Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells.

The sensitivity of lung cancer to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has been found to be associated with mutations in the tyrosine kinase domain of EGFR. However, not all mutations are sensitive to gefitinib. While CpG island methylation in the promoter region of the EGFR gene and transcriptional silencing are common in solid tumors, the role of the EGFR gene promoter methylation in affecting resistance to TKIs in non-small cell lung cancer (NSCLC) remains unknown. In this study, we examined the correlation between EGFR gene promoter methylation and the therapeutic effect of gefitinib in NSCLC cells. Three NSCLC cell lines with different EGFR mutation statuses and levels of sensitivity to EGFR-TKIs were used in this study: H1650 (del E746-A750), H1299 (wild-type EGFR) and PC-9 (del E746-A750). Cells were treated with gefitinib or 5-aza-2'-deoxy cytidine (5-aza-CdR), a methylation inhibitor, alone or in combination. Subsequently, the methylation status of the EGFR gene promoter was examined by methylation-specific PCR (MSP). Cell survival and apoptosis assays were performed using the Cell Counting Kit-8 (CCK-8) and flow cytometry. In addition, western blot analysis and quantitative real-time PCR were used to examine the expression levels of EGFR protein and mRNA. Our study showed that the promoter region of the EGFR gene in PC-9 cells was unmethylated, and that the cells were sensitive to gefitinib. By contrast, the promoter region of the EGFR gene in the H1650 and H1299 cells was methylated, and the cells were resistant to gefitinib. Of note, the combination treatment with 5-aza-CdR and gefitinib further enhanced the growth inhibitory effects and led to the induction of apoptosis, while a significant reduction in the expression of EGFR protein and mRNA was observed in the H1650 and H1299 cells. These results suggest that blockade of DNA methylation may enhance the antitumor effects of EGFR-TKIs and gefitinib in NSCLC cells. Thus, EGFR gene promoter methylation may be a potential mechanism for acquired resistance to gefitinib.

Polymorphisms of dihydropyrimidine dehydrogenase gene and clinical outcomes of gastric cancer patients treated with fluorouracil-based adjuvant chemotherapy in Chinese population.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD), a key enzyme involved in the catabolism of 5-fluorouracil (5-FU), is the attractive candidate for pharmacogenetic research on efficacies and toxicities of 5-FU. The aim of this study is to explore the association between polymorphisms of dihydropyrimidine dehydrogenase gene (DPYD) and clinical outcomes of gastric cancer patients treated with fluorouracil-based adjuvant chemotherapy in the Chinese population. METHODS: Three hundred and sixty-two patients with gastric cancer in the Chinese population were treated with fluorouracil-based adjuvant chemotherapy. The single nucleotide polymorphic genotypes of DPYD were determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) using DNA samples isolated from peripheral blood collected before treatment. RESULTS: The average response rate for chemotherapy was 46.7%. A significantly different distribution of the rs1801159 (c2=8.76, P=0.012) genotypes was observed. Homozygous genotype rs1801159A/A was over-represented in responsive patients. Conversely, carriers of the rs1801159A/G genotype were prevalent in non-responsive patients. In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (c2=3.96, P=0.0465). CONCLUSIONS: These results suggest that polymorphisms of rs1801159 in DPYD may be used as valuable predictors of the response to fluorouracil-based chemotherapy for gastric cancer patients in the Chinese population. Well-designed, comprehensive, and prospective studies on determining these polymorphisms of DPYD as predictive markers for gastric cancer in response to fluorouracil-based therapies are warranted.

[Detection of single nucleotide polymorphisms of mthfr and dpyd genes in leukemia cell lines K562 and K562/A02].

This study was purposed to detect single nucleotide polymorphisms (SNP) of 2 pharmacokinetics-related genes in K562 and K562/A02 cell lines. Leukemia cell line K562 and its resistant line K562/A02 were cultured, the genomic DNA was isolated by QIAamp DNA Blood Mini kit, primers were designed, the related DNA fragments were amplified by PCR. The SNP genotyping of mthfr gene rs1801131, rs1801133 and rs2274976 and dpyd gene rs1801159, rs1801160 and rs17376848 was performed by means of matrix assisted laser desorption ionization-time of flight mass spectrometry method (MALDI-TOFMS). The results showed that the genotype of mthfr gene locus 1801131 was AC, rs1801133 was CC, rs2274976 was GG, genotype of dpyd gene locus 1801159 was GG, rs1801160 was GG, rs17376848 was AA in both K562 and K562/A02 cell lines. It is concluded that the above-mentioned loci of mthfr and dpyd genes in K562 and K562/A02 cell lines are not expressed differently.

[Effects of 5-bromotetrandrine and daunorubicin on apoptosis and expression of survivin in K562/A02 cells].

The aim of this study was to investigate the potential benefit of combination therapy with 5-bromotetrandrine (5-BrTet) and daunorubicin (DNR) on chronic leukemia. The apoptosis of K562/A02 cells treated by DNA, BrTet and BrTet combined with DNR for 48 hours was detected by flow cytometry; the expressions levels of survivin mRNA and protein K562/A02 cells treated by DNR, BrTet and BrTet combined with DNR and in untreated K562 cells for 48 hours were measured by RT-PCR and Western blot respectively. The results showed that the combination of BrTet with DNR increased apoptotic rate of K562/A02, down-regulated the expression levels of survivin mRNA and protein in K562/A02 cells as compared with blank control and cells treated by BrTet or DNR alone, the survivin expression in K562/A02 cells was higher than that in K562 cells. It is concluded that the combination of BrTet with DNR can effectively reverse the multidrug resistance of K562/A02 cells, promote the apoptosis of K562/A02 cells, the mechanism of which may be related with down-regulation of survivin expression. Survivin may be a target for the treatment of MDR in hematopoietic malignancies.

[Inducing apoptosis and reversal effect of nilotinib in combination with tetrandrine on multidrug resistance of K562/A02 cell line].

This study was aimed to investigate the relevance of nilotinib in combination with tetrandrine (Tet) on reversing multidrug resistance and inducing apoptosis of K562/A02 cell line and its mechanism. Methyl-thiazol tetrazolium (MTT) assay was employed to examine the pharmacological effect of nilotinib or Tet alone on K562/A02 cell line, the IC(50) of daunorubicin (DNR) on K562/A02 cell line treated with nilotinib and Tet was calculated; the flow cytometry (FCM) was employed to detect the apoptosis rate of K562/A02. The expression of bax/survivin mRNA was determined by RT-PCR, and the expression of bax/survivin protein was assayed by Western blot. The results showed that after being treated by 5 nmol/L nilotinib or 1.0 µml/L Tet for 48 hours, IC(50) of DNR to K562/A02 was 5.71 ± 0.72 mg/L or 6.52 ± 0.43 mg/L, respectively, while in their combined treatment, IC(50) decreased to 3.12 ± 0.13 mg/L. Nilotinib or Tet alone could increase DNR-inducing apoptosis rate of K562/A02 cell, while the apoptosis rate of K562/A02 increased remarkably in combination treatment of nilotinib with Tet. After being treated with 5 nmol/L nilotinib or 1.0 µml/L Tet alone for 48 hours, the expressions of bax mRNA and BAX protein was up-regulated, while both effects were more obvious in combination treatment of nilotinib with Tet. Treatment with 5 nmol/L nilotinib or 1.0 µmol/L Tet alone for 48 hours down-regulated the expression of survivin mRNA and its protein, while treatment of nilotinib in combination with Tet had more significant effect on down-regulation of their expression. It is concluded that the K562/A02 cells are resistant to DNR, nilotinib or Tet alone both can partially reverse resistance of K562/A02 cells to DNR, increase the apoptosis rate of K562/A02 cells. Combination of nilotinib with Tet shows obvious synergistic action, mechanism of which may associate with up-regulation of bax mRNA and BAX protein expressions and down-regulation of survivin mRNA and its protein expressions.

[Gene expression in patients with myelodysplastic syndrome].

This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in the patients with myelodysplastic syndrome (MDS) and its clinical significance. The mRNA expression of c-FLIPL, c-FLIPS and DLK1 in bone marrow mononuclear cells (BMMNC) of 16 patients with MDS and 3 controls were detected by RT-PCR. The results indicated that the expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls (p < 0.05). There was no significant difference in expression of DLK1 between RA and RAEB patients (p > 0.05); the expression of c-FLIPL mRNA both in RA and RAEB patients was higher than that in controls (p < 0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB patients (p > 0.05); the expression of c-FLIPS mRNA was not significantly different between MDS patients and controls (p > 0.05), but its expression in RAEB patients was significantly higher as compared with RA patients and controls (p < 0.05). It is concluded that the mRNA expressions of DLK1, c-FLIPL and c-FLIPS in MDS patients are abnormal, some of which may be useful as an important indicator for the evaluation of development in MDS.

[Effects of platelet-derived membrane microparticles on angiogenesis in chick chorioallantoic membranes].

This study was purposed to investigate the angiogenesis effect of platelet-derived membrane microparticles (PMPs) in chick chorioallantoic membranes (CAM). Thrombin were adopted to activate the platelets and then PMPs were obtained. PMPs were isolated by high speed centrifugation. Flow cytometry (FCM) was adopted to evaluate the efficiency of thrombin to produce PMPs and BCA method was adopted to evaluate the content of PMPs. PMPs were put into CAM and the effects of PMPs on angiogenesis in CAM were observed. The results indicated that after incubation for 72 hours at the concentration of 80 microg/ml PMPs, the vessel nets in a 'spoked-wheel' pattern were shown around mixed fibrous filter membranes, number of vessel ramification was 112.5 +/- 11.31 and ratio of vessel area/CAM area was 6.19 +/- 1.29%, but there were not localized allantoic vessels developing in the control group, the number of vessel ramification and ratio of vessel area/CAM area in control group were 82.6 +/- 8.05 and 1.78 +/- 0.33 respectively, so there was significant difference between PMP and control groups. In above mentioned conditions, the number of vessel ramification and ratio of vessel area/CAM area in VEGF group were 128.4 +/- 10.02 and 7.44 +/- 1.36 respectively, so there was no difference between PMP and VEGF groups. It is concluded that PMPs show promotive effect on the formation of capillaries in chick chorioallantoic membranes.

[Effects of platelet-derived membrane microparticles on the proliferation and apoptosis of human umbilical vein endothelial cells].

This study was purposed to investigate the effects of platelet-derived membrane microparticles (PMP) on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC). Different concentrations of thrombin were adopted to activate the platelets so as to release PMPs. Flow cytometry (FCM) was adopted to evaluate the efficiencies of different concentrations of thrombin to release PMPs. By using the HUVEC cultivated in vitro as vector, the effects of PMPs on the proliferation and apoptosis of HUVEC were investigated by MTT and FCM. The results showed that the efficiencies releasing PMPs from platelets activated by 2.0, 1.5, 1.0, 0.5 U/ml thrombin were 28.7, 47.7, 50.1 and 43.9% respectively; PMPs induced proliferation of HUVEC in a dose dependent manner. At the concentration of 40 microg/ml PMPs, the proliferation rate of HUVEC was 1.8 +/- 0.3 times as much as blank control, the proliferation rate in group of vascular endothelial growth factor was 1.9 +/- 0.5 times of as much as blank control, there was no statistic difference (p > 0.05). The PMPs inhibited HUVEC apoptosis. Compared with the apoptosis rate of control (9.4 +/- 0.5)%, apoptosis rate in PMP group (40 microg/ml) was (3.9 +/- 0.4)% (p < 0.05). The addition of VEGF (10 microl/ml) did not successfully prevented apoptosis of HUVEC with apoptosis rate of (8.0 +/- 0.8)%. It is concluded that platelets activated by 1 U/ml thrombin gets the best efficiency of PMP release, which stimulates proliferation of HUVEC and inhibits its apoptosis.

[Test of activated plasma clotting time to assess efficacy of platelet transfusion].

The study was aimed to investigate the value of activated plasma clotting time (APCT) for estimating the efficacy of platelet transfusion therapy. There were twenty patients with hematological diseases, who received transfusion of platelet, involved in the test. APCT was determined before and after transfusion of these patients, then APCT was contrasted with corresponding CCI and PPR. The results showed that 1 hour and 24 hour APCTs were shortened obviously. APCT before transfusion was (103.7 +/- 11.3) seconds, but the 1 hour and 24 hour APCTs were shortened to (60.0 +/- 9.7) seconds and (68.5 +/- 9.8) seconds respectively (P < 0.01). According to the judging criteria of CCI and PPR (CCI and PPR values at 1 and 24 hours after transfusion are < 7500, < 5000 and < 30%, < 20% respectively, the transfusion is invalid), two patients received invalid transfusion. Their 1 and 24 hour CCIs were 7415, 2966 and 6913, 4988 respectively. Their 1 and 24 hour PPRs were 28.0%, 11.2% and 25.2%, 14.1% respectively. One patient's PPR reached the standard of invalid transfusion, but his CCI showed a valid transfusion he received. Two patients' PPR reached the standard of invalid transfusion, but their 1 hour CCI reached the standard of valid transfusion, and their 24 hour CCI reached the standard of invalid transfusion. It is concluded that APCT reflects the variations of quantity and quality of platelet simultaneously, and can evaluate precisely the efficacy of platelet transfusion.

[Platelet-derived microparticles stimulate proliferation of granulocyte-macrophage progenitor cells from umbilical cord blood].

This study was aimed to investigate the effect of platelet-derived microparticles (PMP) on stimulating the proliferation of granulocyte-macrophage progenitors (CFU-GM) from umbilical cord blood. Different concentrations of thrombin were adopted to activate the platelets for releasing PMP. Flow cytometry was adopted to evaluate the efficiencies of different concentrations of thrombin to produce PMP. Umbilical cord blood mononuclear cells (MNC) were obtained from healthy donors and the MNC were isolated by Ficoll density gradient centrifugation. MNC were cultured in 2.7% methylcellulose containing different concentration of PMP and colonies were counted under an inverted microscope after 7 days. The result showed that the release rate of PMP activated by 2.0, 1.5, 1.0 and 0.5 U/ml thrombin were 28.7%, 47.7%, 50.1% and 43.9% respectively. The PMP enhanced colony formation in dose-dependent manner. The number of colonies per 2 x 10(5) MNCs in groups of PMP at different concentrations (10, 50 and 100 microg/ml) were 119.8 +/- 32.2,142.8 +/- 45.2 and 180.8 +/- 85.1 respectively. The number of colonies in the groups of PMP at 100 microg/ml and 50 microg/ml were statistically significant when compared with control group (103.0 +/- 24.8) (P < 0.05). The number of colonies per 2 x 10(5) MNC in the group of PMP (10 microg/ml) was 119.8 +/- 32.2 which was higher than that in control group, but there was no statistical significance between two groups. It is concluded that platelet activated with 1.0 U/ml thrombin can get the best release efficiency of PMP and PMP can enhance the proliferation of granulocyte-macrophage progenitor cells of umbilical cord blood.