Objective:To prepare IL-12 modified with polyethylene glycol derivatives and determine its modification sites,and to evaluate it in vitro in terms of stability,hemogram recovery,anti-tumor effects and other aspects.Methods:IL-12 was modified with polyethylene glycol propionaldehyde and maleimide imide derivatives,and modified sites of two different derivatives were deter-mined by ultra performance liquid chromatography(UPLC).Recombinant human IL-12 and PEG-IL-12 were used to stimulate NK92 cells,activity and cytotoxicity of IFN-γ were evaluated by kits.CD34+was stimulated to evaluate its blood picture recovery potential.NK cell killing was stimulated to evaluate its effectiveness in improving immunity and anti-tumor effects.Results:Compared with recombinant human IL-12,PEG-IL-12 had higher stability,blood picture recovery potential and anti-tumor effects.Conclusion:PEG-IL-12 can effectively overcome many drawbacks of recombinant human IL-12,greatly improving possibility of its widespread applica-tion in clinical trials.

PURPOSE: The aim of our study was to explore the relationships between the M2 isoform of pyruvate kinase (PKM2) and the sensitivity of human non-small cell lung cancer (NSCLC) cells to docetaxel in vitro. MATERIALS AND METHODS: With the method of plasmid transfection, we silenced the expression of PKM2 successfully in A549 and H460 cells. Western blotting and real-time PCR were applied to detect PKM2 expression at protein and gene levels. Cell viability was examined by CCK8 assay. Cell cycle distribution and apoptosis were examined by flow cytometry. P21 and Bax were detected. RESULTS: Expression of PKM2 mRNA and protein were significantly decreased by shRNA targeting PKM2. Silencing of PKM2 increased docetaxel sensitivity of human NSCLC A549 and H460 cells in a collaborative manner, resulting in strong suppression of cell viability. The results of flow cytometric assays suggested that knockdown of PKM2 or docetaxel treatment, whether used singly or in combination, blocked the cells in the G2/M phase, which is in consistent with the effect of the two on the expression of p21. Cells with PKM2 silencing were more likely to be induced into apoptosis by docetaxel although knockdown of PKM2 alone can't induce apoptosis significantly, which is in consistent with the effect of the two on Bax expression. CONCLUSION: The results suggest that PKM2 knockdown could serve as a chemosensitizer to docetaxel in non-small lung cancer cells through targeting PKM2, leading to inhibition of cell viability, increase of cell arrest of G2/M phase and apoptosis.
Apoptosis ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; Cell Cycle ; Cell Line* ; Cell Survival ; Drug Therapy ; Flow Cytometry ; Humans* ; In Vitro Techniques* ; Lung Neoplasms ; Methods ; Plasmids ; Pyruvate Kinase* ; Pyruvic Acid* ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering* ; Transfection

AIM:To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS:Sequences of TLR9 siRNAs were designed.A549 and H520 cells were transfected with TLR9 siR-NA by lipofectamine.The expression of TLR9 was detected by Western blot .The cell activity was measured by CCK-8 as-say.The experiments were divided into blank control group , control siRNA group and TLR9 siRNA interference group.The cell cycle distribution and cell apoptosis were analyzed by flow cytometry .The expression of P38 and Bax was determined by Western blot .The cells in each group were exposed to CpG ODN 7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity , G2/M phase arrest and apoptosis , but in-creased the protein expression of P 38 and Bax ( P<0.01) .In addition, there was no significant changes of the above inde-xes in CpG ODN7909 treated-TLR9 siRNA group was observed .DOC alone significantly inhibited the cell activity , higher the G2/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn’t affect the expression of P38 in all 3 groups.Compared with the cells treated with DOC alone , the cells treated with CpG ODN7909 combined with DOC exhibi-ted lower cell activity, higher G2/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no sig-nificant change of P38 expression.In addition, there was no significant change of the above indexes in CpG ODN 7909 com-bined with DOC treated-TLR9 siRNA group was observed .CONCLUSION:CpG ODN7909 may enhance the chemothera-peutic sensitivity of DOC in human lung cancer cells by combining with TLR 9.The mechanism might be related to enhan-cing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G 2/M phase of the cells .

Objective To explore the role of pyruvate kinase M2 (PKM2) siRNA in the radiosensitivity of human lung cancer A549 cells.Methods PKM2 siRNA was synthesized according to the coding sequence of PKM2 mRNA and then was transferred into A549 cells with lipofectamine.The expressions of PKM2 gene and protein was detected by RT-PCR and Western blot,respectively.The experiments were divided into PKM2 siRNA interference group,siRNA negative control group,and blank control group.The cells of each group were exposure to 6 MV X-rays in different dose.Radiosensitivity was evaluated by colony formation assay.Flow cytometry was applied to analyze cell cycle distribution and apoptosis.Data are representative of three independent experiments.Results Ccompared with blank control cells,the expressions of PKM2 gene and protein in the PKM2 siRNA transferred A549 cell was efficiently diminished (t =20.91,47.00,P ＜0.01) with inhibition rates of (70.27 ± 1.38)％ and (70.42 ± 1.18) ％,respectively.Compared with control,PKM2 siRNA transfection significantly decreased the D0,Dq,N and SF2 values (t =43.82,28.44,15.60,29.63,P ＜ 0.01) and hence yield a sensitization enhancement ratio (SER) of 1.27.In addition,the percentage of G2/M phase cells in the siRNA group and irradiated group were both significantly higher than that of the blank control group (t =8.35,27.87,P ＜ 0.01).The combined treatments of PKM2 siRNA interference and irradiation arrested more cells in the G2/M phase compared to either treatment alone.The apoptosis rate of siRNA group was not dramatically different from that of blank control group.The apoptosis rate of irradiation group was higher than that of blank control group (t =23.99,P ＜ 0.01),and the combined treatments of siRNA and irradiation enhanced the apoptotic rate compared to either treatment alone (t=9.42,65.21,P ＜ 0.01).Conclusions Specific blockage of PKM2 expression by gene silencing could enhance the sensitivity of human lung cancer A549 cells to radiotherapy in vitro,which may due to the cell cycle arrest and apoptosis induction after irradiation.

Objective To observe the effect of Oxycontin combined with Gabapentin for treatment of malignant neuropathic pain.Methods Sixty-three cases of malignant neuropathic pain in Jinshan Hospital Affiliated to Fudan University were randomly divided into group A, B and C.Patients of which were given Oxycontin, Gabapentin, Oxycontin combined with Gabapentin respectively for pain treatment.The analgesic effects, toxic reaction side effects, quality of life, and immune function were all compared in three groups.Results Compared with pretherapy, the cancer pain score (NRS), quality of life (QOL) and karnofsky performance status(KPS) scores in all groups were changed significantly after drugs therapy(F=375.852,154.612, 151.838,P＜0.05).The levels of CD3,CD4, CD4/CD8 and NK cells in all groups were higher than before therapy(F=158.935,108.145,366.973,92.090,P＜0.05).After treatment,the NRS, QOL and KPS scores in group C were 2.00± 0.86,44.80± 6.07, 84.50± 6.05, in group A were 3.35 ± 0.67,37.35 ± 5.71,74.50 ±10.99,and in group B were 4.05±0.94,35.85±5.90,72.00±8.34, and the different were significant (F =3.250,10.499,3.465,P＜0.05).The levels of CD3, CD4, CD4/CD8and NK cells in group C were (72.94 ±5.63)％,(41.52±4.19)％, 1.86±0.30, (27.57±6.86)％,in group A were (62.84±5.27)％, (33.84 ±5.40)％,1.35±0.37, (20.49±6.67) ％,and in group B were (62.22±8.10)％, (33.19±6.90)％, 1.32 ± ±0.41, (20.32±5.63) ％, and the different were significant (F =3.377,3.344,3.352,3.386, P＜ 0.05).The patient in group C had less adverse effects than those in group A and B.Conclusion Oxycontin and Gabapentin in treatment of malignant neuropathic pain is effective.

Objective To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A549 cells.Methods Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM),as well as pSilencer2.1-nonspecific,was constructed.Lung adenocarcinoma A549 cells were divided into positive group,negative group,and control group to be transfected with pSilencer2.1-ATM,pSilencer2.1-nonspecific,and no plasmid,respectively.The mRNA and protein expression of ATM was measured by RT-PCR and Western blot,respectively.The change in cell radiosensitivity was observed by colony-forming assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.Results The eukaryotic expression plasmid containing ATM siRNA was successfully constructed.The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group.The sensitization enhancement ratios (D0 ratios) for the positive group and negative group were 1.50 and 1.01,respectively.The flow cytometry revealed that the proportions of A549 cells in G1 and G2/M phases were significantly lower in the positive group than in the control group (51.27％ vs 61.85％,P =0.012;6.34％ vs 10.91％,P =0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31％ vs 13.58％,P=0.000;49.31％ vs 13.17％,P=0.000).Conclusions Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A549 cells,probably by affecting the cell cycle and promoting cell apoptosis.

Objective To explore the protectional function of CpG ODN 1826 against radiation pulmonary fibrosis in rats.Methods The rat left lung was exposed to 20 Gy of 6 MV X-rays for establishing a radiation pulmonary fibrosis model.SD rats were randomly divided into control group,irradiated group and intervention group,with 30 rats in each group.CpG ODN 1826 was intraperitoneally injected into rats at 0,1,2,5 and 7 d post-irradiation.The rats were terminated at 5,15,30 and 90 d post-irradiation,and the lung indexes were recorded.Paraffin sections of the radiated lung were conducted with HE staining and Masson staining,the pulmonary fibrosis scores were recorded.The serum concentrations of TGF-β1 and hydroxyproline (Hyp) were measured.Results The radiation pulmonary fibrosis rat model was successfully established.The lung indexes of the control group were lower than those of the irradiated and intervention groups at 5 d post-irradiation (t =3.046,2.252,P ＜ 0.05).The lung indexes of the intervention group were lower than those of the irradiated group (t =4.120,5.226,5.719,P ＜ 0.05).Pulmonary fibrosis scores of intervention group were lower than those of irradiated group (t =3.212,4.959,P ＜ 0.05).The serum concentrations of TGF-β1 of irradiated group were higher than those of the intervention group (t =4.138,5.924,4.138,5.924,P ＜ 0.05).The Hyp in the lung of irradiated group was higher than that of intervention group (t =7.527,8.416,P ＜ 0.05).Conclusions CpG ODN1826 will not worse the radiation pulmonary fibrosis,on the contrary,it could reduce the serum concentrations of TGF-β1 and the lung content of Hyp in radiation pulmonary fibrosis,and protects rat against radiation pulmonary fibrosis.

Objective To investiGate the efficacy of three-dimensional conformal radiotherapy( 3-DCRT)combined with concurrent S-l for locally advanced esophaGus carcinoma in the elderly. Methods Fifty-four elderly patients with locally advanced esophaGus carcinoma were randomly divided into test Group(27 cases treated with 3-DCRT combined with S-l)and control Group(27 cases treated with 3-DCRT alone). DiaGnosis in all cases was confirmed by patholoGy. Results The complete remission rate and overall efficiency rate in test Group were 62. 96% and 85. l9% respectively,and 33. 33% and 59. 26% in control Group(χ2 =4. 75,P=0. 029;χ2 =4. 52,P=0. 033). All 23 patients with dysphaGia in test Group were in remission,and l6 of 22 patients with dysphaGia in control Group were in remission,and there was statistical siGnificant difference between two Groups(χ2 =7. 24,P=0. 007). The one-and two-year survival rates in test Group were 74. l% and 54. 8%respectively,and 55. 6% and 36. l% in control Group(χ2 =3. 9l,P=0. 047;χ2 =3. 98,P=0. 046). Toxicities of Grade 0-Ⅱ in both Groups were similar,and there was no statistical siGnificant difference(χ2 =l. 08,P=0. 299). Conclusion The complete remission rate,overall efficiency rate and one-and two-year survival rates of 3-DCRT combined with concurrent S-l were siGnificant hiGher than 3-DCRT alone in the treatment for the elderly patients with locally advanced esophaGus carcinoma. And ti’s adverse effects were well tolerated.

Objective To evaluate the efficacy and toxicity of anthracycline and taxane combination regimen in the treatment of advanced breast cancer in patients who were previously treated with anthracycline and taxane.Methods Twenty-six patients who previously failed to respond to anthracycline and taxane based chemotherapy received pemetrexed and tegafur combination regimen (pemetrexed:500 mg/m2,ivgtt on the first day; tegafur:80 mg/(m2 · d),twice daily during day 1-14).The regimen was repeated at least two cycles of three weeks each and the clinical response was recorded at two weeks after treatment.Results Of the 26 patients,he tumor control rate was 65.3％ (17/26),and the overall response rate was 46.2％ (12/26),including 3 patients with complete remission and 9 patients with partial remission.The symptoms were stable in 5 patients and improved in 9 patients.There was no severe complications or death observed.The most frequent treatment-related adverse events were myelosuppression,nausea,vomitting,rash and hepatic function impairment.Conclusion Pemetrexed combined with tegafur in the treatment of patients with advanced metastatic breast cancer who did not respond to anthracycline and taxane treatment is effective with minimal adverse reactions and can be tolerated by patients.

Objective To investigate the relationship between the effect of unmethylated cytosine phosphate-guanine oligodeoxynucleotide ( CpG ODN ) 7909 on X-ray-induced G2/M phase arrest and apoptosis and the phosphorylation of ATM kinase.Methods Human lung adenocarcinoma A549 cells were randomly classified into five groups,control,CpG group,irradiation group,CpG + irradiation group and ATM siRNA + CpG + irradiation group.Cell survival fraction was evaluated by clonogenic assay.Cell cycle and apoptosis were analyzed by flow cytometry.The expressions of ATM,checkpoint kinase-2(Chk2) and p53 were detected with Western blot.Results Compared to the situation under X-ray irradiation alone,decreased cell clonogenic survival,prolonged G2/M arrest,and increased cell apoptosis were observed after the combination treatment with CpG ODN7909 and X-rays ( t =13.41,17.32 and 7.71,P ＜ 0.05).Moreover,the phosphorylations on ATM,Chk2,and p53 were increased in the irradiated A549 cells that had been pre-treated with CpG ODN7909.After ATM siRNA interfering,abrogation of G2/M arrest,reduction of apoptosis,and decrease of Chk2 and p53 phosphorylation were found in A549 cells treated with CpG ODN7909 and X-rays ( t =26.84,2.98,47.24 and 67.47,P ＜0.05).Conclusions CpG ODN7909 can enhance the X-ray-induced phosphorylation of ATM kinase in human lung adenocarcinoma cells in vitro,which might be involved in regulating G2/M phase arrest and apoptosis.