[Enhancing effect of antisense oligonucleotide targeting bFGF on apoptosis in hepatoma cells in vitro].

OBJECTIVE: To investigate the cell cycle changes of hepatoma cells and the effect of antisense oligonucleotide targeting bFGF on apoptosis in the hepatoma cells. METHODS: The oligodeoxynucleotides were transfected with Lipofectin into hepatoma HepG2 cells. Inhibition of bFGF protein expression was assessed by confocal laser scanning microscopy and Western blot under the best condition of transfection of antisense oligonucleotide targeting bFGF, and the apoptosis in those cells was determined by flow cytometry. HepG2 cells were cultured in 24-well culture dish. The cultured cells were divided into 3 groups: group 1, the normal control group without any treatment; group 2, transfected with antisense oligonucleotide targeting bFGF; group 3, transfected with scrambled sequence targeting bFGF. RESULTS: The results from confocal microscopy and Western blot showed an inhibition of expression of bFGF at different levels under the best condition of transfection with antisense oligonucleotide targeting bFGF. The treatment with antisense oligonucleotide of bFGF not only reduced the expression of bFGF revealed by confocal microscopy and Western blotting, but also increased the apoptosis in HepG 2 cells (P < 0. 01). CONCLUSION: Treatment with antisense oligonucleotide of bFGF inhibits expression of bFGF protein and increase apoptosis. bFGF may take part in apoptosis regulation of hepatoma cells and may be used as a target in the treatment of hepatocellular carcinoma.

MicroRNA 25, microRNA 145, and microRNA 210 as biomarkers for predicting the efficacy of maintenance treatment with pemetrexed in lung adenocarcinoma patients who are negative for epidermal growth factor receptor mutations or anaplastic lymphoma kinase translocations.

This study was conducted to evaluate microRNAs (miRNAs) as biomarkers for use in predicting the efficacy of maintenance therapy with pemetrexed in patients with stage IIIb or IV lung adenocarcinoma and who had already received first-line treatment with pemetrexed plus platinum. Patients who were negative for epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) translocations were assigned to a pemetrexed group and an observation group. Patients in the pemetrexed group (n = 76) received maintenance treatment with pemetrexed (500 mg/m(2), once every 21 days) plus best supportive care. Patients in the observation group (n = 72) agreed to receive only best supportive care until disease progression. Blood samples were collected from all patients in both groups before treatment and were used to detect expression levels of various miRNAs in serum by the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. The expression levels of miR-25, miR-145, and miR-210 were significantly different in the 2 groups of patients. Furthermore, the median progression-free survival (PFS) times for patients in the pemetrexed and observation groups were 4.5 and 2.9 months, respectively. The PFS times among patients in the pemetrexed group varied significantly and were related to patient expression levels of miR-25, miR-145, and miR-210, whereas patients in the observation group showed no differences in PFS time. Our data suggest miR-25, miR-145, and miR-210 as predictors for the efficacy of maintenance treatment with pemetrexed in lung adenocarcinoma patients who were negative for EGFR mutations or ALK translocations.

[Prognostic factors in patients with small cell lung cancer].

OBJECTIVE: To investigate the prognostic factors of small cell lung cancer (SCLC) and establish a reliable model of clinical prognostic index. METHODS: Kaplan-Meier and Cox regression were used to analyze the relationship between survival time and prognostic factors in 60 cases of SCLC. The prognostic factors included clinical and laboratory parameters, serum cytokeratin fragment 19 (CYFRA21-1), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), CA125, interleukin-2 (IL-2) and soluble interleukin-2 receptors (sIL-2R). RESULTS: Kaplan-Meier analysis showed that poor prognosis was in patients with KPS < 80 or extensive disease and unrelated to other clinical parameters such as age, sex and smoking index, and in patients with serum NSE > 30 micro g/L, CEA > 5.0 micro g/L, CA125 > 37 KU/L and sIL-2R > 500 KU/L. Serum IL-2 and CYFRA21-1 were also elevated, but had no significant prognostic value. Multivariate analysis indicated that serum NSE, stage and treatment of disease were independent prognostic factors. The three prognostic factors enabled establishment of a prognostic index (PI) based on a simple algorithm: PI = NSE (0 if < or = 30 micro g/L, 1 if > 30 microg/L) + stage (0 = LD, 1 = ED) + CEA (0 if < or = 5.0 microg/L, 1 if > 5.0 microg/L). CONCLUSION: The stage of disease, systemic treatment and the level of serum NSE are independent prognostic factors. Without considering the influence of treatment-related factors on survival, the levels of serum CEA, NSE and stage of disease before treatment are significant independent prognostic factors. PI calculated on the basis of CEA, NSE and stage is recommended to predict the survival of SCLC.

Efficacy of erlotinib plus dendritic cells and cytokine-induced killer cells in maintenance therapy of advanced non-small cell lung cancer.

The aim of this study was to evaluate the safety and effectiveness of erlotinib plus DC/CIK in maintenance therapy of advanced non-small cell lung cancer. After 4 cycles of the 2-drug regimen treatment with platinum, the 54 patients with non-small cell lung cancer in phase IIIb or IV reached stable or beyond stable stages. The patients were then randomly divided into 2 groups. One group was treated with erlotinib therapy (erlotinib group), and the other was treated with DC/CIK plus erlotinib (DC/CIK plus erlotinib group). The progression-free survival of the erlotinib group and the DC/CIK plus erlotinib group was 3.98 months (95% CI, 3.56-4.40) and 5.02 months (95% CI, 4.32-5.72) (P=0.002), respectively. The median overall survival of the erlotinib group and the DC/CIK plus erlotinib group was 9.9 months (95% CI, 9.1-10.6) and 10.5 months (95% CI, 9.6-11.4) (P=0.29), respectively. The levels of CD3, CD4, and CD8 were significantly different before and after the treatment in the DC/CIK plus erlotinib group, but not in the erlotinib group. There was no significant difference in toxicity between the 2 groups. In conclusion, there was no statistically significant difference in overall survival between DC/CIK plus erlotinib and erlotinib as maintenance therapy. DC/CIK plus erlotinib was well tolerated with a manageable safety profile.

Pemetrexed plus dendritic cells as second-line treatment for patients with stage IIIB/IV non-small cell lung cancer who had treatment with TKI.

The aim of this study was to determine the efficacy and toxicity of pemetrexed plus dendritic cells (DCs) in patients suffering from stage IIIB or IV lung adenocarcinoma, who had undergone maintenance treatment with gefitinib or erlotinib. Patients who had failed gefitinib or erlotinib maintenance treatment had ECOG performance statuses ranging from 0 to 2.27 patients received pemetrexed plus DCs as second-line treatment. Dosage: 500 mg/m(2) pemetrexed was administered on day 1 of a 21-day cycle. DCs were given for one cycle of 21 days. Three patients (11.1 %) experienced a partial response and 14 patients (51.9 %) showed stable disease. Ten patients (37.0 %) had progressive disease. The median time to progression-free survival (PFS) was 4.8 months [95 % confidence interval (CI) 4.4-5.2], and the median overall survival was 10.7 months (95 % CI 10.3-11.2). In the subgroup analysis, PFS had a significant difference between the low ratio of CD4/CD8 and normal ratio of CD4/CD8, with 4.5 months (95 % CI 4.2-4.9) and 5.0 months (95 % CI 4.5-5.7), (Log Rank = 0.039), respectively. No one patient experienced grade 4 toxicity. A regimen of pemetrexed combined with DCs is marginally effective and well tolerated in patients with stage IIIB or IV lung adenocarcinoma who had received gefitinib or erlotinib first-line treatment.

S-1 plus CIK as second-line treatment for advanced pancreatic cancer.

This study aimed to evaluate the efficacy and tolerability of S-1 (Tegafur, Gimeracil, and Oteracil Potassium Capsules) plus CIK (Cytokine-induced killer cells) in patients with advanced pancreatic cancer who had previously received gemcitabine-based therapy. In this prospective study, fifty-eight patients were randomly divided into two groups. One group (CT group) was given S-1 alone, and the other group (immuno-CT group) was given S-1 plus CIK. S-1 was administered orally twice a day at 80 mg/m(2)/day on days 1-21 of a 28-day cycle till disease progression or unacceptable toxicity occurred. CIK was given for one cycle of 28 days. The disease control rate for S-1 and CIK was 40.0 and 53.6%, respectively (p = 0.621). The serum CA19-9 level decreased for more than 25% was significantly different (33.3 and 60.7 % in CT group and immuno-CT group, respectively, p = 0.037). The median time to progression was 2.5 (95% CI 2.3-2.8) and 2.9 (95% CI 2.6-3.2) months (p = 0.037) for CT group and immuno-CT group, respectively. The median overall survival was 6.1 (95% CI 5.7-6.5) and 6.6 (95% CI 6.1-7.1) months (p = 0.09) for CT group and immuno-CT group, respectively. The difference in hematological toxicity, including leukocytopenia, anemia, and neutropenia, was insignificant between the two groups. In contrast, the differences in non-hematological toxicity, fatigue, and non-infective fever were significantly different between the two groups (p < 0.05). The S-1 plus CIK regimen was well tolerated in a second-line setting in patients with gemcitabine-refractory and advanced pancreatic cancer.

Treatment of stage IIIb/IV non-small cell lung cancer with Pemetrexed plus Oxaliplatin after failure of Erlotinib as second-line treatment.

To determine the efficacy and toxicity of Pemetrexed plus Oxaliplatin in patients suffering from stage IIIb or IV lung adenocarcinoma and being treated with Erlotinib as second-line treatment, a total of 45 patients were randomly divided into two groups. One group was treated with 500 mg/m(2) Pemetrexed plus 100 mg/m(2) Oxaliplatin, and the other was treated with 500 mg/m(2) Pemetrexed plus 75 mg/m(2) Cisplatin. All drugs were administered on day one of a 21-day cycle. In the Oxaliplatin group, 3 patients (13.6 %) experienced partial response (PR), 9 patients (41.0 %) showed stable disease (SD), and 10 patients (45.5 %) had progressive disease (PD). In the Cisplatin group, 2 patients (8.7 %) experienced PR, 7 patients (30.4 %) showed SD, and 14 patients (60.9 %) had PD. The PFS of the Oxaliplatin group and the Cisplatin group was 4.45 months (95 % CI 4.10-4.80) and 3.96 months (95 % CI 3.68-4.24) (P = 0.03), respectively. The median overall survival (OS) was 10.8 months (95 % CI 10.2-11.5) and 10.7 months (95 % CI 10.2-11.3) (P = 0.72), respectively. There was no statistically significant difference in the occurrence rate of grades 3 and 4 myelotoxicity between the two groups. However, there was a significant difference in the occurrence rate of grades 3 and 4 gastrointestinal reactions and peripheral neurotoxicity between the two groups (P < 0.05). A regime combining Pemetrexed and Oxaliplatin was marginally effective and well tolerated in patients with stage IIIb or IV lung adenocarcinoma who have received Erlotinib as second-line treatment.

[Antisense oligonucleotide targeting endostatin enhances hematopoiesis reconstitution in BMT mice].

OBJECTIVE: To explore the effect of antisense oligonucleotide targeting endostatin (endostatin-ASON) transfecting bone marrow stromal cells ( BMSC) on hematopoiesis reconstitution in BMT mice. METHODS: Inhibition of endostatin / VCAM-1 protein and mRNA expression was investigated by transfection of antisense oligonucleotide targeting endostatin with confocal microscopy, Western blot and RT-PCR. Bone marrow stromal cells were cultured and divided into 4 groups: group (1) without any treatment; group (2) BMT only; group (3) BMT + endostatin-ASON transfection; group (4) BMT + endostatin scrambled sequence transfection. RESULTS: (1) Endostatin-ASON was successfully introduced into BMSC in vitro, and the transfecting rate was 86% ;(2) After Endostatin-ASON transfected into BMSC, the expression of Endostatin mRNA and its protein on the BMSC was signficantly inhibited at different time point after BMT [the grey value of Endostatin was (0.09 +/- 0.03) - (1.44 +/- 1.19) and (0.02 + 0.02) - (0.14 +/- 0.05), respectively] (P < 0.01 and P < 0.05); (3) Transfecting with Endostatin-ASON effectively promoted the expression of VCAM-1 mRNA and its protein on the BMSC [the gray value of VCAM-1 was (1.60 +/- 0. 92) - (8.05 +/- 0.87) and (0.07 +/- 0.02) - (0.67 +/- 0.09) , respectively] (P <0.01 and P <0.05) ; (4) There was no effects of transfecting Endostatin scrambled sequence on the expression of Endostatin and VCAM-1 on the BMSC (P > 0.05). CONCLUSION: Endostatin-ASON could inhibit Endostatin expression and enhance VCAM-1 expression in BMSC after syngeneic-BMT in mice, which might be one of the mechanisms underlying the endostatin-ASON accelerating hematopoiesis reconstitution after allogeneic-BMT.

[Effect of ligustrazine on the expression of bFGF in bone marrow stromal cells of mice after BMT].

This study was purposed to investigate the effect of ligustrazine on the expression of bFGF in bone marrow stromal cells (BMSC) and to explore the mechanism of hematopoietic reconstitution after bone marrow transplantation (BMT). The mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. BMT mouse models were established. The mice of normal group were not treated, the mice of saline group were given normal saline (0.2 ml/mouse, twice a day) through gastric tube, while the mice of ligustrazine group were given ligustrazine (0.2 ml/mouse, twice a day) through gastric tube. On day 7, 14, 21 and 28 after BMT, the femora were taken and the bone marrow mononuclear cell (BMMNC) suspensions were used for the cultivation of bone marrow stromal cells according to Dexter's culture method. The mRNA and protein expressions of bFGF in BMSC were assayed by RT-PCR and Western blot respectively. The results showed that the expression of bFGF in BMSC on the level of mRNA and protein were all reduced significantly after BMT, and increased slowly with the time. On day 7, 14 and 21 after BMT, the expressions of bFGF mRNA and protein in bone marrow stromal cells of ligustrazine group and saline group were lower than that in bone marrow stromal cells of normal group, but the expressions of bFGF mRNA and protein in ligustrazine group were obviously higher than that in saline group (P < 0.01 or P < 0.05). On day 28 after BMT, the expressions of bFGF mRNA and protein in ligustrazine group returned to normal level, while the expressions of bFGF mRNA and protein in saline group not returned to normal level, there was significant difference between these two groups. It is concluded that ligustrazine can enhance bFGF expression level in bone marrow stromal cells after syngeneic bone marrow transplantation in mice, which confirms that ligustrazine can enhance the repair of bone marrow microvessels, improve bone marrow microenvironment and promote hematopoietic reconstitution.

[Relationship between endostatin and vascular cell adhesion molecule-1 expressions on bone marrow stromal cells in BMT-mice].

This study was aimed to investigate the relationship between endostatin and vascular cell adhesion molecule-1 (VCAM-1) expressions on bone marrow stromal cells (BMSC) in mice after bone marrow transplantation (BMT) and effect of ligustrazine on their expressions. The mice were randomly divided into 3 groups: normal group (without treatment), saline group (control of BMT) and ligustrazine group (BMT + ligustrazine). BMT mouse models were established. The normal group was not treated, the saline group was given normal saline (0.2 ml/mouse, twice a day) through gastric tube, while the ligustrazine group was given ligustrazine (0.2 ml/mouse, twice a day) also through gastric tube. On day 7, 14, 21 and 28 after BMT, mice were killed by euthanasia. The expression levels of endostatin and VCAM-1 in bone marrow stromal cells were detected by immunohistochemistry and RT-PCR analysis respectively. The results showed that the endostatin protein mainly expressed in nuclei of BMSCs, the VCAM-1 protein mainly expressed in plasma of BMSCs. On day 7, 14, 21 after BMT the expression levels of endostatin mRNA and protein in ligustrazine and saline groups were significantly lower than that in normal group (P < 0.01 or P < 0.05), while their expression levels in ligustrazine group were lower than that in saline group. On day 28 the expression levels in saline group returned to normal, while the expression levels in ligustrazine group not were normalized. On day 7, 14, 21 after BMT the expression levels of VCAM-1 mRNA and protein in ligustrazine and saline groups were significantly lower than that in normal group (P < 0.01 or P < 0.05), but their expression levels in ligustrazine group were significantly lighter than that in saline group (P < 0.01 or P < 0.05). On day 28 the VCAM-1 expression level in ligustrazine group returned to normal, while its expression level in saline group not were normalized. The difference between these two groups was significant (P < 0.01). Correlation analysis revealed that there was a negative correlation between endostatin and VCAM-1 expression in saline group, there was a positive correlation between endostatin and VCAM-1 expression in ligustrazine group. It is concluded that the endostatin can influence hematopoiesis in bone marrow by affecting VCAM-1 expression on BMSC and hindering connection between stromal cells and hematopoietic cells as well as extracellular stroma and hematopoietic cells, while ligustrazine can enhance the adhesion molecule expression on stromal cell surface of bone marrow in BMT-mice, accelerate the homing and proliferation of HSPC in bone marrow after BMT, meanwhile can promote the repair of bone marrow microenvironment, accelerate hematopoietic reconstitution of bone marrow after BMT through feedback regulation of endostatin expression of BMSC in BMT-mice.