Captopril-polyethyleneimine conjugate modified gold nanoparticles for co-delivery of drug and gene in anti-angiogenesis breast cancer therapy

Captopril-polyethyleneimine (CP) containing low molecular weight polyethyleneimine and anti-angiogenesis drug captopril conjugated via an amide bond was fabricated to modify gold nanoparticles and complex with siRNA to construct siRNA/CP/GNP complexes for the co-delivery of drug and siRNA in anti-angiogenesis breast cancer therapy. The self-assembled siRNA/CP/GNP complexes exhibited desirable and homogenous particle size, reasonable positive charges and condensation ability, and effective gene-silencing property in vitro. In addition, siRNA/CP/GNP complexes co-delivering captopril and siRNA achieved combined angiogenesis suppression by more effectively downregulating the expression of vascular endothelial growth factor mRNA and protein via different pathways in vitro, as compared to mono-delivery systems. In vivo investigation on nude mice bearing MDA-MB435 tumor xenografts revealed that siRNA/CP/GNP complexes possessed satisfying tumor homing ability and strong antitumor activity. These findings suggested that siRNA/CP/GNP complexes could be an ideal system for simultaneous transfer of drug and siRNA, which might be a new promising strategy for effective breast cancer therapy.     

Genetic deletion and overexpression of angiotensin II receptors

Angiotensin II receptors are essential components of the renin-angiotensin system transducing angiotensin II mediated signals across the plasma membrane of many cell types in the cardiovascular system. To date, three subtypes of angiotensin II receptors have been identified by molecular cloning, termed angiotensin II type 1 (AT_1A, AT_1B) and type 2 (AT₂) receptors. This review focuses on recent transgenic animal models which have been generated to study the in vivo significance of angiotensin receptor diversity. AT_1A receptors are the major blood pressure regulators and have a potent growth-stimulatory effect on cardiac myocytes in vivo. The AT_1B receptor subtype may participate in the control of vascular tone if AT_1A receptors are absent. AT₂ receptors are abundantly expressed during embryonic development and may also play a role in blood pressure regulation by influencing vascular development and differentiation. Received: 16 February 1998 / Accepted: 10 August 1998     

Supramolecular self-assembly forming a multifunctional synergistic system for targeted co-delivery of gene and drug

For developing a multifunctional bioreducible targeted and synergistic co-delivery system for anticancer drug paclitaxel (PTX) and p53 gene for potential cancer therapy, supramolecular self-assembled inclusion complex was prepared from PTX and star-shaped cationic polymer containing γ-cyclodextrin (γ-CD) and multiple oligoethylenimine (OEI) arms with folic acid (FA) conjugated via a disulfide linker. The inclusion complex, termed as γ-CD-OEI-SS-FA/PTX, was formed between PTX and the hydrophobic cavity of γ-CD core of the star polymer. The γ-CD-OEI-SS-FA/PTX complex further formed polyplexes with pDNA to give positively charged nanoparticles, becoming multifunctional supramolecular self-assembled co-delivery system for PTX and pDNA targeting to cancer cells that overexpress folate receptors (FRs). The results showed that the FA-targeted function induced higher gene transfection efficiency in the FR-positive KB cells. The redox-sensitive disulfide linker in the self-assembly system led to the detachment of the FA groups from the carrier after the FR-mediated endocytosis, which resulted in the release of the bound FRs followed by the recycling of the FRs from the cytosol onto the cell membrane surface, facilitating continuous FR-mediated endocytosis to achieve enhanced gene transfection. In addition, the complexed PTX was co-delivered to the cells with pDNA, which further enhanced the gene transfection even at low N/P ratios in the FR-positive KB cells. Further, the efficient delivery of wild-type p53 gene resulted in large cell population at sub G1 and G2/M phases, inducing significant cell apoptosis. Therefore, the multifunctional γ-CD-OEI-SS-FA/PTX self-assembly system with the synergistic effects of redox-sensitive FA-targeted and PTX-enhanced p53 gene delivery may be promising for cancer therapeutic application.     

pH-Responsive polymer-liposomes for intracellular drug delivery and tumor extracellular matrix switched-on targeted cancer therapy

This study presents a tumor-extracellular matrix pH-induced targeting liposome (ECM-targeting liposomes), crosslinked from methoxy-poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide-co-histidine)-cholesterol copolymers and biotin2-polyethylene glycol crosslinkers by hydrogen bonds to overcome the defects of liposomes. In this study, ECM-targeting liposomes were completely investigated their pH-responsibility, drug releasing behaviors, anticancer efficiencies and the time-dependent organ distribution and toxic effects. Experimental results indicate that ECM-targeting liposomes showed rapid drug releasing profiles in acidic conditions. Because the ECM-targeting liposomes accumulated preferentially in tumor, the ECM-targeting liposomes exhibited exceptional anticancer activity in vivo and lower hepatic and renal toxicity. The ECM-targeting liposomes which are switched on the targeting ability in tumor ECM possess potential for future application in anticancer therapy.     

Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer

Metastatic relapse, development of drug resistance in cancer cells and adverse side effects of chemotherapeutic agents are the major obstacles for effective chemotherapy against triple-negative breast cancer. To address these problems, miR-34a, a potent endogenous tumor suppressive molecule in breast cancer, was co-encapsulated with doxorubicin (DOX) into hyaluronic acid (HA)-chitosan (CS) nanoparticles (NPs) and simultaneously delivered into breast cancer cells for improved therapeutic effects of drug. DOX-miR-34a co-loaded HA-CS NPs were successfully prepared through ionotropic gelation method in water. In vitro and in vivo experiments showed that miR-34a and DOX can be efficiently encapsulated into HA-CS NPs and delivered into tumor cells or tumor tissues and enhance anti-tumor effects of DOX by suppressing the expression of non-pump resistance and anti-apoptosis proto-oncogene Bcl-2. In addition, intracellular restoration of miR-34a inhibited breast cancer cell migration via targeting Notch-1 signaling. The obtained data suggest that co-delivery of DOX and miR-34a could achieve synergistic effects on tumor suppression and nanosystem-based co-delivery of tumor suppressive miRNAs and chemotherapeutic agents may be a promising combined therapeutic strategy for enhanced anti-tumor therapy.     

Co-delivery of doxorubicin and tumor-suppressing p53 gene using a POSS-based star-shaped polymer for cancer therapy

In this work, a star-shaped polymer consisting of a cationic poly[2-(dimethylamino) ethyl methacrylate] (PDMAEMA) shell and a zwitterionic poly[N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide] (PMPD) corona was grafted from a polyhedral oligomeric silsesquioxanes (POSS)-based initiator via atomic transfer radical polymerization (ATRP). The reported star-shaped polymer could form stable micelles in aqueous solutions even in the presence of serum. In addition, anti-cancer drug doxorubicin and tumor-suppressing p53 gene were loaded in the process of micelle formation. The formed polyplex was biocompatible and highly efficient for both drug and gene delivery. Furthermore, the polyplex was able to cause a high apoptotic rate of tumor cells both in vitro and in vivo. This combination delivery strategy offers a promising method for cancer therapy and can be used for further clinical applications.     

用于肿瘤联合治疗的基因和化疗药物纳米共载体系的研究进展

化学药物治疗（化疗）或基因治疗单独使用治疗肿瘤均具有较多缺陷,而将两者联合应用能协同治疗肿瘤,克服单一疗法的不足.纳米载体既能包载化疗药物又能递送基因,其用于肿瘤的联合治疗,可减少化疗药物的剂量,增加药物在靶器官的分布量,减轻毒副作用,从而提高抗肿瘤效果;同时保护携带基因的稳定性和完整性,一定程度上提高基因的转染效率,以达到减轻毒副作用及提高疗效的协同目的.基因和化疗药物纳米共载体系用于肿瘤的联合治疗是近年来肿瘤治疗的研究热点.就基因和化疗药物纳米共载体系的类型及负载基因类型,特别是纳米共载体系用于肿瘤联合治疗的研究进行总结和展望.     

Sequence variants of ACE, AGT, AT1R, and PAI-1 as genetic risk factors for vascular dementia

Sequence variants of angiotensin converting enzyme (ACE) insertion/deletion (I/D), angiotensinogen (AGT) T235M, angiotensin II type 1 receptor (AT1R) A1166C, and plasminogen activator inhibitor-1 (PAI-1) 4G/5G were analyzed to see their genetic associations with vascular dementia as its candidate genetic risk factors involving renin-angiotensin and fibrin systems. While the ACE I/D, AT1R A1166C, and PAI-1 4G/5G did not contribute to the genetic susceptibility to vascular dementia (P>0.05), a significant association with vascular dementia was shown in the T235M polymorphism of AGT. The frequency of the M allele in patients was higher than in controls with the odds ratio (OR) estimate of 1.51 (P<0.05). In a dominant model, the TM+MM genotypes increased the risk of vascular dementia compared to the TT genotype (OR=2.01; P<0.001). The current results suggested that AGT T235M polymorphism might be a risk factor of vascular dementia.     

Gold nanoparticles functionalized with therapeutic and targeted peptides for cancer treatment

Functionalization of nanostructures such as gold nanoparticles (AuNPs) with different biological molecules has many applications in biomedical imaging, clinical diagnosis and therapy. Researchers mostly employed AuNPs larger than 10 nm for different biological and medicinal applications in previous studies. Herein, we synthesized a novel small (2 nm) AuNPs, which were functionalized with the therapeutic peptide, PMI (p12), and a targeted peptide, CRGDK for selective binding to neuropilin-1(Nrp-1) receptors which overexpressed on the cancer cells and regulated the process of membrane receptor-mediated internalization. It was found that CRGDK peptides increased intracellular uptake of AuNPs compared to other surface conjugations quantified by ICP-MS. Interestingly, CRGDK functionalized AuNPs resulted in maximal binding interaction between the CRGDK peptide and targeted Nrp-1 receptor overexpressed on MDA-MB-321 cell surface, which improved the delivery of therapeutic P12 peptide inside targeted cells. Au@p12 + CRGDK nanoparticles indicated with highly effective cancer treatment by increasing p53 expression upregulated with intracellular enhanced p12 therapeutic peptide. These results have implications to design and functionalize different molecules onto AuNPs surfaces to make hybrid model system for selective target binding as well as therapeutic effects for cancer treatment.     

非小细胞肺癌表皮生长因子受体及其靶向治疗研究进展

非小细胞肺癌是目前全球最常见的恶性肿瘤之一,尽管手术治疗和化学治疗技术不断发展,但非小细胞肺癌患者生存率却没有明显改变。表皮生长因子受体 (epidermal growth factor receptor,EGFR)是一种受体型酪氨酸激酶,在非小细胞肺癌中有过表达,且与非小细胞肺癌的发生、发展、侵袭等方面密切相关,是最有前途的特异性肿瘤靶向治疗分子之一。此外,在非小细胞肺癌中常检测到EGFR基因突变,尤其是在女性、非吸烟者、肺腺癌和亚洲人种中,这与非小细胞肺癌患者对吉非替尼治疗的敏感性密切相关。     

Identification of lineariifolianoid A as a novel dual NFAT1 and MDM2 inhibitor for human cancer therapy

There is an increasing interest in development of novel anticancer agents that target oncogenes.We have recently discovered that nuclear factor of activated T cells 1(NFAT1) is a novel regulator of the Mouse Double Minute 2(MDM2) oncogene and the NFAT1-MDM2 pathway has been implicated in human cancer development and progression,justifying that targeting the NFAT1-MDM2 pathway could be a novel strategy for discovery and development of novel cancer therapeutics.The present study was designed to examine the anticancer activity and underlying mechanisms of action of lineariifolianoid A(LinA),a novel natural product inhibitor of the NFAT1-MDM2 pathway.The cytotoxicity of LinA was first tested in various human cancer cell lines in comparison with normal cell lines.The results showed that the breast cancer cells were highly sensitive to LinA treatment.We next demonstrated the effects of LinA on cell proliferation,colony formation,cell cycle progression,and apoptosis in breast cancer MCF7 and MDA-MB-231 cells,in dose-dependent and p53-independent manners.LinA also inhibited the migration and invasion of these cancer cells.Our mechanistic studies further indicated that its anticancer activities were attributed to its inhibitory effects on the NFAT1-MDM2 pathway and modulatory effects on the expression of key proteins involved in cell cycle progression,apoptosis,and DNA damage.In summary,LinA is a novel NFAT1-MDM2 inhibitor and may be developed as a preventive and therapeutic agent against human cancer.     

Primed in situ labeling (PRINS) for evaluation of gene deletions in cancer

Rearrangements involving the 13q14 and 17p13 chromosomal regions are often observed in leukemias and lymphomas. These rearrangements are not always identifiable cytogenetically. In more than 50% of cases, deletions occur at the submicroscopic level and the karyotypes appear normal. Molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) have accordingly contributed to the identification of a variety of subtle rearrangements such as those involving submicroscopic deletions. However, FISH is expensive, time consuming, technically burdensome, and requires cloned DNA probes. A newer technique, primed in situ labeling (PRINS), has been tested as a possible alternative to FISH. To assess the utility and efficiency of the PRINS method in the detection of RB1 and p53 deletions, we evaluated 10 patients with hematological disorders and known rearrangements, i.e., deletions involving 13q14 and 17p13 regions. The data in these cases were validated against data obtained with standard FISH probes. Our results indicate that PRINS could be used with relative ease in cytogenetics laboratories and could serve as an alternative to conventional FISH for defining deletions involving unique sequences. © 2001 Wiley‐Liss, Inc.     

Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients

The amplification of cyclin D1, located on chromosome 11q13, in breast cancer patients has been found to be associated with reduced relapse-free and overall survival; however, there still exists strong controversy about these findings. In order to evaluate the prognostic value of cyclin D1 and other prognostic variables in human breast cancers, we have assessed estrogen receptor (ER) status, cyclin D1, c-erbB2 and p53 overexpression in 175 primary breast carcinomas, and investigated the relationships of prognostic variables to the patient clinical outcome and the association between cyclin D1 overexpression and other prognostic variables. There was some degree of variability in staining intensities and proportions within the same tumor. The overexpression of both cyclin D1 and ER revealed a significantly prolonged survival in univariate analysis (P = 0.020). Among the various prognostic variables, distant metastasis showed a statistically significant association with overall survival. A significant correlation was observed between cyclin D1 overexpression and small size of the primary tumor (P = 0.031), low Bloom and Richardson's histological grade (P = 0.001), and positive ER status (P = 0.000). In contrast to what was previously expected, the present study suggests that the overexpression of cyclin D1 has a tendency to have a positive clinical outcome and a potential role in identifying a subset of patients predicting a good prognosis, particularly when ER is coexpressed.     

沉默Notch1基因促进人乳腺癌MCF-7细胞JNK1和p53磷酸化

 目的：探究沉默Notch1基因对人乳腺癌MCF-7细胞JNK1和p53磷酸化的影响。方法：选取人乳腺癌MCF-7细胞作为研究对象,构建shRNA-Notch1真核表达质粒用于转染MCF-7细胞使Notch1基因沉默。采用Western blotting方法检测MCF-7细胞Notch1、Hes-1、PUMA和NOXA蛋白的表达,JNK1和p53蛋白磷酸化水平以及caspase-3活化水平的改变。应用流式细胞术检测细胞凋亡和线粒体膜电位的变化。结果：人乳腺癌MCF-7细胞Notch1基因被沉默后,Notch1和Hes-1蛋白表达量明显减少(P<0.01),细胞凋亡率显著升高(P<0.01),JNK1和p53的磷酸化水平明显高于对照组(P<0.01),PUMA和NOXA表达量显著升高(P<0.05),cleaved caspase-3蛋白明显多于对照组(P<0.01),线粒体膜电位明显下降(P<0.05)。结论：沉默Notch1基因可能通过激活JNK1信号通路活化p53,促进PUMA和NOXA蛋白表达,进而通过线粒体途径导致人乳腺癌MCF-7细胞凋亡。     

PI3K/Akt/mTOR信号通路及其相关基因突变与子宫内膜癌靶向性药物治疗的研究进展

子宫内膜癌(EC)是最常见的女性生殖道恶性肿瘤之一,其发生发展的分子生物学机制十分复杂。近年来研究发现,磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路调节异常与子宫内膜癌密切相关。PI3K/Akt/mTOR信号通路中的多种受体及激酶的突变和异常激活,可能成为子宫内膜癌治疗的靶点。     

Comparison of in vitro micronucleus and gene mutation assay results for p53-competent versus p53-deficient human lymphoblastoid cells

The high frequency of false or irrelevant positive results in in vitro mammalian cell genotoxicity tests is a critical concern for regulators. Here, we tested whether such results may be due to the mammalian cells used in the tests being deficient in p53, which is involved in the maintenance of genomic stability. We compared the in vitro responses of two human lymphoblastoid cell lines derived from the same progenitor cell-p53-competent (TK6) and p53-deficient (WTK-1) cells-in a micronucleus (MN) test and a thymidine kinase gene (TK) mutation assay. We tested 14 chemicals including three mutagens and 11 clastogens and spindle poisons. The three mutagens evoked clear positive responses in both assays in both cell lines. The responses to the clastogens and spindle poisons, on the other hand, depended on the assay endpoint and/or the cell line. Most of clastogens and spindle poisons were positive in the MN test in both cell lines. In the TK mutation assay, on the other hand, WTK-1 cells but not TK6 cells detected spindle poisons, which may have been due to the disturbance of the spindle checkpoint and lack of apoptosis in the p53-deficient cells. Some chemicals that induced chromosome aberrations in rodent cells were negative in both TK6 and WTK-1 cells, indicating that a species-specific factor rather than p53 status was associated with the response. In conclusion, the p53 status did not seriously influence the MN test results but it did influence the TK mutation assay results.     

Genomic and bioinformatics analysis of HAdV-7, a human adenovirus of species B1 that causes acute respiratory disease: implications for vector development in human gene therapy

Human adenovirus serotype 7 (HAdV-7) is a reemerging pathogen identified in acute respiratory disease (ARD), particularly in epidemics affecting basic military trainee populations of otherwise healthy young adults. The genome has been sequenced and annotated (GenBank accession no. ). Comparative genomics and bioinformatics analyses of the HAdV-7 genome sequence provide insight into its natural history and phylogenetic relationships. A putative origin of HAdV-7 from a chimpanzee host is observed. This has implications within the current biotechnological interest of using chimpanzee adenoviruses as vectors for human gene therapy and DNA vaccine delivery. Rapid genome sequencing and analyses of this species B1 member provide an example of exploiting accurate low-pass DNA sequencing technology in pathogen characterization and epidemic outbreak surveillance through the identification, validation, and application of unique pathogen genome signatures.     

Expression of proliferating cell nuclear antigen,Ki-67 antigen,estrogen receptor protein,and tumor suppressor p53 gene in cytologic samples of breast cancer: An immunochemical study with clinical,pathobiological, and histologic correlations

Sixty-six unselected breast cancers were analyzed in cytologic smears and histologic sections for the expression of Ki-67, proliferating cell nuclear antigen (PCNA). estrogen receptor protein (ERP), and p53 protein using a standard immunochemical method. The results, expressed as both positive cases and labelling index (LI), were compared with clinical and pathobiological variables. Ki-67 and PCNA immunostaining was seen in all cases, whereas ERP was detectable in 46/63 cases and p53 protein in 20/66 cases. The expression of these markers was generally lower in cytology than in histology, though the differences were not statistically significant. PCNA-LI and Ki-67-LI were closely correlated (P < 0.001), the mean PCNA:Ki-67 ratio being 0.92 ± 0.57. Occasional discrepancies, however, were found. PCNA and Ki-67 expression was associated with an increase in histologic grade and a decrease in ERP content of tumors, whereas p53 was statistically associated with no clinical or pathobiological variables. The data suggest that proliferative activity and oncogene overexpression may be reliably evaluated in breast cancer by FNA cytology, though PCNA is not a suitable indicator for cell proliferation. The results do not resolve the issue as to whether immunostaining for p53 protein constitutes a dedifferentiation product of the tumor, or is a fundamental aspect of the malignant progression. Survival studies in a larger series of tumors are thus needed to elucidate this point. Diagn Cytopathol 1994; 11:131–140. © 1994 Wiley-Liss, Inc.     

Clinical evaluation and COL2A1 gene analysis in 21 Brazilian families with Stickler syndrome: identification of novel mutations, further genotype/phenotype correlation, and its implications for the diagnosis

We present clinical and molecular evaluation from a large cohort of patients with Stickler syndrome: 78 individuals from 21 unrelated Brazilian families. The patients were selected in a Hospital with a craniofacial dysmorphology assistance service and clinical diagnosis was based on the presence of cleft palate associated to facial and ocular anomalies of Stickler syndrome. Analysis of COL2A1 gene revealed 9 novel and 4 previously described pathogenic mutations. Except for the mutation c.556G>T (p.Gly186X), all the others were located in the triple helical domain. We did not find genotype/phenotype correlation in relation to type and position of the mutation in the triple helical domain. However, a significantly higher proportion of myopia in patients with mutations located in this domain was observed in relation to those with the mutation in the non-tripe helical domain (c.556G>T; P < 0.04). A trend towards a higher prevalence of glaucoma, although not statistically significant, was observed in the presence of the mutation c.556G>T. It is possible that this mutation alters the splicing of the mRNA instead of only creating a premature stop codon and therefore it can lead to protein products of different ocular effects. One novel DNA variation (c.1266+7G>C) occurs near a splice site and it was observed to co-segregate with the phenotype in one of the two families with this DNA variation. As in silico analysis predicted that the c.1266+7G>C DNA variation can affect the efficiency of the splicing, we still cannot rule it out as non-pathogenic. Our study also showed that ascertainment through cleft palate associated to other craniofacial signs can be very efficient for identification of Stickler syndrome patients. Still, high frequency of familial cases and high frequency of underdevelopment of distal lateral tibial epiphyses observed in our patients suggested that the inclusion of this information can improve the clinical diagnosis of Stickler syndrome.