Single Drug Treatment Group Research Articles

Therapeutic effect of in vitro 5-aza-2'-deoxycytidine combined with imatinib on gastrointestinal stromal tumor

To investigate the impact of 5-aza-2'-deoxycytidine(5-aza-CdR) combined with imatinib on the proliferation, motility, invasion, and apoptosis of gastrointestinal stromal tumors(GIST) cells in vitro. MTT assay was used to investigate the effect of the two agents on proliferation of GIST882. Plate colony forming assay was used to determine the number of colony-forming. Motility and invasion abilities were tested to evaluate the inhibitory effect of each agent. Flow cytometry was used to observe apoptosis and cell cycle. 5-aza-CdR or imatinib effectively inhibited the growth of GIST882 cells in concentration- and time-dependent manner. The inhibitory rate of combined treatment using 5-aza-CdR and imatinib was significantly higher than that of 5-aza-CdR or imatinib alone(P<0.05). After treatment for 48 h, the apoptosis rates of 5-aza-CdR group (1000 μg/L) and imatinib group (100 μmol/L) were (11.7±1.2)% and (14.6±0.8)%, respectively. Compared with the control group (2.8±0.3)%, the difference was statistically significant(P=0.000). Furthermore, the difference in apoptosis rate was significant between combined treatment group (19.4±1.1)% and single drug treatment group(vs. 5-aza-CdR group, P=0.000, vs. imatinib group, P=0.013). 5-aza-CdR raised G0/G1 ratio and reduced S ratio of GIST882. Imatinib and combined group had no apparent influence on the cell cycle of GIST882 cells. 5-aza-CdR may be a potential agent of GIST treatment in the near future.

Increased Expression of P-Glycoprotein Is Associated with Doxorubicin Chemoresistance in the Metastatic 4T1 Breast Cancer Model

Development of drug resistance is one of the major causes of breast cancer treatment failure. The goal of this study was to understand the chemoresistance mechanism using the highly metastatic 4T1 breast cancer model, which emulates stage IV breast cancer in humans. The metastatic 4T1 breast cancer cell line treated with either doxorubicin or 5-FU showed a concentration-dependent reduced cell proliferation, with induced G2-phase growth arrest (doxorubicin) or G1-phase growth arrest (5-FU). Doxorubicin treatment partially suppressed the multiorgan metastasis of 4T1 breast cancer cells in the lung, heart, liver, and bone, compared with either 5-FU or cyclophosphamide. We isolated and characterized 4T1 breast cancer cells from doxorubicin-resistant metastatic tumors (cell line 4T1-R). Multiorgan metastasis of drug-resistant 4T1 breast tumors was totally resistant to doxorubicin treatment. Our results indicate that doxorubicin is localized exclusively in the cytoplasm of resistant 4T1 breast cancer cells and that it cannot reach the nucleus because of increased nuclear expression of P-glycoprotein. Pretreatment of doxorubicin-resistant 4T1-R breast cancer cells with verapamil, a general inhibitor of P-glycoprotein, increased nuclear translocation of doxorubicin and cellular cytotoxicity. Thus, impaired nuclear translocation of doxorubicin due to increased expression of P-glycoprotein is associated with doxorubicin resistance of highly metastatic 4T1 breast cancer.

Study on mechanism of thalidomide combined with temozolomide to suppress proliferation of U251 glioma cell in vitro

Objective To provide a more reasonable regimen of temozolomide and thalidomide, and study the mechanism of these 2 drugs in inhibiting the proliferation and growth of U251 glioma cell in vitro. Methods Human glioma cell line U251 was cultured in vitro and divided into different treatment groups for 3 d: temozolomide group (100 μmol/L), thalidomide group (100 μg/L), temozolomide (100 μmol/L) with thalidomide group (100 μg/L) and vehicle control group. After different treatment for 3 d, 3⁃(4, 5⁃ dimethylthiazol⁃2⁃yl)⁃2, 5⁃diphenyltetrazolium bromide (MTT) was adopted for the determination of cell viability, and cell cycle was analysed by flow cytometry. After labeled with acridine orange (AO), autophagy vesicles were quantitatively detected by flow cytometry. TdT⁃ mediated dUTP⁃biotin nick end labeling (TUNEL) was employed in observing and detecting the apoptosis of treated cells. Western blotting was used in examining the autophagy and apoptosis⁃related proteins. Results Compared with the 2 drugs used alone, temozolomide with thalidomide imposed more obvious inhibition on tumor cell growth (P = 0.000, for all). Combination of 2 drugs induced tumor cell cycle arrest in G0-G1. Both of autophagic and apoptotic cell death could be induced by temozolomide with thalidomide in U251. In two⁃drug treatment group, the expression of autophagy⁃related microtubule⁃associated protein 1 light chain 3 (MAP1LC3) and apoptosis⁃related Caspase⁃3 was significantly higher in transcriptional level in comparing with single drug treatment group (P = 0.000, for all). Conclusion Temozolomide combined with thalidomide may induce 2 types of programmed cell death—apoptotic and autophagic cell death by up ⁃regulating the expression of related genes in U251 glioma cell, thereby, the combination of these 2 drugs may suppress the proliferation and growth of U251 glioma cell in vitro. DOI：10.3969/j.issn.1672-6731.2010.05.007

Combined Use of Acid Fibroblast Growth Factor, Granulocyte Colony-stimulating Factor and Zinc Sulphate Accelerates Diabetic Ulcer Healing

Our previous studies demonstrated that topic application of recombinant human acid fibroblast growth factor (aFGF) significantly, but still not completely, improved in diabetic ulcer healing. To obtain a maximal therapy for diabetic ulcer healing, a combined protocol containing aFGF, anti-oxidative reagent zinc (Zn) and stem cell stimulator granulocyte colony-stimulating factor (G-CSF), i.e.: aFGF/G-CSF/zinc sulphate (ZnSO4), was explored in the present study. Diabetes was induced by a single dose of streptozotocin (STZ, 55 mg/kg) in Sprague Dawley rats, and full thickness skin wound was made in diabetic rats at 2 months after diabetes onset. Diabetic ulcer rats were treated with aFGF, G-CSF, ZnSO4, aFGF/G-CSF/ZnSO4 or vehicle control, respectively and 3, 6, 9, 12, 15, 18, 21 and 28 days later, therapeutic effects were evaluated by calculating ulcer area. On day 7, 14, 21 after treatment, rats were sacrificed to collect 2 pieces of dorsal skin from 3 different sites (wound center, edge and healed area) to perform histopathological examination. Results showed that treatment with aFGF/G-CSF/ZnSO4 significantly enhanced ulcer healing compared with single drug treatment groups at different time points. Healing times for 100% of the wound in the aFGF/G-CSF/ZnSO4 group was 20.00 ± 1.15 days, and significantly shorter than those in single treatment groups (p < 0.05). Histopathological and immunohistochemical analysis disclosed significant increases in capillary density, proliferating cells, the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and the ratios of TIMP-1 to matrix metalloproteinase 1 (MMP-1) in aFGF/G-CSF/ZnSO4 group as compared to other single treatments. Collectively, the combinative protocol of aFGF/G-CSF/ZnSO4 significantly enhanced the therapeutic effect on diabetic ulcer wound, probably through the promotion of fibroblast proliferation and differentiation, enhancement of blood vessel regeneration, and up-regulation of TIMP-1 and down-regulation of MMP-1 expression in diabetic ulcer healing process.