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 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 evaluate the therapeutic effect and side effects of three-dimensional conformal re-irradiation combined with nedaplation plus 5-fluorouracil synchronously in treatment of recurrent esophageal carcinoma.Methods Fifty-four patients with recurrentesophageal carcinoma were randomly divided into two groups:study group with 27 patients who were treated with three-dimensional conformal re-irradiation (50 ～ 56Gy/25 ～ 28 F) combined with chemotherapy synchronously and control group with 27 patients who were treated with re-irradiation (50 ～56 Gy/25 ～ 28 F)combined with chemotherapy orderly.The patients of study group were treated with nedaplation80 mg/(m2 · d)plus5-FU 350 mg/( m2 · d),from 1 to 5 day synchronously in radiotherapy and after radiotherapy.The patients of control group were treated with nedaplation80 mg/( m2 · d)plus 5-FU 350 mg/(m2 · d)d from 1 to 5 day orderly after radiotherapy.The patients were treated every 3 -4weeks as a cycle with total 2 - 4 cycles in these two groups.Results ( 1 ) The short-term effect:the short-term effect(CR + PR)of the two groups were 59.2％ (16/27) and 55.5％ (15/27) respectively,there was no statistical difference between the two groups( x2 =0.076,P =0.5 ).( 2 ) The survival rate:the survival rate of one,two and three years were 54.2％,26.4％,15.7％ in study group and 40.3％,19.7％,10.8％ in control group respectively The survival rate in study group was significantly higher than that in control group ( x2 =6.87,P =0.032 ).( 3 ) The study of side effects:there was significant difference on the hematologic toxicity between the study group and the control group ( x2 =5.882,P =0.043 ).And there was no significant difference on the non-hematologic toxicity between the two groups ( x2 =4.25,P =0.75 ).(4) The acute radiationesophagitis:There was no statistical difference on radiation-esophagitis between the two groups ( x2 =0.869,P =0.661 ).Conclusion Three-dimensional conformal re-irradiation combined with nedaplation plus 5-fluorouracil synchronously for recurrent esophageal carcinoma has a good efficacy and can also improve the survival rate.

Objective To explore the combination effect of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) 1826 and X-rays on Lewis lung cancer in mouse and the dose response of CpG ODN.Methods The tumor-bearing mouse model was established by injecting Lewis lung cancer cells into the right infra-axillary dermis of mouse.Sixty-four C57BL /6 J mice were evenly randomized into eight groups with 8 mice each:control group,IR group,CpG OND1826 0.15 mg group,CpG OND1826 0.3 mg group,CpG OND1826 0.45 mg group,CpG OND1826 0.15 mg + IR group,CpG OND1826 0.30 mg+ IR group,and CpG OND1826 0.45 mg + IR group.On the 1st,2nd,and 9th days,CpG ODN was injected into mouse.After 3 hours of injection,the mice were start to irradiate with X-rays once a day on the 2nd-6th days,and the total dose was 12.50 Gy.Tumor growth and TGD were measured,and the apoptosis of tumor cells were examined with TUNEL.Results The Lewis lung cancer-bearing model was successfully established in all mice.Under the treatments of CpG OND1826 and irradiation,the tumor volumes were smaller than that of control group,and the tumor volumes of CpG OND1826 0.45 mg+IR group was the smallest.TUNEL results revealed that the apoptosis rate were (2.40 ± 0.51 )％ in control group,(5.62 ±0.50)％ in IR,(7.13±0.83)％ in CpG OND1826 0.15 mg,(11.63±1.06)％ in CpG OND1826 0.3 mg,(19.13 ±0.83)％ in CpG OND1826 0.45 rag,( 12.88±0.83)％ in CpG OND1826 0.15 mg+ IR,(20.57±2.37)％ in CpG OND1826 0.3 mg+ IR,and (28.17 ±3.31)％ in CpG OND1826 0.45 mg + IR group,and thus the apoptosis rate of every therapy group was higher than that in control ( t=11.15,7.91,17.82,39.48,24.73,16.61 and 17.05,P＜0.05).The apoptosis rates of CpG ODN1826 plus X-ray irradiation group were significantly higher than those in IR alone ( t =13.78,15.08 and 17.47,P＜0.05 ) or CpG ODN group (t=18.53,9.66and7.51,P＜0.05).Conclusions CpG ODN1826 can dramatically increase the efficiency of radiotherapy by inhibiting tumor growth and promoting lumor 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.

Background and purpose:As the second general platinum antineoplastic agent, carboplatin has been applied in treating more and more malignant tumors clinically. But it has severe bone marrow suppression when applied in large doses. The purpose of this research was to explore the influence of carboplatin of fractional dose, combined with X ray,on the immunity of Lewis lung cancer in mice. Methods:The model of tumor-bearing mice was induced by injecting Lewis lung cancer cells into the right infra-axillary dermis. In cases given the same total dose, the tumor growth and the immune suppression effect of carboplatin of single dose or of fractional dose , combined with X ray radiation, on the mouse with Lewis lung cancer were observed. Results:The concentration of IL-10 and TNF-? in serum,the spleen exponent were respectively(144.9?48.4)ng/ml,(194.63?64.4)ng/ml,(12.07?2.2)mg/ g in the control group;(279.0?46.9)ng/ml,(71.5?8.4)ng/ml,(5.52?1.31)mg/g in carboplatin of single dose with X ray radiation group, which ,compared with the control group, can dramatically increase the concentration of IL-10,decrease the concentration of TNF-? in the serum ,and dramatically decrease the spleen exponent(P

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.

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