Molecular analysis and clinical significance of hemoglobin Quong Sze in Huizhou city, Southern China.

OBJECTIVE: Hemoglobin Quong Sze (Hb QS) is one of the most common non-deletional α-thalassemia (α-thal), which is prevalent in the Southern Chinese population. However, there are still few comprehensive researches on the molecular characterization of Hb QS. So it is important to find out appropriate diagnosis and characterization of Hb QS carrier for genetic counseling. MATERIALS AND METHODS: A hematological screening including hematological indices and hemoglobin analysis was performed in 113,400 individuals from Huizhou city, Southern China. Then, suspected thalassemia carriers were detected by a suspension-array system and DNA sequencing for α- and ß-thal. RESULTS: In our study, we identified 521 subjects who were Hb QS carriers, including fourteen different genotypes. Among them, 445 Hb QS heterozygotes showed a decrease in the mean corpuscular hemoglobin (MCH), 16 compound heterozygotes for Hb QS/α+-thal presented mild thalassemia, 28 Hb QS in combination with --SEA/αα manifested as Hb H disease, varying clinical symptoms from only moderate anemia to severe anemia and requiring blood transfusion, and 29 double heterozygotes for Hb QS and ß-thal behaved as ß-thal trait. The mean corpuscular volume (MCV) and MCH were significantly reduced and no Hb H peak could be detected in one patient with Hb H-Hb QS and ß-thal. Meanwhile, we identified two homozygous Hb QS carriers, who showed mild to moderate anemia and increased Hb A2 level but negative results from a sequencing analysis for the first time. Additionally, Comparison of hematological parameters among the major four genotype groups showed significant differences in most box-whisker plots. CONCLUSION: People who originated from Huizhou city showed many genotypes and diversity in the clinical manifestations of Hb QS carriers. This study enlarges the mutation spectrum of α-thal and emphasizes that reliable detection of the gene mutations is important for genetic counseling. It also strengthens the prevention and control of thalassemia.

[Association Analysis between Genotype and Phenotype of α,ß-Thalassaemia Carriers in Huizhou Area of Guangdong Province].

OBJECTIVE: To analyze the prevalence, genotype distribution and hematological characteristics of α,ß-thalassaemia carriers in Huizhou area of Guangdong Province. METHODS: 10 809 carriers of simple ß-thalassaemia and 1 757 carriers of α,ß-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. RESULTS: The prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, ßCD41-42/ ßN (19.29%), the next was --SEA/ αα, ßIVS-II-654/ ßN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple ß-thalassaemia and α,ß-thalassaemia. Violin plots showed that carriers with co-inheritance of ß-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of ß-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. CONCLUSION: There is a high prevalence of α,ß-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,ß-thalassaemia, it is necessary to distinguish it from simple ß-thalassaemia by genotyping of α- and ß-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

Case Report: The third-generation sequencing confirmed a novel 7.2 Kb deletion at ß-globin gene in a patient with rare ß-thalassemia.

Background: Thalassemia was the most common monogenic diseases worldwide, which was caused by mutations, deletions or duplications in human globin genes which disturbed the synthesis balance between α- and ß-globin chains of hemoglobin. There were many classics methods to diagnose thalassemia, but all of them had limitations. Although variations in the human ß-globin gene cluster were mainly point mutations, novel large deletions had been described in recent years along with the development of DNA sequencing technology. Case report: We present a case of 32-year-old male with abnormal hematological results. However, 23 genotypes of the most common thalassemia were not detected by two independent conventional platforms. Finally, using multiplex ligation-dependent probe amplification (MLPA), third-generation sequencing (TGS) and Gap PCR detection methods, we first confirmed the case with a novel 7.2 Kb deletion (Chr11:5222800-5230034, hg38) located at HBB gene. Conclusion: Our results showed that TGS technology was a powerful tool for thalassemia breakpoint detection, had promising potentiality in genetic screening of novel thalassemia, especially for the novel deletions in globin genes.

A novel 15.8 kb deletion α-thalassemia confirmed by long-read single-molecule real-time sequencing: Hematological phenotypes and molecular characterization.

BACKGROUND: Thalassemia is the most frequent recessive Mendelian inherited monogenic disease worldwide, and is characterized by the impaired synthesis of globin chains due to disease-causing variants in α- or ß-globin genes. There are many conventional methods to diagnose thalassemia but all of them have limitations. CASE REPORT: We present the case of a 37-year-old female with abnormal values of routine hematological indices who was admitted for genetic screening of thalassemia. Genomic DNA was extracted and used for genetic assays covering the known and potential novel genotypes in HBA and HBB genes using a suspension-array system, gap-polymerase chain reaction (Gap-PCR), PCR-reverse dot blot (PCR-RDB) and multiplex ligation-dependent probe amplification (MLPA). Finally, using long-read single-molecule real-time (SMRT) sequencing, we first confirmed the case with a novel 15.8 kb deletion located in the HBA gene (Chr16:163886-179768, GRch38/hg38). CONCLUSIONS: Our results showed that long-read SMRT sequencing has great advantages in the detection of rare α-globin gene variants. This study may provide a reference protocol for the use of long-read SMRT sequencing for the detection of known and potential novel genotypes of thalassemia in the population and improve the accuracy of genetic counseling and prenatal diagnosis.

Genetic counseling and prenatal decision for hemoglobin H disease caused by the rare α2 codon 30 (-GAG) (HBA2: c.91_93delGAG) mutation and the SEA deletion: Case series study.

OBJECTIVE: We report a rare mutation on the α2-globin gene, HBA2: c.91_93delGAG and its potential functions. CASE REPORT: We mainly described four patients with hemoglobin (Hb) H disease caused by the rare mutation and the SEA deletion but diversity in clinical presentation. Two had survived to adulthood with normal physical and mental development, except for mild anemia. However, two were children, who had more severe clinical manifestations. One child had developmental disorders of speech and language and mild growth retardation, and the other child suffered from severe hemolytic crises precipitated by infection and received blood transfusion. CONCLUSION: This study is of great significance for clinicians to provide genetic counseling to couples at-risk of having offspring with Hb H disease and let them make the pregnancy decision, particularly reduce the occurrence of severe Hb H disease.

[Diagnosis and clinical phenotype analysis of a case with large fragment homozygous deletion of rare ß gene cluster].

OBJECTIVE: To report on a case with homozygous deletion of large ß gene cluster and its clinical characteristics. METHODS: A total of 71 001 peripheral blood samples were subjected to capillary electrophoresis and conventional testing for common thalassemia mutations. The genotypes of suspected ß gene cluster deletions were analyzed by Gap-PCR and multiplex ligation-dependent probe amplification (MLPA). Their hematological characteristics were compared by statistical analysis R software. RESULTS: Eighty-nine cases were detected with Chinese Gγ(Aγδß) 0-deletion of the ß gene cluster, which gave a detection rate of 0.13%. Among these, there were 70 Chinese Gγ(Aγδß) 0-deletion heterozygotes and 18 Chinese Gγ(Aγδß) 0-deletion heterozygotes in conjunct with α thalassemia. There were 13 683 samples with normal findings. A significant difference was detected in 6 groups of hematological parameters between the heterozygous carriers (P<0.05) by box plotting. One case of Chinese Gγ(Aγδß) 0-deletion homozygote was discovered for the first time. The clinical phenotype was mild anemia. Hemoglobin electrophoresis showed that the value of HbF was 100%. CONCLUSION: The carrier rate for large fragment deletions of ß gene cluster in Huizhou region is rather high, for which the value of HbF is significantly increased. Attention should be paid to screening and diagnosis of rare genotype to prevent missed diagnosis and/or misdiagnosis.

Correction to: The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer.

AbstractAfter the publication of this article [1] it came to our attention that there were some errors in two of the figures.

[Phenotypic and genotypic analysis of 45 cases with Hemoglobin Q-Thailand].

OBJECTIVE: To explore hematological and molecular characteristics of Hemoglobin Q-Thailand in Huizhou area of Guangdong Province. METHODS: A total of 34 977 samples were screened by capillary and agarose gel electrophoresis. Samples suspected with HbQ strips were subjected to blood cell count and DNA sequencing. Twenty three common mutations associated with α- and ß-thalassemia were identified by liquid phase chip and diversion hybridization technique. RESULTS: The carrier rate of Hb Q-Thailand in Huizhou area was 0.13%. Pedigree analysis indicated that the Hb Q-Thailand allele is linked with a leftward single a-globin gene deletion (-α4.2). Hematological index (HGB, MCV, MCH, HbA, HbA2, HbQ) of 45 heterozygous carriers of Hb Q-Thailand were (130.25±17.37) g/L, (79.81±4.97) fl, (26.38±1.48) pg, (71.37±5.07)%, (1.65±0.45)%, (26.87±4.95)%, respectively. A statistical difference was also found in their hematological index of HbA and HbA2 compared with 408 heterozygous carriers of -α4.2 mutation (P<0.05). CONCLUSION: Hb Q-Thailand has a high detection rate in Huizhou area. The allele is mainly in a heterozygous status and linked with -α4.2. The Hb Q strip can be detected by hemoglobin electrophoresis. When combined with other types of thalassemia, the heterozygotes will show unique hematological parameters.

Tailor-made ion-imprinted polymer based on functionalized graphene oxide for the preconcentration and determination of trace copper in food samples.

A tailor-made Cu(II) ion-imprinted polymer based on large-surface-area graphene oxide sheets has been synthesized for the preconcentration and determination of trace copper from food samples by solid-phase extraction. Attributed to the ultrahigh surface area and hydrophilicity of graphene oxide, the Cu(II) ion-imprinted polymer prepared by the surface ion-imprinting technique exhibited a high binding capacity and a fast adsorption rate under the optimized experimental conditions. In the static adsorption experiments, the maximum adsorption capacity of Cu(II) ion-imprinted polymer is 109.38 mg/g at 25°C, which is much higher than that of the nonimprinted polymer (32.12 mg/g). Meanwhile, the adsorption is very rapid and equilibrium is reached after approximately 30 min. The adsorption mechanism is found to follow Langmuir adsorption model and the pseudo-second-order adsorption process. The Cu(II) ion-imprinted polymer was used for extracting and detecting Cu(II) in food samples combined with graphite flame atomic adsorption spectrometry with high recoveries in the range of 97.6-103.3%. The relative standard deviation and limit of detection of the method were evaluated as 1.2% and 0.37 µg/L, respectively. The results showed that the novel absorbent can be utilized as an effective material for the selective enrichment and determination of Cu(II) from food samples.

Preparation of a Two-Dimensional Ion-Imprinted Polymer Based on a Graphene Oxide/SiO2 Composite for the Selective Adsorption of Nickel Ions.

In the present work, a novel two-dimensional (2D) nickel ion-imprinted polymer (RAFT-IIP) has been successfully synthesized based on the graphene oxide/SiO2 composite by reversible addition-fragmentation chain-transfer (RAFT) polymerization. The imprinted materials obtained are characterized by Fourier transmission infrared spectrometry (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). The results show that the thermal stability of the graphene oxide/SiO2 composite is obviously higher than that of graphene oxide. RAFT-IIP possesses an excellent 2D homogeneous imprinted polymer layer, which is a well-preserved unique structure of graphene oxide/SiO2. Owing to the intrinsic advantages of RAFT polymerization and 2D imprinted material, RAFT-IIP demonstrate a superior specific adsorption capacity (81.73 mg/g) and faster adsorption kinetics (30 min) for Ni(II) in comparison to the ion-imprinted polymer prepared by traditional radical polymerization and based on the common carbon material. Furthermore, the adsorption isotherm and selectivity toward Ni(II) onto RAFT-IIP and nonimprinted polymer (NIP) are investigated, indicating that RAFT-IIP has splendid recognizing ability and a nearly 3 times larger adsorption capacity than that of NIP (30.94 mg/g). Moreover, a three-level Box-Behnken experimental design with three factors combining the response surface method is utilized to optimize the desorption process. The optimal conditions for the desorption of Ni(II) from RAFT-IIP are as follows: an HCl-type eluent, an eluent concentration of 2.0 mol/L, and an eluent volume of 10 mL.

The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer.

BACKGROUND: SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown. RESULTS: Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway. CONCLUSIONS: For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.

Significantly inhibitory effects of low molecular weight heparin (Fraxiparine) on the motility of lung cancer cells and its related mechanism.

Low molecular weight heparin (LMWH) improving the cancer survival has been attracting attention for many years. Our previous study found that LMWH (Fraxiparine) strongly downregulated the invasive, migratory, and adhesive ability of human lung adenocarcinoma A549 cells. Here, we aimed to further identify the antitumor effects and possible mechanisms of Fraxiparine on A549 cells and human highly metastatic lung cancer 95D cells. The ability of cell invasion, migration, and adhesion were measured by Transwell, Millicell, and MTT assays. FITC-labeled phalloidin was used to detect F-actin bundles in cells. Chemotactic migration was analyzed in a modified Transwell assay. Measurement of protein expression and phosphorylation activity of PI3K, Akt, and mTOR was performed with Western blot. Our studies found that Fraxiparine significantly inhibited the invasive, migratory, and adhesive characteristics of A549 and 95D cells after 24 h incubation and showed a dose-dependent manner. Fraxiparine influenced the actin cytoskeleton rearrangement of A549 and 95D cells by preventing F-actin polymerization. Moreover, Fraxiparine could significantly inhibit CXCL12-mediated chemotactic migration of A549 and 95D cells in a concentration-dependent manner. Furthermore, Fraxiparine might destroy the interaction between CXCL12-CXCR4 axis, then suppress the PI3K-Akt-mTOR signaling pathway in lung cancer cells. For the first time, our data indicated that Fraxiparine could significantly inhibit the motility of lung cancer cells by restraining the actin cytoskeleton reorganization, and its related mechanism might be through inhibiting PI3K-Akt-mTOR signaling pathway mediated by CXCL12-CXCR4 axis. Therefore, Fraxiparine would be a potential drug for lung cancer metastasis therapy.

Elevated expression of SLC34A2 inhibits the viability and invasion of A549 cells.

Abnormal expression of solute carrier family 34 (sodium phosphate), member 2 (SLC34A2) in the lung may induce abnormal alveolar type II (AT II) cells to transform into lung adenocarcinoma cells, and may also be important in biological process of lung adenocarcinoma. However, at present, the effects and molecular mechanisms of SLC34A2 in the initiation and progression of lung cancer remain to be elucidated. To the best of our knowledge, the present study revealed for the first time that the expression levels of SLC34A2 were downregulated in the A549 and H1299 lung adenocarcinoma cell lines. Further investigation demonstrated that the elevated expression of SLC34A2 in A549 cells was able to significantly inhibit cell viability and invasion in vitro. In addition, 10 upregulated genes between the A549­P­S cell line stably expressing SLC34A2 and the control cell line A549­P were identified by microarray analysis and quantitative polymerase chain reaction, including seven tumor suppressor genes and three complement genes. Furthermore, the upregulation of complement gene C3 and complement 4B preproprotein (C4b) in A549­P­S cells was confirmed by ELISA analysis and was identified to be correlated with recovering Pi absorption in A549 cells by the phosphomolybdic acid method by enhancing the expression of SLC34A2. Therefore, it was hypothesized that the mechanisms underlying the effect of SLC34A2 on A549 cells might be associated with the activation of the complement alternative pathway (C3 and C4b) and upregulation of the expression of selenium binding protein 1, thioredoxin­interacting protein, PDZK1­interacting protein 1 and dual specificity protein phosphatase 6. Downregulation of SLC34A2 may primarily cause abnormal AT II cells to escape from complement­associated immunosurveillance and abnormally express certain tumor­suppressor genes inducing AT II cells to develop into lung adenocarcinoma. The present study further elucidated the effects and mechanisms of SLC34A2 in the generation and development of lung cancer.

Synergistic effects of eukaryotic coexpression plasmid carrying LKB1 and FUS1 genes on lung cancer in vitro and in vivo.

PURPOSE: LKB1 and FUS1 are two kinds of new tumor suppressor genes as well as early-stage genes in lung cancer. Recent studies showed that LKB1 and FUS1 play important roles in lung carcinogenesis process. We hypothesized that combined gene therapy with LKB1 and FUS1 could inhibit lung cancer growth and development synergistically. METHODS: In this study, two kinds of tumor suppressor genes, LKB1 and FUS1, were constructed in an eukaryotic coexpression plasmid pVITRO(2), and then, we evaluated the synergistic effects of the two genes on anticancer activity and explored the relevant molecular mechanisms. RESULTS: We defined coexpression of LKB1 and FUS1 could synergistically inhibited lung cancer cells growth,invasion and migration and induced the cell apoptosis and arrested cell cycle in vitro. Intratumoral administration of liposomes: pVITRO(2)­LKB1­FUS1 complex (LPs­pVITRO(2)­LKB1­FUS1) into subcutaneous lung tumor xenograft resulted in more significant inhibition of tumor growth. Furthermore, intravenous injection of LPs­pVITRO(2)­LKB1­FUS1 into mice bearing experimental A549 lung metastasis demonstrated synergistic decrease in the number of metastatic tumor nodules. Finally, combined treatment with LKB1 and FUS1 prolonged overall survival in lung tumor-bearing mice. Further study showed tha tthe synergistic anti-lung cancer effects of coexpression ofLKB1 and FUS1 might be related to upregulation of p-p53, p-AMPK and downregulation of p-mTOR, p-FAK, MMPs, NEDD9, VEGF/R and PDGF/R. CONCLUSIONS: Our results suggest that combined therapy with eukaryotic coexpression plasmid carrying LKB1 and FUS1 genes may be a novel and efficient treatment strategy for human lung cancer.

Cationic liposome mediated delivery of FUS1 and hIL-12 coexpression plasmid demonstrates enhanced activity against human lung cancer.

FUS1 is one of the most important tumor suppressor genes in lung cancer, as well as an important immunomodulatory molecule. Interleukin (IL)-12 has attracted considerable interest as a potential anti-tumor cytokine. Cationic liposome has been shown to effectively deliver therapeutic genes to the lungs and control metastatic lung tumors when administered intravenously. Here we evaluated the enhanced efficacy of cationic liposome-mediated delivery of FUS1 and human IL (hIL)-12 eukaryotic coexpression plasmid (pVITRO2-FUS1-hIL-12) against the human lung cancer in HuPBL-NOD/SCID mice model by local and systemic administration, and explored the related molecular mechanism. Our study demonstrated that FUS1-hIL-12 coexpression could more sufficiently inhibit tumor growth and experimental lung metastasis, significantly prolong the survival of experimental lung metastasis mice. Moreover, FUS1-hIL-12 coexpression performed higher antitumor activity and lower toxicity in the inhibition of experimental lung metastatic tumor compared to cisplatin. We further identified that FUS1-hIL-12 coexpression could induce strong antitumor immune response by secreting much higher levels of human interferon-γ (hIFN-γ) and hIL-15, enhancing expression of MHC-I and Fas, increasing infiltration of activated human CD4+ and CD8+ T lymphocytes. FUS1-hIL-12 coexpression could also obviously induce tumor cell apoptosis and inhibit tumor cell proliferation partly by higher activation of STAT1 signal pathway and upregulation of p53. In addition, FUS1-hIL-12 coexpression also superiorly reduced the angiogenesis in tumors, which might be associated with downregulation of VEGF and VEGFR, and upregulation of human IP-10. Our results therefore suggest that cationic liposome-mediated FUS1-hIL-12 coexpression may be a new promising strategy for lung cancer treatment in clinical studies.

MicroRNA-143 inhibits migration and invasion of human non-small-cell lung cancer and its relative mechanism.

BACKGROUND: MicroRNAs (miRNAs) play important roles in many biological processes, including cancer development. Among those miRNAs, miR-143 shows tumor-suppressive activity in some human cancers. However, the function and mechanism of miR-143 in lung cancer cells remains unknown. Here we explored the role of miR-143 in lung cancer. RESULTS: According to qRT-PCR, we found that miR-143 was notably down-regulated in 19 NSCLC tissues and 5 cell lines. In vitro experiments showed us that miR-143 could significantly suppress the migration and invasion of NSCLC cell lines while it had no effects on the growth of NSCLC cell lines, and in vivo metastasis assay showed the same results. Finally, we found that the mechanism of miR-143 inhibiting the migration and invasion of NSCLC might be through targeting CD44v3. CONCLUSIONS: The up-regulated miR-143 in lung cancer could significantly inhibit cell migration and invasion, and this might work through targeting CD44v3, which was newly identified by us.

Cationic liposome-mediated nitric oxide synthase gene therapy enhances the antitumor effects of cisplatin in lung cancer.

Cisplatin is one of the most effective antitumor drugs for non-small cell lung carcinoma (NSCLC) patients. However, its efficacy has encountered a plateau due to its side effects and drug resistance. Inducible nitric oxide (NO) synthase (iNOS) gene therapy has been reported to have antitumor effects in several types of cancers and enhances sensitivity to cisplatin, but the effects of iNOS gene therapy alone or its combination with cisplatin in lung cancer remain unclear. In the current study, we evaluated the effects of cationic liposome (LP)-mediated iNOS gene transfection on enhancing low-dose cisplatin-mediated antitumor effects in the A549 human lung adenocarcinoma cell line in vitro. Furthermore, we examined whether iNOS gene therapy enhances the antitumor effects of low-dose cisplatin in two A549 human lung cancer cell xenograft mouse models. The results revealed that iNOS gene therapy may significantly enhance low-dose cisplatin-mediated inhibition of cell proliferation, invasion, migration and promotion of cell apoptosis in A549 cells. Intratumoral administration of the LP-pVAX-iNOS complex significantly enhanced low-dose cisplatin-mediated suppression of subcutaneous tumor growth. Moreover, intravenous injection of the LP-pVAX-iNOS complex greatly enhanced low-dose cisplatin-mediated inhibition of experimental lung metastasis and prolonged the life span of mice without significant organ-related toxicity in a nude mouse model of lung metastasis compared to the cisplatin alone-treated group. Furthermore, iNOS gene-mediated enhancement of cisplatin-mediated antitumor effects in lung cancer may be related to the attenuation of p-mTOR, MMP2 and the activation of p-p53. Thus, the combination treatment with iNOS gene therapy and cisplatin may be a novel and effective therapeutic strategy for lung cancer.

Cyclen-based cationic lipids for highly efficient gene delivery towards tumor cells.

BACKGROUND: Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. METHODS: In this report, we designed and synthesized three amphiphilic molecules (L1-L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. RESULTS: Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1-L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. CONCLUSION: Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen.