transcription was regulated by NF-κB. The NF-κB binding site located upstream of the gene ABCB1 and was demonstrated by the application of gene transfer technology. ABCB1 gene was the downstream gene of NF-κB. NF-κB can regulate ABCB1 encoding P-glycoprotein (P-gp) expression. Studies have shown the P-gp encoded by ABCB1 gene down-regulated through inhibiting the expression of NF-κB in human colorectal adenocarcinoma cell HCT15, and P-gp was confirmed a direct relationship with the drug resistance of paclitaxel. The NF-κB plays an important role in paclitaxel-resistant, and is an important transcriptional regulator in paclitaxel resistance.Eukaryotie transcription machinery is very complex. a variety of protein factors involved in assisting the transcription apart from transcriptional regulation factors. Another important factor is the RNA polymerase II (Pol II). Recent studies have further found that TPL has a wide range of tumor cell transcriptional inhibition.The RNA synthesis could be inhibited widely by TPL in NSCLC A549 cells (including total RNA, mRNA).98% of gene expression of A549 cells was down-regulated by TPL treated, especially the expression those short-lived transcription factors, cell cycle regulators (CDC25A), and proto-oncogenes (Myc, Src) was significantly down-regulated. This effect of TPL depends on its inhibition of RNA polymerase II function.Previous studies have indicated TPL might have the multidrug resistance reversal effect, such as Chen found that multidrug resistance gene (multidrug resistance 1, MDR1) expression can be inhibited by TPL in human oral carcinoma tumor KB cells, and the sensitivity of 5-FU could be increased; Li et al found that the sensitivity of doxorubicin-resistant human leukemia K562/A02 cells to adriamycin could be improved by TPL inhibiting the expression of miR-21 as well as regulating PTEN level. Such evidence suggests that TPL may have potential resistance reversal effect, but the molecular mechanism is unclear. And there is no related research in the effect of TPL in paclitaxel-resistant lung cancer. Therefore, we propose a hypothesis that TPL inhibit transcription of a broad spectrum of resistance genes by inhibiting NF-κB signaling pathway and the RNA polymerase II, and thus play a role in resistance reversal.We will investigate the effect of TPL in apoptosis and cell cycle of paclitaxel-resistant human lung adenocarcinoma cell A549/Taxol in this study, research on the resistance reversal effect of TPL in lung adenocarcinoma paclitaxel-resistant in terms of the molecular mechanisms, elucidate the drug reversal effect and mechanism of TPL in lung adenocarcinoma paclitaxel-resistant. The results of this study will provide a theoretical basis in further study of paclitaxel resistance reversal agents in NSCLC.Materials and methodsPart one:Effect of triptolide on paclitaxel-resistant human lung adenocarcinoma cell line A549/Taxol1. Establishment of the Taxol-resistant lung adenocarcinoma cell line, A549/Taxol.The A549 lung adenocarcinoma cell line was purchased from the Typical Culture Preservation Commission Cell Bank, Chinese Academy of Sciences (Shanghai, China). The Taxol-resistant lung adenocarcinoma cell line, A549/Taxol, was established by the method of increasing the drug concentration gradient. Human lung adenocarcinoma A549 cells in the logarithmic growth phase were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum,100 U/ml penicillin and 100 ig/ml streptomycin at 37℃ in an atmosphere containing 5% CO2. Subsequently, Taxol was added to the culture medium at a minimum concentration of 20 ng/ml. The cells were cultured for 24 h. Sensitive cells died as the drug induced apoptosis. The surviving cells were cultivated to the next logarithmic phase in the culture medium without Taxol. In the next cycle, the cells were cultured andinduced by Taxol using the same process, however, the concentration of Taxol was increased from 20 ng/1 to 400 ng/1 (the concentration gradient was 20,40,60,80,100,120,200, 30 and 400 ng/ml). The process was repeated until the A549 cells grew steadily in the medium with 400 ng/1 Taxol. These cells were Taxol-resistant lung adenocarcinoma cells (A549/Taxol). On completion of the initial stage of the assessment of the cells, the resistance index of the cell line was calculated, according to the half maximal inhibitory concentration (IC50) of A549/Taxol/IC50 of A549, to be 51.87.2. Effect of TPL on A549/Taxol cell apoptosis.Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used for the detection of apoptosis. A549/Taxol cells in thelogarithmic growth phase were inoculated into 6-well plates at a density of 1x105 cells/cm2 and cultured for 24 h. Once the cells had adhered, different concentrations of TPL (0.03, 0.3 or 3 imol/1) were added to the cells, and the negative control group was established. The cells were incubated for 2,4,6 or 12 h, and were subsequently trypsinized and collected using 0.25%trypsin (excluding EDTA). The cells were washed twice with phosphate-buffered saline (PBS), prior to centrifugation at 870 x g for 5 min, and 5x105 cells were collected. Subsequently,500 il binding buffer suspen-sion and 5 il annexin V-FITC were added to the cells prior to mixing, and then 5 il PI was added and mixed. The incubation was performed for 5-15 min at room temperature in the dark. The extent of apoptosis was assessed using a flow cytometer (excitatory wavelength (eex) 488 nm; emission wavelength, (eem) 530 nm).3. Effect of TPL on the A549/Taxol cell cycle.PI staining was used to detect the cell cycle. A549/Taxol cells in the logarithmic growth phase were inoculated into 6-well plates at a density of 1x105cells/cm2, prior to culture for 24 h. Once the cells had adhered, different concentrations of TPL (0.03, 0.3 or 3 imol/1) were added to the cells for 2,4,6 or 12 h. The cells were subsequently harvested with 0.25% trypsin (excluding EDTA) and washed with PBS, prior to centrifugation at 870 x g for 5 min, and 5x105cells were collected. Single cell suspen-sions were fixed with the volume fraction of 70% ethanol overnight, preserved at 4℃, and subsequently washed with PBS fixative prior to staining. An aliquot of 100 il RNase A was added and the cells were incubated in a water bath for 30 min at 37℃ Subsequently,400 il PI was added to stain the cells prior to blending, and the cells were maintained in the dark for 30 min at 4℃. The quantity of red fluorescence at an excitation wavelength of 488 nm was assessed using a flow cytometer.Part two:The inhibition of NF-eB signaling pathway and it mediated drug resistance related gene expression by TPL on A549/ Taxol cell1. Western blotting.The extracted protein concentration was measured using a conventional bicinchoninic acid method. Samples of 40 ig protein were cooled on ice following an incubation at 95-100℃ for 5 min, and the samples were subsequently electrophoresed using 8% SDS-PAGE. For the electrophoresis, a stacking gel was used at a constant voltage (80V) for 20 min, followed by a separating gel at 100 V for-80 min. The gel was removed and placed in transfer buffer to equilibrate for 15 min. The filter paper and the polyvinylidene fluoride (PVDF) membrane were prepared and placed in transfer buffer and deionized water, respectively. For the wet electrotransfer stage, the bottom electrode (anode) was laid flat, with the filter paper, PVDF membrane, the gel and a filter paper placed on top. The top electrode (cathode) was placed on the interlayer following the exclusion of air bubbles. For the electrotransfer, the apparatus waspowered by a constant current (200 mA) for 1 h. The PVDF membranes were blocked with 5% skimmed milk blocking buffer (incubated at room temperature for 1 h), and subsequently the blocking solution was discarded. The primary antibody, rabbit anti-CK19 (1:5,000) and a rabbit anti-a-actin antibody (1:4,000) were added (-0.1 ml/cm2), followed by an incubation with agitation at 4℃ overnight. The membrane was rinsed with PBS with Tween(?) 20 (PBST) four times, each time for 5 min. The membrane and secondary goat anti-rabbit immunoglobulin G antibody conjugated with HRP (horseradish peroxidase-labeled antibody and secondary antibody in blocking buffer; 1:5,000) were incubated with agitation at room temperature for 1-2 h. The membrane was subsequently washed with PBST, and rinsed five times (5 min each time). The calculated quantity of developer (EMD Millipore) was 0.1 ml/cm2, and the developer was applied to the PVDF membrane and placed at room temperature for 1 min. The PVDF membrane was wrapped in plastic in order to avoid air bubbles. The membrane proteins were attached to the X-ray film for quick exposure in a darkroom and developed. The exposure time was adjusted to enable the best development of the protein bands to occur.2. Immunofluorescence staining.The A549/Taxol cells were treated with TPL (3 iM) for 12 h. The cells on the slides were subsequently fixed with 4% paraformaldehyde. A total of two drops of 3% H2O2/methanol solution was added to each slice at room temperature (15-25℃) for 10 min, prior to immersion in PBS three times. Aliquots of 50-100 il ready-to-use goat serum were added dropwise and the cells were incubated at room temperature for 20 min. Subsequently,50-100 il primary rabbit anti-CK19 antibody (1:200 dilution) was added. The cells were incubated in a wet box for 2 h at 37℃ and were subsequently immersed in PBS three times. Subsequently,50-100 il FITC tetramethylrhodamine (1:200 dilution) secondary antibody was added, and the cells were incubated in the dark for 1 h at 37℃, prior to immersion in PBS three times. Aliquots of 50-100 il formulated dye,4’,6-diamidino-2-phenylindole, was added to each slice, which was subsequently placed in the dark at room temperature for 5 min. The expression of the proteins was observed under a fluorescent microscope, and images were captured at three areas where high levels of expression had occurred.ResultsPart one:Effect of triptolide on Taxol-resistant human lung adenocarcinoma cell line A549/Taxol1. The establishment of paclitaxel-resistant human lung adenocarcinoma cell line A549/TaxolConcentration gradient cultured human lung adenocarcinoma paclitaxel-resistant cell A549/Taxol, measured resistance index (RI)= IC50 (A549 /T)/IC50 (A549)= 51.87.2. Apoptosis was detectedby flow cytometry, the result showed that TPL can induce A549/Taxol cell apoptosis, this effect is time-dose dependent manner.3. The cell cycle was detected by flow cytometry, the result showed that TPL can adjust A549/Taxol cell cycle and cell cycle arrest in S/G2 phase, and thus make tumor cell death.Part two:The inhibition of NF-eB signaling pathway and it mediated drug resistance related gene expression by TPL on A549/Taxol cell1. TPL can inhibit the transferred of NF-κB into the nucleus in the A549/ Taxol cell, can inhibit the expression of NF-κB.2. TPL can inhibit the expression of the largest subunit Rpblof RNA polymerase II.3. TPL can inhibit the downstream apoptosis related genes FLIP, XIAP, Bcl-2, Bcl-xL, and COX-2 expression regulated by NF-κB.Conclusions1. TPL can induce paclitaxel-resistant human lung adenocarcinoma cell line A549/Taxol apoptosis, indicated TPL having a resistance reversal effect in paclitaxel-resistant human lung adenocarcinoma cell.2. TPL can inhibit the transferred of NF-κB into the nucleus in the A549/ Taxol cell, can inhibit the function of RNA polymerase II. The result indicated TPL inhibited the function of gene transcription in paclitaxel-resistant human lung adenocarcinoma cell A549/Taxol.3. TPL can inhibit the expression of apoptosis related genes FLIP, XIAP, Bcl-2, Bcl-xL, and COX-2. The result indicated TPL could inhibit drug resistance-related gene expression.4. TPL could inhibit transcription of a broad spectrum of resistance genes by inhibiting NF-κB signaling pathway and the RNA polymerase II, and thus play a role in resistance reversal. |
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