ABSTRACT
<p><b>OBJECTIVE</b>To study the chemosensitivity of lung adenocarcinoma cell line A549 cells to liposome-encapsulated paclitaxel after treatment by nm23-H1-small interference RNA (nm23-H1-siRNA) in vitro.</p><p><b>METHODS</b>The A549 cells were divided into two groups: non-transfected group and nm23-H1-siRNA-transfected group. Western blot analysis was used to detect the expression of nm23-H1. MTT and flow cytometry were used to determine the cell mortality rate, apoptosis rate and cell cycle after liposome-encapsulated paclitaxel treatment in both groups.</p><p><b>RESULTS</b>The expression of nm23-H1 in A549 cells was significantly decreased after transfection with nm23-H1-siRNA. After treatment for 48 hours with liposome-encapsulated paclitaxel, the cell mortality rate was increased with the increasing concentration of liposome-encapsulated paclitaxel in both groups, but increased higher in the nm23-H1-siRNA-transfected group. When the concentration of liposome-encapsulated paclitaxel was above 5 µg/ml, the cell mortality rate was significantly higher than that in the non-transfected group (P < 0.05). The proportion of apoptotic cells also increased in the nm23-H1-siRNA-transfected group, compared with that of the non-transfected group (t = 3.812, P < 0.05), while the proportion of cells at S and G(2)/M phase decreased after transfection with nm23-H1-siRNA (S phase:t = 8.356, P < 0.05; G(2)/M phase:t = 7.256, P < 0.05).</p><p><b>CONCLUSIONS</b>Nm23-H1 is related with the chemoresistance to liposome-encapsulated paclitaxel in lung adenocarcinoma cell line A549 cells. Inhibition of the expression of nm23-H1 by nm23-H1-siRNA can improve the in vitro chemosensitivity of A549 cells to liposome-encapsulated paclitaxel.</p>
Subject(s)
Humans , Adenocarcinoma , Metabolism , Pathology , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Dose-Response Relationship, Drug , Lung Neoplasms , Metabolism , Pathology , NM23 Nucleoside Diphosphate Kinases , Genetics , Metabolism , Paclitaxel , Pharmacology , RNA, Small Interfering , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To construct a multiple myeloma (MM)-specific APE1siRNA expression vector, and detect the specific knock-down effect of the siRNA on expression of APE1 protein.</p><p><b>METHODS</b>APE1siRNA cDNA sequence was designed, synthesized and inserted into pSilencer 2.0-U6 linear expression vector. pSilencer APE1siRNA was digested by enzyme EcoRI and BamHI, then linear vector and IgP fragments were conjugated by T4 DNA ligase. pSilencer IgP-APE1siRNA and pSilencer IE-IgP-APE1siRNA were digested by enzyme EcoRI or XhoI. Linear vector and IE or Kappa fragments were conjugated by T4 DNA ligase. Then a MM specific pSilencer K-IE-IgP-APE1siRNA was cloned. The recombinant products were identified by DNA sequencing and enzyme digestions at each step. pSilencer K-IE-IgP-APE1siRNA plasmid was transfected to KM3, HOS, MDA-231 cells by liposome. APE1 gene silence induced by RNAi was analysed by Western blot.</p><p><b>RESULTS</b>APE1 protein in KM3 cells could be knocked down effectively and specifically by pSilencer K-IE-IgP-APE1siRNA vector. After 2 days, the level of APE1 protein in KM3 cells transfected with siRNA was 0.118 +/- 0.047, while that transfected with plasmid only was 0.988 +/- 0.029. The efficiency of gene silence was 90%.</p><p><b>CONCLUSION</b>A MM specific APE1siRNA expression vector was successfully constructed.</p>
Subject(s)
Humans , Base Sequence , Cell Line, Tumor , Cloning, Molecular , DNA-(Apurinic or Apyrimidinic Site) Lyase , Genetics , Genetic Vectors , Genetics , Molecular Sequence Data , Multiple Myeloma , Genetics , RNA Interference , RNA, Small Interfering , Genetics , TransfectionABSTRACT
Objective To investigate the expression of TGF-β and TGF-β receptor in human breast cancer cell Bcap-37 inhibited by soybean isoflavones. Methods mRNA and protein of TGF-β1、TGF-βRⅠ in Bcap-37 cells were examined with reverse transcription ploymerase chain reaction(RT-PCR) and immunohistochemistry after cells were treated with daidein or genistein for 1-4 d.The expression of TGF-β1 and TGF-β2 was determined with TGF-β resistance test. Results The TGF-β1, TGF-β2 and TGF-β recepor increased in Bcap-37 cells at a concentration of 3×10-5 mol/L of genistein. No changes was found when treated with daidzein. Conclusion Genistein may inhibit the proliferation of Bcap-37 cells and accompany with increasing expression of TGF-β1, TGF-β2 and TGF-β receptor.
ABSTRACT
Objective To investigate the expression of TGF-β and TGF-β receptor in human breast cancer cell Bcap-37 inhibited by soybean isoflavones. Methods mRNA and protein of TGF-β1、TGF-βRⅠ in Bcap-37 cells were examined with reverse transcription ploymerase chain reaction(RT-PCR) and immunohistochemistry after cells were treated with daidein or genistein for 1-4 d.The expression of TGF-β1 and TGF-β2 was determined with TGF-β resistance test. Results The TGF-β1, TGF-β2 and TGF-β recepor increased in Bcap-37 cells at a concentration of 3×10-5 mol/L of genistein. No changes was found when treated with daidzein. Conclusion Genistein may inhibit the proliferation of Bcap-37 cells and accompany with increasing expression of TGF-β1, TGF-β2 and TGF-β receptor.